DeGroot's Endocrinology: Basic Science and Clinical Practice [1-2, 8 ed.] 0323694128, 9780323694124

Table of contents :
Front Matter
DeGroot’s ENDOCRINOLOGY: Basic Science and Clinical Practice
Copyright
sTOC
Vol I
Chapter (1)
1 - Principles of Endocrinology
Definition and Scope of Endocrinology
Historical Perspectives
Principles of Hormone Action
Hormone Biosynthesis and Secretion
Feedback Regulation
Paracrine and Autocrine Regulation
Hormonal Rhythms and Pulsatility
Hormone Transport and Degradation
Hormone Action Through Receptors
Membrane Receptors
Nuclear Receptors
Role of the Clinical Endocrinologist
Major Unsolved Problems
References
Chapter (2)
2 - Principles of Endocrine Measurements
Principles of Endocrine Measurements
Introduction and Brief History of Endocrine Assays
Radioimmunoassay
Nonisotopic Immunoassays
Monoclonal Antibodies
Structural Assays
Preanalytic Methods and Harmonization of Assays
Preanalytic Variables
Analytical Parameters
Clinical Parameters
Reference Intervals
General Interferences
Summary
Principle
Modern Example Analytes
Advantages
Disadvantages
Principle
Advantages
Disadvantages
Special Circumstances
Methods for Measurement of Free Hormones
Additional Techniques
Special Circumstances
Plasma Renin Activity Assay and its Modifications
Advantages
Disadvantages
Direct Renin Concentration
Gas Chromatography—Mass Spectrometry
Liquid Chromatography—Tandem Mass Spectrometry
Special Circumstances
Summary and Future Directions
References
Chapter (3)
3 - Endocrine Rhythms, the Sleep-­Wake Cycle, and Biological Clocks
Endocrine Rhythms, the Sleep-­Wake Cycle, and Biological Clocks
Introduction
What Are Biological Rhythms
The Sleep-­Wake Cycle as a Rhythm
Clocks in the Brain and the Periphery
Endocrine Rhythms
Examples of Endocrine Rhythms in Humans
Glucocorticoid Pulsatility
Rhythms in Endocrine Disease
Limitations of Single Timepoint Assessments
Cell Clocks
The Sleep-­Wake Cycle
Sleep Regulation
Sleep Stages
Sleep Mistiming and Human Health
Jet Lag and Social Jet Lag
Summary
Conclusions
References
Chapter (4)
4 - Prolactinomas and Disorders of Prolactin Secretion
History
Epidemiology
Pathogenesis
Pathology
Nontumoral Lesions Associated With Hyperprolactinemia
Pituitary Prolactin-­Secreting Carcinomas
Physiologic Hyperprolactinemia
Pharmacologic Hyperprolactinemia
Pathologic Hyperprolactinemia
Diagnosis of Prolactinomas
Clinical Features
Laboratory Evaluation
Imaging
Treatment
Medical Therapy
Surgical Therapy
Radiotherapy
Therapeutic Advances and Perspectives
Prolactinomas and Pregnancy
Treatment Planning and Follow-­Up
References
Chapter (5)
5 - Hypopituitarism Including Growth Hormone Deficiency
HISTORY
ETIOLOGY OF HYPOPITUITARISM
Hypophysitis
MORBIDITY AND MORTALITY
CLINICAL FEATURES
DIAGNOSIS AND ENDOCRINE EVALUATION
Adrenocorticotropic Hormone Deficiency: Secondary Adrenal Insufficiency
Thyroid-Stimulating Hormone Deficiency: Central Hypothyroidism
Luteinizing Hormone/Follicle-Stimulating Hormone Deficiency: Hypogonadotropic Hypogonadism
Growth Hormone Deficiency
Hypopituitarism Following Pituitary Surgery
HORMONE REPLACEMENT THERAPY
Adrenocorticotropic Hormone Deficiency: Secondary Adrenal Insufficiency
Thyroid-Stimulating Hormone Deficiency Central Hypothyroidism
Luteinizing Hormone/Follicle-Stimulating Hormone Deficiency: Hypogonadotropic Hypogonadism
Growth Hormone Deficiency
Childhood-Onset Growth Hormone Deficiency in Adults
Hormone–Hormone Interactions
PREGNANCY
REFERENCES
Chapter (6)
6 - Acromegaly
Excess Growth Hormone Secretion
Acromegaly
6
Excess Growth Hormone–Releasing Hormone Secretion
Role of the Hypothalamus in the Etiology of Acromegaly
Intrinsic Pituitary Lesions
Candidate Genes in the Etiology of Acromegaly
Epidemiology
Diagnosis
Documenting Growth Hormone Hypersecretion
Localizing the Source of Excess Growth Hormone
Clinical Manifestations
Laboratory Findings
Mortality
Management
Treatment Goals
Surgery
Pituitary Irradiation
Pharmacologic Management (Fig. 6.5)
Integrated Treatment Approach to the Management of Acromegaly
Patients With Likelihood of Good Surgical Outcome
Follow-­Up
Additional Management Considerations
References
Chapter (7)
7 - Cushing Disease
Etiology of Cushing Disease
Cushing Disease
7
Pathophysiology
Clinical Features
Clinical Aspects
Biochemical Diagnosis of Cushing Syndrome
General Considerations
First-­Line Biochemical Investigations
Second-­Line Biochemical Investigations
Other Biochemical Investigations
Establishing Adrenocorticotropic Hormone–Dependency of Cushing Syndrome
Differential Diagnosis of Adrenocorticotropic Hormone–Dependent Cushing Syndrome
Hypercortisolemic States Without Cushing Syndrome
Differentiating Cushing disease From Ectopic Adrenocorticotropic Hormone Syndrome and Ectopic Corticotropin-­Releasing Hormone S...
Treatment of Cushing Disease
General Considerations
Somatostatin Analogs
Dopamine Agonists
Glucocorticoid Receptor–Directed Drugs
Combination of Medical Treatments
Future Medical Treatments
Metabolic Syndrome and Cardiovascular Risk
Muscle
Bone Disease
Mood and Cognitive Function
Autoimmunity
Quality of Life
Mortality in Cushing Disease
Implications for Treatment and Long-­Term Care
Special Conditions
Cushing Disease as an Emergency Condition
Cyclic Cushing Syndrome
Pediatric Cushing Disease
Cushing Disease and Pregnancy
Cushing Disease and Chronic Renal Failure
Future Directions
References
Chapter (8)
8 - Clinically Nonfunctioning Sellar Masses
Clinically Nonfunctioning Sellar Masses
Pituitary Adenomas
Etiology of Pituitary Adenomas
Other Benign Tumors
Malignant Tumors
Pituitary Hyperplasia
Hypophysitis
Other Sellar Masses
Clinical Presentations
Neurologic Presentations
Incidental Finding
Endocrinologic Presentations
Pituitary Apoplexy
Diagnosis
Imaging of the Sellar Region
Endocrinologic Tests
Pathologic Evaluation
Treatment
Surgery
Radiation
Pharmacologic Treatment
Observation
References
Chapter (9)
9 - Thyroid-­Stimulating Hormone–Producing Pituitary Tumors
Epidemiology
Thyroid-­Stimulating Hormone–Producing Pituitary Tumors
9
Etiopathogenesis and Pathology
Etiopathogenesis
Pathology
Clinical Presentation
Differential Diagnosis
Investigation
Step 1 – Clinical Assessment
Step 2 – Exclusion of Laboratory Assay Interference
Step 3 – Distinguishing a Thyroid-­Stimulating Hormone–Secreting Pituitary Adenoma From TRβ Resistance to Thyroid Hormone
Step 4 – Pituitary Imaging
Management
Conclusions
References
Chapter (10)
10 - Pituitary Surgery
Pituitary Adenoma
Pituitary Surgery
10
Rathke’s Cleft Cyst
Craniopharyngioma (see also Chapter 11)
Other Lesions
Preoperative Management
Endocrine Status
Imaging Evaluation
Surgical Technique
Endoscopic Endonasal Approach for Pituitary Adenoma, Rathke’s Cleft Cyst, and Craniopharyngioma
Diabetes Insipidus
Syndrome of Inappropriate Antidiuretic Hormone
Early Remission
Major Complications
Permanent Hypopituitarism
Cerebrospinal Fluid Leak and Meningitis
Cranial Nerve Injury
Postoperative Intrasellar Hematoma
Internal Carotid Artery Injury
Resection And Remission rates
Pituitary Adenoma
Rathke’s Cleft Cyst
Craniopharyngioma (see also Chapter 11)
Pituitary Centers Of Excellence
Conclusion
References
Chapter (11)
11 - Craniopharyngiomas and Parasellar Masses
Craniopharyngiomas and Parasellar Masses
Types of Parasellar Masses
Craniopharyngiomas
Epidemiology
Pathology
Pathogenesis
Imaging Features
Management
Long-­Term Morbidity and Mortality
Rathke’s Cleft Cysts
Epidemiology
Pathology
Pathogenesis
Imaging Features
Management
Meningiomas
Gliomas
Germ Cell Tumors
Langerhans Cell Histiocytosis
Hamartomas
Pituitary Metastasis
References
Chapter (12)
12 - Disorders of Sodium, Diabetes Insipidus and Hyponatremia
Physiology of Sodium and Water Homeostasis
Synthesis and Secretion of Vasopressin
Disorders of Sodium, Diabetes Insipidus and Hyponatremia
12
Cellular Action of Vasopressin
Osmoregulation of Vasopressin Release
Baroregulation of Vasopressin Release
Other Mechanisms Regulating Vasopressin Release
Secretion of Copeptin
Diabetes Insipidus
Hypothalamic Diabetes Insipidus
Nephrogenic Diabetes Insipidus
Primary Polydipsia
Investigation of Diabetes Insipidus
Adipsic Diabetes Insipidus
Treatment of Diabetes Insipidus
Hyponatremia
Morbidity and Mortality
Differential Diagnosis of Hyponatremia
The Syndrome of Inappropriate Antidiuresis
Introduction
Causes of Syndrome of Inappropriate Antidiuresis
Pathophysiology of Syndrome of Inappropriate Antidiuresis
Diagnosis of Syndrome of Inappropriate Antidiuresis
Differential Diagnosis of Syndrome of Inappropriate Antidiuresis
Treatment of Hyponatremia
Factors Affecting Therapy
Treatment of Chronic Hyponatremia Owing to Syndrome of Inappropriate Antidiuresis
Treatment of Acute Hyponatremia
Conclusions
References
Chapter (13)
13. Anatomy and Physiology of the Hypothalamus and Pituitary
Anatomy and Physiology of the Hypothalamus and Pituitary
Anatomy of the Hypothalamus
Blood Supply of the Hypothalamus
The Median Eminence and Tanycytes
Anatomy and Histology of the Pituitary
The Blood Supply of the Pituitary
The Pars Intermedia
The Pars Tuberalis
The Pars Distalis (Anterior Pituitary)
Posterior Pituitary Gland (Neurohypophysis)
Conclusions and Future Directions
References
Chapter (14)
14 - Genetic Disorders of the Pituitary
Genetic Disorders of the Pituitary
Introduction
Familial Isolated Pituitary Adenoma
AIP-­Related Familial Isolated Pituitary Adenoma
X-­Linked Acrogigantism
Syndromic Pituitary Tumors
Multiple Endocrine Neoplasia Type 1
Multiple Endocrine Neoplasia Type 4
Carney Complex
McCune–Albright Syndrome
DICER1 syndrome
Lynch Syndrome
Neurofibromatosis 1 and Tuberous Sclerosis Complex
Somatic Mutations in Pituitary Tumors
Conclusion
References
Chapter (15)
15. Aggressive Pituitary Tumors
Aggressive Pituitary Tumors
Aggressive Pituitary Tumors
Definitions
Pituitary Carcinomas
Aggressive Pituitary Tumors
Epidemiology
Natural History of Pituitary Carcinomas
Predictive Factors of Aggressiveness
Pathological Markers of Aggressiveness
Molecular Markers of Aggressiveness
Treatment
Temozolomide
Therapeutic Perspectives
Conclusion
References
Chapter (16)
16. Radiotherapy in Pituitary Tumors
Radiotherapy in Pituitary Tumors
Introduction
Indications for Pituitary Radiotherapy
Three-Dimensional Conformal Radiotherapy
Stereotactic Radiotherapy Techniques
Radiotherapy Fractionation
Linear Accelerator–Based Stereotactic Radiosurgery
Gamma Knife Stereotactic Radiosurgery
CyberKnife Stereotactic Radiosurgery
Proton Therapy
Development of Radiosurgery
Efficacy of Radiotherapy
Overall Survival
Radiological Tumor Control: Progression-Free Survival
Hormone Normalization
Visual Function
Toxicity of Radiotherapy
Hypopituitarism
Visual Pathway Damage and other Cranial Nerve Injuries
Radionecrosis
Cerebrovascular Disease
Second Brain Tumors
Mortality
Proton Therapy
Summary
Reirradiation
Conclusions and Future Directions
References
Chapter (17)
17. Somatic Growth and Maturation: Growth Hormone and Other Growth Factors
Somatic Growth and Maturation: Growth Hormone and Other Growth Factors
Introduction
Prenatal Considerations
Chronological Age
Growth Status
Growth Rate
Maturity Status
Skeletal Maturity Status
Secondary Sex Characteristics
Maturity Timing
Tempo of Maturation
Evaluation of Children for Disorders of Growth
Growth Charts
Evaluation of Children With Short Stature
History
Physical Examination
Preliminary Laboratory Evaluation
Evaluation for Endocrine Disorders of Growth
Growth Hormone/Insulin Growth Factor-1 Axis
Growth Hormone Release and Action
Diagnostic Approach
Growth Hormone Stimulation Tests
Limitations of Growth Hormone Stimulation Tests
Growth Hormone Treatment (See Also Chapter 18)
Dosing and Monitoring of Growth Hormone Therapy
Evaluation of Children With Tall Stature
Endocrine Causes of Tall Stature
Treatment of Children/Adolescents With Tall Stature
Growth Evaluation of Children With Obesity
Conclusions
References
Chapter (18)
18 - Growth Hormone Deficiency in Children
INTRODUCTION
EPIDEMIOLOGY
PATHOGENESIS
Genetic and Structural Abnormalities
Acquired Defects
The Hypothalamo-Pituitary-Somatotroph Axis
CLINICAL FEATURES
Neonatal Presentation
Infant And Childhood Years
Guidance Derived From Clinical Assessment
Principles of Testing
Diagnosis of Growth Hormone Deficiency
A Practical Approach to Diagnostic Evaluation
TREATMENT
Growth Hormone
Transition to Adult Care
CONCLUSIONS
REFEERENCES
Chapter (19)
19. Basic and Clinical Appetite Regulation and Energy Expenditure
Introduction
Components of Eating Behavior
Appetite Regulation
Leptin, Growth Differentiation Factor 15, and Endocannabinoids
The Role of Genetics and Epigenetics
Components of Energy Expenditure
Resting Metabolic Rate
Thermic Effect of Food
Physical Activity Energy Expenditure
Brown Adipose Tissue and Uncoupled Respiration
Body Weight Regulation
Body Weight Regulation and Eating Behavior: Beyond Homeostasis
Contributions of Components of Total Daily Energy Expenditure With Prospective Weight Change
Physiological Responses to Weight Loss and Weight Regain
The Effects of Exercise on Energy Balance
Conclusions
References
Chapter (20)
20. Obesity: Risk, Risk Factors, and Medical Management
Obesity: Risk, Risk Factors, and Medical Management
20
Diseases Associated With Obesity
Cardiovascular and Cerebrovascular Disease
Type 2 Diabetes Mellitus
Nonalcoholic Fatty Liver Disease
Gastrointestinal Complications
Cancer
Reproductive Health
Obstructive Sleep Apnea
Osteoarthritis
Cognitive Dysfunction and Alzheimer Disease
Therapy for Obesity
Lifestyle Intervention
Pharmacotherapy
Bariatric Surgery
References
Chapter (21)
21. Genetic Syndromes Associated With Obesity
Genetic Syndromes Associated With Obesity
Introduction
Monogenic Obesity Syndromes
Congenital Leptin and Leptin Receptor Deficiency
Proopiomelanocortin and PCSK1 Deficiency
MC4R Deficiency
SIM1 Deficiency
BDNF, TRKB, and sh2b1 Deficiency
Pleiotropic Obesity Syndromes
Prader–Willi Syndrome
Albright’s Hereditary Osteodystrophy
Bardet–Biedl Syndrome
Alstrom Syndrome
Cohen Syndrome
Rare Variants Associated with Obesity
Conclusions
References
Chapter (22)
22. Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise
Introduction
Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise
22
Postabsorptive Glucose Utilization
Postabsorptive Fatty Acid Utilization
Fatty Acid Reesterification as a Component of Flux
Postabsorptive Glucose Production
Hormonal Governance of Glucose Production
Glucose Counterregulation
Prandial And Postprandial Intermediary Metabolism
Introduction
Digestion, Absorption, and Enteroendocrine Cells
Insulin and Glucagon Responses
Effects on Energy Expenditure and Respiratory Quotient
Hepatic Glucose Metabolism
Postprandial Adipose Tissue Metabolism
Hepatic Metabolism of Fatty Acids and Lipoproteins
Protein And Amino Acid Metabolism
Exercise Metabolism
Exercise Dramatically Increases Demand For Energy
Energy Metabolism During Exercise
Glucose Metabolism During Exercise
Fatty Acid Metabolism During Exercise
Effects of Exercise Training on Intermediary Metabolism
Summary And Concluding Remarks
References
Chapter (23)
23. Adipose Tissue Function: Metabolic and Endocrine
Different Types of Obesity and Associated Health Risks
Adipose Tissue Function: Metabolic and Endocrine
23
Summary
Adipogenesis And Its Regulation
Adipose Cell Differentiation and Role of Genetic Factors
Role of Aging and Cell Senescence
Summary
Immunometabolism: Its Regulation And Role In Insulin Resistance, Inflammation, And Disease
Linking Inflammation to Insulin Resistance
Summary
Lipid Metabolism In White Adipose Tissue
Lipid Turnover
Methodology
Lipid Uptake and Lipogenesis in Fat Cells
Regulation of Lipolysis
Glucose Metabolism
Summary
Brown versus Beige/Brown Adipose Cells
Aging Reduces Brown and Beige Adipose Cells
Summary
Summary
Adipose Tissue As A Therapeutic Target
Pharmacotherapy in Obesity
Summary
References
Chapter (24)
24. Lipodystrophy Syndromes
Lipodystrophy Syndromes
24
Pathophysiology of Lipodystrophy
Generalized Lipodystrophies
Partial Lipodystrophies
Localized Lipodystrophies
Fat Redistribution and Fat Metabolism
Adipocytokines
Inflammation
Mitochondrial Stress, Oxidative Stress, and the Endoplasmic Reticulum
Other Mechanisms
Lifestyle Modification
Management of Insulin Resistance
Management of Dyslipidemia
Management of Human Immunodeficiency Virus–Infected Patients With Highly Active Antiretroviral Therapy–Induced Metabolic Syndrom...
Management of Cosmetic Appearance
Adipokines in Lipodystrophy
Future Perspectives
Adiponectin
References
Chapter (25)
25. Lipoprotein Metabolism and the Treatment of Lipid Disorders
LIPOPROTEIN METABOLISM
Lipoproteins
Metabolism of Lipids and Lipoproteins
DISORDERS OF LIPID METABOLISM IN PATIENTS WITH DIABETES
Type 1 Diabetes
Type 2 Diabetes
GENETIC BASIS OF LIPID DISORDERS
Monogenic Low-Density Lipoprotein Disorders
Monogenic High-Density Lipoprotein Disorders
Polygenic Lipid Disorders
DIAGNOSIS OF LIPID DISORDERS
MANAGEMENT/TREATMENT
Dietary and Drug Treatment of Lipid Disorders
REFERENCES
Chapter (26)
26. Metabolic Syndrome
DIAGNOSIS AND PREVALENCE
HISTORY
Obesity, Type 2 Diabetes, Insulin Resistance, and Cardiovascular Disease
The Lipid Theory
Inflammation and Oxidative and Endoplasmic Reticulum Stress
Specific Tissues
AMPK and SIRT1
AMPK and Metabolic Syndrome in Humans
DIAGNOSIS
CORONARY HEART DISEASE AND TYPE 2 DIABETES
Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis
Polycystic Ovarian Syndrome
Certain Cancers
Alzheimer Disease
Cushing Syndrome and Related Disorders
Lipodystrophy
Hyperalimentation
TREATMENT OF METABOLIC SYNDROME
Lifestyle Modification (Weight Loss and Physical Activity)
Drug Therapy
Special Considerations
CHILDREN AND ADOLESCENTS
ACKNOWLEDGMENTS
REFERENCES
Chapter (27)
27. Bariatric Procedures and Operations
Bariatric Procedures and Operations
Introduction/Overview
Commonly Performed Bariatric Operations
The Physiology Of Weight Loss After Bariatric Operations And Procedures
Physiologic Effects Of Bariatric Surgery On Cardiometabolic Health And Mortality
Late and Chronic Complications
Weight Regain
Dumping Syndrome
Summary
Chapter (28)
28. Development of the Endocrine Pancreas
Pancreas Morphogenesis
Early Organ Specification And Bud Formation
Tissue Interaction and Signaling Pathways
Induction of the Pancreatic Gene Expression Program
Cell Type Differentiation
Signaling Pathways
Transcription Factors
Islet Formation
References
Chapter (29)
29. Regulation of Islet Hormone Synthesis and Secretion
Hormonal Synthesis
Insulin
Regulation of Islet Hormone Synthesis and Secretion
29
Glucagon
Hormonal Secretion and Action
Insulin
Glucagon
Glucagon-Like Peptide-1 Secretion and Signaling
References
Chapter (30)
30. Mechanisms of Insulin Action
30
Introduction
Insulin
Introduction
Regulation of the Insulin Receptor Tyrosine Kinase
The Insulin Receptor–Related Receptor
The Insulin Signaling Cascade
Introduction
Insulin Receptor Tyrosine Protein Scaffolds
The IRS→PI3K→AKT Cascade
Insulin-Regulated Proteostasis
Insulin-Regulated Glucose Transport
Heterologous Regulation of Insulin Signaling Cascades
Proximal Insulin Signals
Transcriptional Control of Insulin Receptor Substrate 1
Transcriptional Control of Insulin Receptor Substrate 2
Regulation of Insulin Receptor Substrate Degradation
Regulation by Protein and Lipid Phosphatases
miRNA-Mediated Posttranscriptional Regulation
Mouse Genetics Reveal Tissue-Integrated Insulin Signaling
Introduction
Systemic Insulin Receptor Signaling Cascade
Hepatic InsR─┤FoxO1 Signaling
Adipose Insulin Signaling
Insulin/IGF Signaling in Neurodegenerative Disease
Insulin Resistance and Metabolic Disease
Introduction
Chronic Hyperinsulinemia
Nutrient Excess
Proinflammatory Cytokines
References
Chapter (31)
31. Classification and Diagnosis of Diabetes Mellitus
Classification and Diagnosis of Diabetes Mellitus
Definition
Classification
Type 1 Diabetes
Type 2 Diabetes
Other Specific Types of Diabetes
Genetic Defects in β Cell Function
Genetic Defects in Insulin Action
Diseases of the Exocrine Pancreas
Endocrinopathies
Drug- or Chemical-Induced Diabetes
Infections
Uncommon Forms of Immune-Mediated Diabetes
Other Genetic Syndromes Sometimes Associated With Diabetes
Gestational Diabetes
Diagnosis
Diagnostic Criteria
What Level of Fasting Plasma Glucose Constitutes Diabetes
What’s New
References
Chapter (32)
32. Autoimmune (Type 1) Diabetes Mellitus: Etiology, Pathogenesis, Prediction, and Prevention
Epidemiology
Prevalence
Autoimmune (Type 1) Diabetes Mellitus: Etiology, Pathogenesis, Prediction, and Prevention
32
Incidence
Geographical Distribution
Variation with Age
Variation With Gender
Seasonal Variation
Inheritance
Natural History and Disease Staging
Etiology
Genetic Etiology
Environmental Etiology
Pathogenesis
Humoral Immunopathophysiology
Cellular Immunopathophysiology
Diagnostic Criteria
Prediction
Prevention
Primary Prevention
Secondary Prevention
Intervention
Summary and Future Directions
References
Chapter (33)
33. Type 2 Diabetes Mellitus: Etiology, Pathogenesis, and Natural History
Epidemiology
Type 2 Diabetes Mellitus: Etiology, Pathogenesis, and Natural History
33
Origins of the Disease: Etiologic Factors
Region, Race, Ethnicity, and Genetic Factors
Environment and the Social Determinants of Health
Behavior and Culture
Biological Factors
Natural History of Diabetes as an Evolving Glucoregulatory Defect
Diagnosis of Type 2 Diabetes
Overview of the Evolving Glucoregulatory Defect
Progression of Abnormal Glucose Tolerance
Mechanisms Causing Pancreatic β Cell Dysfunction
The Pathophysiology of Insulin Resistance
Measurement of Insulin Sensitivity in Humans: Defect in Muscle Glucose Uptake
Hepatic Insulin Resistance
The Molecular Basis of Human Insulin Resistance in Target Tissues
General Principles
Insulin Resistance at the Level of the Insulin Receptor
Insulin Resistance at the Level of Signal Transduction
Cell Stress Responses and Insulin Action
Nutrient Sensors and Nutrient Stress
Syndromes of Severe Insulin Resistance
Heterogeneity of Type 2 Diabetes
Severe Insulin Resistance
The Spectrum of Cardiometabolic Disease
Role of Obesity
References
Chapter (34)
34. Diabetes Mellitus and Pregnancy
Metabolic Effects of Pregnancy
Diabetes Mellitus and Pregnancy
34
Classification of Diabetes in Pregnancy
Diagnostic Criteria for Gestational Diabetes Mellitus
Glucokinase Monogenic Diabetes and Pregnancy
Bariatric Surgery and Gestational Diabetes
Diabetes and Pregnancy and the Offspring
Congenital Malformations and Early Fetal Loss
Disturbances of Metabolism and Fetal Growth
Maternal Obesity and the Offspring
Management of Diabetes in Pregnancy
Contraception and Preconception Management
Management of Type 1 Diabetes Mellitus in Pregnancy
Glycemia Monitoring
Diet and Physical Activity
Insulin Management
Management of Diabetes Complications in Pregnancy
Management of Gestational Diabetes Mellitus
Prevention of Gestational Diabetes
Obstetric Management of Diabetes in Pregnancy
Obstetric Surveillance
Antenatal Steroids for Preterm delivery
Hypertensive Disorders of Pregnancy
Decrease in Insulin Requirements
Peripartum Management and Birth
Long-Term Outcomes and Management of Gestational Diabetes
Long-Term Risks of Gestational Diabetes to the Mother
Benefits of Breastfeeding for the Mother
Postpartum Follow-up of Mothers
Postpartum Follow-Up of Offspring
Risk of Obesity to the Offspring
Potential Mechanisms for Long-term Effects of Preexisting Maternal Diabetes and Gestational Diabetes on the Offspring
Summary and Future Directions
References
Chapter (35)
35. Hyperglycemia Secondary to Non-Traditional Diabetic Conditions
Hyperglycemia Secondary to Non-Traditional Diabetic Conditions
Pancreatectomy
Acute Pancreatitis
Chronic Pancreatitis
Pancreatic Cancer
Autoimmune Pancreatitis
Hemochromatosis
Hemosiderosis
Cystic Fibrosis
Acromegaly
Growth Hormone Treatment
Cushing Syndrome
Glucagonoma Syndrome
Somatostatinoma
Pheochromocytoma
Hyperglycemia in Critical Care Patients
Drugs That can Cause Hyperglycemia
Drugs That Directly Adversely Affect β Cell Function
Drugs That Inhibit Increases in β Cell Cytosolic Calcium
Drugs That Cause Potassium Depletion
Antiimmune and Cancer-Treatment Drugs
Drugs That Cause Insulin Resistance
Oral Contraceptives and Sex Hormones
Nicotinic Acid
Lipid-Lowering Agents and New-Onset Diabetes Mellitus
Statins
Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors
Cholesterol Ester Transfer Protein Inhibitors
Acquired Immunodeficiency Syndrome and Antiretroviral Drugs
Atypical Antipsychotic Agents
Conclusion
References
Chapter (36)
36. β Cell Glucose Toxicity and Oxidative Stress
36
Introduction
Brief Overview of the Contributions of Oxidative Stress to Secondary Complications of Diabetes
References
Chapter (37)
37. Diabetic Ketoacidosis and Hyperglycemic Hyperosmolar State
Epidemiology
Diabetic Ketoacidosis and Hyperglycemic Hyperosmolar State
37
Pathogenesis of Diabetic Ketoacidosis
Precipitating Factors
Diagnosis of Diabetic Ketoacidosis
Treatment
Complications of Therapy
Prevention
Hyperglycemic Hyperosmolar State
Epidemiology
Pathophysiology
Precipitating Events
Diagnosis
Treatment
Complications
Prevention
References
Chapter (38)
38. Hypoglycemia and Hypoglycemic Syndromes
Accurate Measurement of Blood or Plasma Glucose
Physiology of Hypoglycemia
Brain Fuel Metabolism
Hypoglycemia and Hypoglycemic Syndromes
38
General Approach to the Patient
Acute Treatment of Hypoglycemia
Evaluation of the Hypoglycemic Patient
Hypoglycemia in Diabetes
Mortality and Cardiovascular Disease
Strategies to Reduce Hypoglycemia
Hypoglycemia in the Absence Of Diabetes
Hypoglycemia in Medicated or Ill Individuals
Hypoglycemia in Apparently Well Individuals
Endogenous Hyperinsulinism
Neonatal Hypoglycemia
Conclusion and Future Directions
References
Chapter (39)
39. Treatment of Type 1 Diabetes Mellitus in Adults
39
Goals Of Management
Glycemic Goals
Acute Complications
Chronic Complications
Quality Of Life
Team Approach To Management
Monitoring
Glycemic Control
Ketone Testing
Complication Surveillance
Insulin Therapy
Principles of Insulin Replacement
Insulin Preparations
Intensive Insulin Therapy Regimens
Adjustment of Insulin Therapy in Special Situations
Nutrition
Other Therapies For Type 1 Diabetes
Metformin, DPP-IV Inhibitors, and GLP-1 RAs
Future Perspectives
References
Chapter (40)
40. Clinical Management of Type 2 Diabetes Mellitus
Diabetes Education
Clinical Management of Type 2 Diabetes Mellitus
40
Individualization of the Diabetes Education Care Plan
Nutrition
Nutrition Therapy––the Evidence
Individualization of the Nutrition Prescription
Summary
Physical Activity
Pharmacotherapy Of Type 2 Diabetes
Metformin
Thiazolidinediones
Insulin Secretagogues
Incretin-Based Therapies
Glucagon-Like Peptide-1 Receptor Agonists
Dipeptidyl Peptidase Inhibitors
α-Glucosidase Inhibitors
Insulin Therapy In Type 2 Diabetes
Short-Acting Insulin Analogs
Long-Acting Insulin Analogs (Table 40.5)
Initial Therapy (Fig. 40.1)
Initial Combination Therapy
Diabetes Technology
Self-Monitoring of Blood Glucose
Digital Health Technology
Telemedicine
Population Management
Conclusion
References
Chapter (41)
41. Pancreas and Islet Transplantation
Pancreas and Islet Transplantation
Introduction
Historical Perspective
Pancreas Transplantation
Patient Selection for Pancreas Transplantation
Surgical Approaches to Pancreas Transplantation
Patient and Graft Survival After Pancreas Transplant
Metabolic Outcomes
Allogeneic Islet (Alloislet) Transplantation
Patient Selection for Alloislet Transplantation
Surgical Approaches to Alloislet Transplantation
Patient and Graft Survival After Alloislet Transplant
Metabolic Outcomes
Immunosuppressive Drugs
Autologous Islet (Autoislet) Transplantation
Patient Selection for Autoislet Transplantation
Surgical Approaches to Autoislet Transplantation
Patient and Graft Survival After Autoislet Transplant
Metabolic Outcomes
Effect of Successful Pancreas or Islet Transplantation on Complications of Diabetes
Future Directions and Conclusions
References
Chapter (42)
42. Diabetes and Long-Term Complications
Diabetes and Long-Term Complications
History
Classification
Rheumatic, Dermatologic, and Infectious Manifestations
Epidemiology
Pathophysiology
Can We Prevent Macrovascular Disease
Effects of Specific Hypoglycemic Agents on Cardiovascular Disease and Renal Outcomes in Patients With Type 2 Diabetes
Conclusion
References
Chapter (43)
43. Diabetic Eye Disease
Diabetic Eye Disease
43
Natural History of Diabetic Retinopathy
Proliferative Diabetic Retinopathy
Diabetic Macular Edema, Ischemia, and Traction
Nonproliferative Diabetic Retinopathy Levels
Proliferative Diabetic Retinopathy Levels
Epidemiology
Prevalence and Incidence of Retinopathy
Risk Factors for Diabetic Retinopathy
Diabetes-Related Risk Factors
Other Risk Factors
Other Ocular Diseases Associated With Diabetes
Glaucoma
Visual Impairment
Detection
Telemedicine Programs for Diabetic Retinopathy
Management
Ophthalmic
Surgical
Rehabilitation
Protective and Risk Factors for Diabetic Retinopathy
Novel Therapies
References
Chapter (44)
44. Diabetic Neuropathy
Diabetic Neuropathy
Definition of Diabetic Neuropathies
Classification of Diabetic Neuropathies
Pathogenesis
Distal Symmetrical Polyneuropathy
Epidemiology
Screening and Diagnosis in Clinical Care
Secondary Distal Symmetric Polyneuropathy Complications: Diabetic Foot Ulcers/Charcot Neuroarthropathy
Distal Symmetric Polyneuropathy Treatment
Diabetic Autonomic Neuropathies
Cardiovascular Autonomic Neuropathy
Cardiovascular Autonomic Neuropathy Evaluation in Clinical Research
Treatment
Gastrointestinal Autonomic Neuropathy
Urogenital Autonomic Neuropathy
Sudomotor Dysfunction
Atypical Neuropathies
Mononeuropathies
Diabetic Radiculoplexus Neuropathy
Conclusions
References
Chapter (45)
45. Diabetic Kidney Disease
Diabetic Kidney Disease
Epidemiology
Risk Factors
Diagnosis and Classification
Biomarkers
Natural History
Structural Changes of the Kidney
Pathophysiology
Increased Glucose Uptake in the Diabetic Kidney
Hemodynamic Abnormalities
Mechanotransduction
Inflammation and Fibrosis
Epigenetic Changes
Treatment
Glycemic Control
Glucose-Lowering Agents
Blood Pressure Control
Cardiovascular Risk Reduction
Nutrition Management
Additional Interventions and Management Approaches
Summary and Future Directions
References
Chapter (46)
46. Diabetic Foot and Vascular Complications
Pathophysiology
Diabetic Foot and Vascular Complications
46
Neuropathy
Infection
Ischemia
Bone and Joint Abnormalities
Presentation and Diagnosis
Diabetic Foot Ulcers
Infection/Osteomyelitis
Peripheral Arterial Disease
Medical Management
Prevention and Screening
Antibiotics
Wound Care
Offloading
Surgical Management
Drainage Procedures
Lower Extremity Arterial Reconstruction
Endovascular Procedures
Amputations
Multidisciplinary Approach
Future Trends
References
Chapter (47)
47. Parathyroid Hormone and the Parathyroid Hormone Receptor Type 1 in the Regulation of Calcium and Phosphate Homeostasis and Bone Metabolism
Parathyroid Hormone
Parathyroid Hormone Chemistry
Evolution of Parathyroid Hormone Ligands
The Parathyroid Hormone Gene and its mRNA
Regulation of Parathyroid Hormone Gene Expression
Regulation of Parathyroid Hormone Secretion
Mechanism of Extracellular Calcium Ion Sensing by Parathyroid Cells and Other Cells Involved in Mineral Ion Homeostasis
Metabolism of Parathyroid Hormone
Actions of Parathyroid Hormone on the Kidney
Renal Calcium Reabsorption
Regulation of 1α- and 24-Hydroxylase Activity
Renal Phosphate Transport
Actions of Parathyroid Hormone on Bone
Molecular Properties Of The Parathyroid Hormone Receptor Type-1
Gene Structure, Protein Topology, and Evolution
Disease Mutations in the PTHR-1 and its Ligands
Summary
References
Chapter (48)
48. Parathyroid Hormone-Related Protein
Parathyroid Hormone-Related Protein
The Skeleton
The Mammary Gland
The Skin
Placenta
Endocrine Pancreas
Smooth Muscle and the Cardiovascular System
Teeth
The Nervous System
The Immune System
Cancer
Skeletal Disease
Skin Disease
Diabetes
Conclusion
References
Chapter (49)
49. Calcitonin
49
Calcitonin Chemistry
Calcitonin Gene
Calcitonin Gene Alternative Product
Calcitonin Receptors
Receptor Gene
Receptor Isoforms
Receptor Polymorphisms
Receptor Distribution
Calcitonin–Calcitonin Receptor Signaling
Receptor Regulation
Calcitonin Physiology
Calcitonin in Growth and Development
Calcitonin in Calcium Stress: Pregnancy and Lactation
Renal Actions of Calcitonin
Calcitonin in the Central Nervous System
Peptides Related to Calcitonin
Calcitonin and its Receptors in Cancer
Medullary Carcinoma of the Thyroid
Calcitonin Receptors in Other Cancers
Calcitonin As a Therapeutic
Hypercalcemia
Osteoporosis and Paget Disease of Bone
Osteoarthritis
Conclusion
References
Chapter (50)
50. Vitamin D: From Photosynthesis, Metabolism, and Action to Clinical Applications
Vitamin D: From Photosynthesis, Metabolism, and Action to Clinical Applications
Historic Overview
Metabolism of Vitamin D
25-Hydroxylation
1α-Hydroxylation
24-Hydroxylation: Catabolism or Specific Function
Vitamin D Transport
Action and Mode of Action
General Characteristics of the Vitamin D Receptor
Classic Target Tissues
Clinical Consequences of Vitamin D Status
Skeletal Effects of the Vitamin D Endocrine System
Extraskeletal Actions of the Vitamin D Endocrine System
Clinical Use of Vitamin D
Optimal Serum 25OHD Concentration
Worldwide Vitamin D Status
Vitamin D Requirements
Summary
References
Chapter (51)
51. Bone Development and Remodeling
Intramembranous Ossification
Bone Development and Remodeling
51
Endochondral Ossification
Principles of Bone Remodeling and Skeletal Homeostasis
The Bone Remodeling Cycle
Cellular and Molecular Control of Skeletal Development and Bone Remodeling
Chondrogenesis and Growth Plate Biology
Skeletal Vascularization
Skeletal Stem and Progenitor Cells
Osteoblastogenesis
Osteoclastogenesis and Skeletal Tissue Resorption
Regulation of Bone Remodeling and Bone Mass
Coupling of Bone Resorption and Bone Formation During Remodeling
Conclusion
References
Chapter (52)
52. Regulation of Calcium Homeostasis and Genetic Disorders That Affect Calcium Metabolism
Distribution and Metabolic Actions of Calcium
Regulation of Calcium Homeostasis and Genetic Disorders That Affect Calcium Metabolism
52
Calcium Absorption
Mechanisms and Sites of Calcium Absorption
Renal Calcium Excretion
Sites and Mechanisms of Renal Calcium Reabsorption
Regulation of Renal Calcium Reabsorption
PTH Gene Structure and Function
Hypercalcemic Diseases
Parathyroid Tumors
Syndromic Forms of Primary Hyperparathyroidism
Nonsyndromic Forms of Primary Hyperparathyroidism
Other Hereditary Hypercalcemic Disorders
Hypocalcemic Disorders
Hypoparathyroidism
PTH Gene Abnormalities
GCM2 Abnormalities
X-Linked Recessive Hypoparathyroidism
Pluriglandular Autoimmune Hypoparathyroidism
DiGeorge Syndrome
Charge Syndrome
Calcium-Sensing Receptor Abnormalities
Blomstrand Disease
Conclusion
References
Chapter (53)
53. Genetic Disorders of Phosphate Homeostasis
Regulation of Phosphate Absorption
Phosphate Excretion
Genetic Disorders of Phosphate Homeostasis
53
Regulation of Phosphate Reabsorption
Fibroblast Growth Factor-23
Fibroblast Growth Factor-23 Activity
Regulation of Fibroblast Growth Factor-23 Production
Disorders Associated with Increased FGF23 Bioactivity
Disorders Associated With Reduced Fibroblast Growth Factor-23 Bioactivity
Possible Future Therapeutic Avenues
References
Chapter (54)
54. Primary Hyperparathyroidism
Primary Hyperparathyroidism
Introduction
Epidemiology, Diagnosis, and Overview of Clinical Features
Incidence
Diagnosis and Differential Diagnosis
Other Biochemical Features
Clinical Presentation
The Skeleton
Bone Turnover Markers
Bone Densitometry
Bone Histomorphometry
New Imaging Technologies
Fractures
Nephrolithiasis and Renal Function
Other Organ Involvement
Cardiovascular System
Neurological, Psychologic and Cognitive Features
Gastrointestinal Manifestations
Other Systemic Involvement
Unusual Presentations
Neonatal Primary Hyperparathyroidism
Primary Hyperparathyroidism in Pregnancy
Acute Primary Hyperparathyroidism
Parathyroid Cancer
Evaluation
Natural History
Natural History With Surgery
Natural History Without Surgery
Guidelines for Parathyroidectomy
Surgery
Surgical Approach
Immediate Postoperative Course
Medical Management
General Measures
Diet and Supplements
Pharmaceuticals
Hydrochlorothiazide
Treatment of Parathyroid Cancer
Summary
References
Chapter (55)
55. Malignancy-Associated Hypercalcemia
Malignancy-Associated Hypercalcemia
Introduction and History
Normal Physiology of Calcium Metabolism
Parathyroid Hormone and the Calcium-Sensing Receptor
Vitamin D
Parathyroid Hormone–Related Protein
Malignancy-Associated Hypercalcemia
Humoral Hypercalcemia of Malignancy
Local Osteolytic Hypercalcemia
Overproduction of 1,25-Dihydroxyvitamin D
Ectopic Production of Parathyroid Hormone
Treatment
Hydration
Inhibition of Bone Resorption
Glucocorticoids
Dialysis
Other treatments
References
Chapter (56)
56. Surgical Management of Hyperparathyroidism
Clinical Presentation
Indications for Surgery
Surgical Management of Hyperparathyroidism
56
Preoperative Preparation
Operative Approaches
Intraoperative Adjuncts
Postoperative Management
Outcomes of Parathyroid Surgery
Reoperative Parathyroid Surgery
Hypercalcemic Crisis
Inherited Syndromes
Parathyroid Carcinoma
Pediatric Parathyroid Surgery
Hyperparathyroidism in Pregnancy
Secondary Hyperparathyroidism
Tertiary Hyperparathyroidism
COVID-19 and Parathyroid Surgery
Summary
References
Chapter (57)
57. Pseudohypoparathyroidism, Albright’s Hereditary Osteodystrophy, and Progressive Osseous Heteroplasia: Disorders Caused by GNAS Mutations that Reduce Gsα Activity
Pseudohypoparathyroidism, Albright’s Hereditary Osteodystrophy, and Progressive Osseous Heteroplasia: Disorders Caused by GNAS M...
Pseudohypoparathyroidism Type-Ia
The Complex Gnas Locus
The Alpha Subunit of the Stimulatory G Protein (Gsα)
Pseudopseudohypoparathyroidism
Progressive Osseous Heteroplasia
Disorders of Concurrent Gsα Inactivity and Hyperactivity
Pseudohypoparathyroidism Type-Ib
Autosomal Dominant PHP1B
Sporadic Pseudohypoparathyroidism Type-1B
Pseudohypoparathyroidism Type-II
Acrodysostosis With Hormonal Resistance
Treatment
Summary
References
Chapter (58)
58. Genetic Defects in Vitamin D Metabolism and Action
Genetic Defects in Vitamin D Metabolism and Action
Overview of Vitamin D Metabolism
Rickets and Osteomalacia
Vitamin D–Deficiency Rickets, Vitamin D Hydroxylation–Deficient Rickets, and Vitamin D–Dependent Rickets
CYP2R1 Mutations
CYP27B1 Mutations
Vitamin D Receptor Mutations
Treatment OF Vitamin D-Dependent Rickets, Type 2A
Idiopathic Infantile Hypercalcemia
CYP24A1 Mutations
Future Directions
References
Chapter (59)
59. Hereditary Disorders of the Skeleton
Hereditary Disorders of the Skeleton
Dense Bone Disorders
Osteopetroses
Pycnodysostosis
Kenny–Caffey Syndrome
Infantile Cortical Hyperostosis (Caffey Disease)
Endosteal Hyperostoses
Osteopoikilosis
Osteopathia Striata
Pachydermoperiostosis
Paget Disease of Bone
Familial Expansile Osteolysis
Osteoprotegerin Deficiency
References
Chapter (60)
60. Osteoporosis
Osteoporosis
What Is Osteoporosis
Origins of Skeletal Fragility
Bone Density
Bone Quality
Falls and Frailty
Nutrition
Genetics
Epidemiology
Bone Mineral Density
Fractures
Assessing Patients at Risk of Fracture
Fracture Risk Assessment
Bone Turnover Markers
Skeletal Imaging
Other Investigations
Lifestyle
Antiresorptive Medications
Anabolic Medications
Treatment Decisions
Whom to Treat
Selection of Treatment
How Long to Treat
Follow-Up
Management of Subtypes of Osteoporosis
References
Chapter (61)
61. CKD-MBD Syndrome: Chronic Kidney Disease Produced Disorders in Skeletal, Vascular, and Cardiac Tissues and Mineral Metabolism
Vascular
CKD-MBD Syndrome: Chronic Kidney Disease Produced Disorders in Skeletal, Vascular, and Cardiac Tissues and Mineral Metabolism
61
Cardiac
Skeletal
Plasma
Pathogenesis
Pathogenic Factors in the Chronic Kidney Disease–Mineral and Bone Disorder
FGF23
Klotho
Sclerostin
Hyperphosphatemia
Calcitriol Deficiency
Hypocalcemia
Hyperparathyroidism
Hypogonadism
Other Factors
Pathology of Renal Osteodystrophy
Low-Turnover Bone Disease, Adynamic Bone Disorder
Associated Features
Osteoporosis and Osteosclerosis
Management of Osteoporosis in Chronic Kidney Disease
Clinical Manifestations
Bone Pain, Fractures, and Skeletal Deformities
Conclusion
References
Chapter (62)
62. Disorders of Mineralization
Disorders of Mineralization
62
Radiologic Features
Nutritional Osteomalacia and Rickets
Gastrointestinal Diseases
Disorders of Vitamin D Metabolism
Hypophosphatemia
FGF23-Mediated Hypophosphatemia
Non–FGF23-mediated Hypophosphatemia
Miscellaneous Causes of Impaired Mineralization
Acidosis
Fanconi Syndrome
Chronic Kidney Disease
Aluminum, Fluoride, and Heavy Metal Intoxication
Hypophosphatasia
Osteopetrosis
Fibrogenesis Imperfecta Ossium
Treatment
Conclusion
References
Chapter (63)
63. Paget Disease of Bone
Paget Disease of Bone
63
Pathophysiology
Clinical Features
Regional Manifestations
Biochemical Features
Indices of Bone Resorption
Indices of Bone Formation
Calciotropic Hormones
Systemic Complications and Associated Diseases
Primary Hyperparathyroidism, Nephrolithiasis, Hypercalciuria, Hypercalcemia
Hyperuricemia and Gout
Cardiovascular Abnormalities
Drug Treatment
Indications for Treatment
Pretreatment Laboratory Evaluation
Calcitonin
Bisphosphonates
Surgery
Causes
Animal Models of Paget Disease
References
Vol II
Chapter (64)
64. Anatomy and Development of the Thyroid
The Anatomy Of The Thyroid Gland
Gross Anatomy
Blood Supply
Lymphatics
Innervation
Anatomic Variants
Other Thyroid Cells
C Cells
Ultimobranchial Body-Derived Epithelial Cells
Thyroid Stem Cells
The Development Of The Thyroid Gland
Differentiation of C Cells
Functional Genomics of Thyroid-Enriched Transcription Factors
From Animal Models to Human Diseases
Generation of Functional Thyroid Follicles In Vitro
Phylogenesis Of The Thyroid Gland
References
Chapter (65)
65. Thyroid-Stimulating Hormone and TSH Receptor
Thyroid-Stimulating Hormone and TSH Receptor
65
Regulation of Thyroid-Stimulating Hormone Secretion
Hypothalamic Regulation of Thyroid-Stimulating Hormone Secretion
Thyrostimulin
Thyroid-Stimulating Hormone Receptor
Protein Structure
Thyroidal Actions of Thyroid-Stimulating Hormone
Regulation of Thyroid-Specific Gene Expression
Thyroid-Stimulating Hormone Receptor/Insulin-Like Growth Factor 1 Receptor Crosstalk
Regulation of Thyroid Hormone Synthesis
Regulation of Thyroid Hormone Secretion
References
Chapter (66)
66. Thyroid Hormone Transporters and Metabolism
Thyroid Hormone Transporters and Metabolism
Disorders Of Thyroid Hormone Transport
MCT8 Deficiency
Disorders Of Thyroid Hormone Metabolism
Iodothyronine Deiodinase I Deficiency
SBP2 Deficiency
Sec-tRNA[Ser]Sec
References
Chapter (67)
67. Thyroid Hormone Action
Thyroid Hormone Action
Components Of Thyroid Hormone Action
Thyroid Hormone Receptor Coregulatory Proteins
Thyroid Hormone Receptor Posttranslational Modifications
Nongenomic Thyroid Hormone Action
Thyroid Hormone Tissue-Specific Actions
Thyroid Hormone Receptor Isoform–Specific Expression
Growth
Stem Cell Proliferation
Thyroid Hormone and Somatic Growth
Brain
Heart and Blood Vessels
Skeletal Muscle
Skeletal System
Small Intestine
Metabolic Regulation and Actions in Liver
White and Brown Adipose Tissue
Skin
Resistance to Thyroid Hormone Beta
Resistance to Thyroid Hormone Alpha
References
Chapter (68)
68. Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine)
Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine)
Introduction
THYROTROPIN
Immunoassay for Thyrotropin
Thyrotropin Reference Intervals
Standardization of Thyrotropin Immunoassays
Free Thyroid Hormones
Indirect Estimation of Free Thyroid Hormones
Free Thyroid Hormones by Equilibrium Dialysis
Immunoassay of Free Thyroid Hormones
Free Thyroid Hormone Reference Intervals
Standardization of Free Thyroid Hormones
Total Thyroxine And Triiodothyronine
Future Directions
References
Chapter (69)
69. Thyroid Imaging
Indications for Sonographic Assessment
Physics of Ultrasonography
Thyroid Imaging
69
Diffuse Thyroid Disorders
Primary Thyroid Lymphoma
Subacute Thyroiditis
Sonographic Risk Stratification Systems
Korean Thyroid Imaging Reporting and Data System
Specialized Sonographic Techniques
Scintigraphy
Thyroid Nodules
Diffuse Thyroid Disorders
Congenital Hypothyroidism
Perchlorate Discharge Test
Incidentalomas
Future Directions
References
Chapter (70)
70. Pathology of the Thyroid Gland
Pathology of the Thyroid Gland
Thyroid Gland
Pathologic Assessment Of Thyroid Lesions
Pathology of Thyroid Lesions
Diffuse Enlargements of Thyroid Gland
Nodular Enlargements and Nodules
Benign Neoplasms
Malignant Neoplasms
Papillary Thyroid Carcinoma
Variants of Papillary Cancer
Follicular Carcinoma
Hürthle Cell (Oncocytic Follicular) Tumors
High-Grade Thyroid Carcinoma
Poorly Differentiated Carcinoma
Anaplastic Carcinoma
Familial Follicular-Derived Thyroid Tumors
Medullary Carcinoma
Rare Tumors of Thyroid Gland
Malignant Tumors Arising in Ectopic Thyroid Tissue
Secondary Tumors
Summary and Future Directions
References
Chapter (71)
Chapter (72)
72. Graves’ Orbitopathy
Graves’ Orbitopathy
Epidemiology
Risk Factors
Age
Gender
Ethnicity
Smoking
High Thyroid Stimulating Hormone Receptor Antibody Titer
Radioactive Iodine Therapy
Cholesterol Level and Statin Use
Pathogenesis
Key Pathological Changes in Graves’ Orbitopathy
Thyroid Stimulating Hormone Receptor as the Putative Autoantigen
Immune Dysregulation
Signaling Pathways Mediating Hyaluronan Production and Adipogenesis (Fig. 72.2)
Orbital Infiltration of CD34+ Fibrocytes
Thyroid Stimulating Hormone Receptor and Insulin-Like Growth Factor-1 Receptor Crosstalk (Fig. 72.3)
Genetic Susceptibility
Clinical Manifestations And Diagnosis
Clinical Evaluation
Thyroid Function Test
Thyroid Stimulating Hormone Receptor Antibody
Orbital Imaging
Quality of Life Assessment
Management
General Measures
Mild Graves’ Orbitopathy
Active Moderate-to-Severe Graves’ Orbitopathy
Sight-Threatening Graves’ Orbitopathy
Inactive Graves’ Orbitopathy and the Role of Rehabilitative Surgery
Novel Treatment Approaches for Immunosuppression in Graves’ Orbitopathy
Summary And Perspectives
References
Chapter (73)
73. Thyroiditis
Thyroiditis
Subtypes of Thyroiditis
Hashimoto’s Thyroiditis
Definition and Incidence
Etiopathogenesis and Physiopathologic Mechanisms in Autoimmune Thyroid Disease
Natural History and Clinical Features
Relationship With Other Autoimmune Diseases
Diagnosis
Treatment and Follow-Up
Hashimoto’s Encephalopathy
Acute/Infectious Thyroiditis
Definition and Incidence
Clinical and Diagnostic Features
Treatment and Follow-Up
Subacute Thyroiditis
Definition and Incidence
Pathology
Treatment and Follow-Up
Silent Thyroiditis
Definition and Incidence
Clinical Features
Diagnosis: Clinical, Laboratory, and Imaging Findings
Treatment
Riedel’s Thyroiditis
Definition and Incidence
Etiology
Clinical Features
Diagnosis: Laboratory and Imaging Findings
Pathology
Treatment
IgG4 Thyroiditis
Definition, Incidence, and Relationships in the Setting of Other Subtypes of Thyroiditis
Clinical Aspects
Diagnosis
Treatment and Follow-Up
Summary and Future Directions
References
Chapter (74)
74. Hypothyroidism
Hypothyroidism
Brief History
Hypothalamic-Pituitary-Thyroid Axis
Epidemiology
Clinical Manifestations
Symptoms
Signs
Etiology
Primary Hypothyroidism
Central Hypothyroidism
Peripheral Causes of Hypothyroidism
Diagnosis
Reference Intervals
Serum Thyroid-Stimulating Hormone
Log-Linear Relationship
Free Thyroxine Levels
Triiodothyronine Levels
Thyroid Antibody Testing
Detection of Central Hypothyroidism
Subclinical Versus Overt Hypothyroidism
Screening
Treatment
Levothyroxine Therapy
Therapy Other Than Levothyroxine
Summary and Areas of Future Research
References
Chapter (75)
75. Thyroid and Pregnancy
Epidemiology
Physiology
Thyroid and Pregnancy
75
Physiology During Assisted Reproduction Technology
Clinical Outcomes and Management
Maternal and Fetal Physiology in Pregnancy
Thyroid Function Tests in Pregnancy
Hyperthyroidism in Pregnancy
Diagnosis
Treatment and Monitoring
Lactation
Hypothyroidism and Hypothyroxinemia In Pregnancy
Overt Hypothyroidism
Subclinical Hypothyroidism
Isolated Hypothyroxinemia
Thyroid Autoimmunity in Pregnancy
Associated Obstetric and Fetal Outcomes
Potential Mechanisms for Associations
Implications for Therapy and Monitoring
Screening for Thyroid Dysfunction in Pregnancy
Thyroid Nodules and Cancer During Pregnancy
Thyroid Disorders in the Postpartum Period
Postpartum Thyroiditis
Considerations in Lactating Women
Summary and Future Directions
References
Chapter (76)
76. Iodine Nutrition and the Thyroid
Iodine Nutrition and the Thyroid
Dietary Sources Of Iodine
Absorption and Bioavailability of Iodine
Thyroidal Iodine Uptake
Goitrogens
Metabolism
Thyroidal Adaptation to Iodine Deficiency
Iodine Requirements
Methods To Assess Iodine Status
Urinary Iodine Concentration
Thyroid Size
Thyroid-Stimulating Hormone
Thyroglobulin
Effects of Iodine Deficiency on the Thyroid and Brain
Goiter
Pregnancy and Infancy
Childhood
Adulthood
Epidemiology of Iodine Deficiency
Treatment And Prevention Of Iodine Deficiency
Salt Fortification with Iodine
Iodine Supplementation
Iodine Excess
References
Chapter (77)
77. Euthyroid and Hyperthyroid Nodules and Goiter
Definition and Clinical Manifestation
Environment and Heredity
Euthyroid and Hyperthyroid Nodules and Goiter
77
Hot Thyroid Nodules
Cold Thyroid Nodules
Euthyroid Nodules
Iodine-Induced Hyperthyroidism
Epidemiology, Occurrence, and Natural History of Euthyroid and Hot Thyroid Nodules
Thyroid Nodule Diagnostic Pathway to Distinguish Benign and Malignant Thyroid Nodules
Clinical Examination
Laboratory Investigations
Diagnosis of Thyroid Autonomy
Malignancy Risk Stratification using Thyroid Ultrasound
Fine Needle Aspiration Cytology
Treatment of Autonomous Thyroid Nodules
Surgery
Radioiodine Therapy
Follow-Up
Treatment of Euthyroid Nodules (see Table 77.3)
Iodine Supplementation
Thyroid Hormone Treatment
Surgery
Radioiodine Therapy
Nonsurgical Thermal Ablation of Thyroid Nodules
References
Chapter (78)
78. Differentiated Thyroid Cancer – Streamlining Diagnosis and Optimizing Management
Differentiated Thyroid Cancer – Streamlining Diagnosis and Optimizing Management
Epidemiology
Pathology
Papillary Thyroid Carcinoma Variants
Follicular Thyroid Carcinoma
Hurthle Cell (Oncocytic) Tumors
Poorly Differentiated Thyroid Carcinoma
Causes
Somatic Gene Alterations
Germline Genetic Factors
Ionizing Radiation
Diagnosis, Clinical Features, And Course
Response to Therapy
Prognostic Factors
Age and Gender
Histopathological Factors
Size of the Primary Tumor and Extrathyroidal Extension
Extrathyroidal Extension
Lymph Node Metastases
Therapy And Monitoring
Radioiodine Therapy
Levothyroxine Therapy
Monitoring for Disease Recurrence
Treatment of Metastatic Disease
References
Chapter (79)
79. Medullary Thyroid Cancer
Clinical Presentation And Diagnosis
Sporadic Medullary Thyroid Cancer
Medullary Thyroid Cancer
79
Hereditary Forms (Multiple Endocrine Neoplasia Type II)
Gene Carriers
Pathology And Pathogenesis
Histology
Genetics
Initial Therapy
Postsurgical Follow-Up
Follow-Up of Cured Medullary Thyroid Cancer Patients
Follow-Up of Patients with Biochemical Persistent Disease
Follow-Up of Patients with Structural Disease
Other Therapies
Local Treatment of Recurrent or Persistent Neck Disease
Conclusions
References
Chapter (80)
80. Anaplastic (Undifferentiated) Thyroid Carcinoma
Epidemiology
Clinical Characteristics
Diagnosis
Biopsy
Histopathology Subtypes
Anaplastic (Undifferentiated) Thyroid Carcinoma
80
Differential Diagnoses
Molecular Genetics
Evaluation
Clinical and Labs
Imaging
Staging
Establish Goals of Care
Multidisciplinary Team
Prognosis
Predictors of Survival
Median and Overall Survival
Therapy
Treatment Strategies by Stage
Surgery
Radiation Therapy
Multimodal Therapy
Future Directions
Conclusions
Acknowledgments
References
Chapter (81)
81. Resistance to Thyroid Hormone and Genetic Defects of the Thyroid
Resistance to Thyroid Hormone and Genetic Defects of the Thyroid
Congenital Primary Hypothyroidism
Epidemiology and Screening
Genetic Causes of Thyroid Dysgenesis
Genetic Causes of Dyshormonogenesis
Resistance to Thyroid Hormone β
Clinical Features
Differential Diagnosis
Molecular Genetics
Management
Resistance to Thyroid Hormone Alpha
Background
Clinical Features
Molecular Genetics
Management
References
Chapter (82)
82. Surgical Management of Thyroid Disease
Surgical Management of Thyroid Disease
82
Relevant Surgical Anatomy
The Recurrent Laryngeal Nerve
The External Branch of the Superior Laryngeal Nerve
Parathyroid Glands
Lymphatics
Imaging For Surgical Planning
Ultrasound
Axial Imaging
Molecular Testing And Its Use For Surgical Planning
Indeterminate Nodules
Planning the Extent of Surgery
Diseases Of The Thyroid And Surgical Procedures To Consider
Graves Disease
Hashimoto Thyroiditis
Thyroglossal Duct Cyst and Surgical Excision
Thyroid Hemiagenesis and Agenesis
Ectopic Thyroid and Surgical Excision
Lingual Thyroid
Suprahyoid and Infrahyoid Thyroid
Substernal Goiter
Papillary Thyroid Cancer
Follicular Thyroid Cancer
Hurthle Cell Thyroid Cancer
Medullary Thyroid Cancer
Anaplastic Thyroid Cancer
Radioactive Iodine Therapy
Indications For Surgery Of The Thyroid Gland
Indications for Lobectomy versus Total Thyroidectomy
Indications for Active Surveillance
Indications for Remote Access Surgery
Indications for Prophylactic Central Neck Dissection
Indications for Therapeutic Central Neck Dissection
Indications for Lateral Neck Dissection
Transoral Approach To Thyroidectomy
Open Surgical Techniques
Lobectomy and Total Thyroidectomy
Retrograde Medial Approach to the Thyroid Dissection
Central Neck Dissection
Lateral Neck Dissection
Minimally Invasive Techniques
Radiofrequency Ablation Technique
Surgical Complications
Recurrent Laryngeal Nerve Injury
Hypoparathyroidism
Spinal Accessory Nerve Injury
Greater Auricular Nerve Injury and Sensory Cervical Rootlets
Other Cranial Neuropathies
Chyle Leak
Cosmesis
Complications Associated with Transoral Surgery
Complications Associated with Radiofrequency Ablation
References
Chapter (83)
83. Nonthyroidal Illness Syndrome
Nonthyroidal Illness Syndrome
Low Triiodothyronine States
Serum Hormone Levels
Serum Total and Free Triiodothyronine
Serum Reverse Triiodothyronine
Serum Total and Free Thyroxine
Serum Thyroid-Stimulating Hormone
Thyroid Hormone Transport
Thyroid Hormone Metabolism and Action
Hypothalamus
Pituitary
Liver
Muscle
Adipose Tissue
Neutrophils and Macrophages
Mechanism of Altered Thyroid Hormone Metabolism in Nonthyroidal Illness Syndrome
Cyrokines
Nuclear Factor κB
Alternative Mechanisms
Energy Status
Conclusion
References
Chapter (84)
84. Drugs and Thyroid
Drugs and Thyroid
84
Drugs That Enhance Thyroid Autoimmunity
Drugs Causing Direct Thyroid Damage
Drugs Affecting Protein Binding Of Thyroid Hormone
Changes in Binding Proteins
Displacement of Thyroid Hormone From Binding Proteins
Drugs Affecting Thyroid Hormone Activation, Metabolism, And Excretion
Thyroxine-to-Triiodothyronine Conversion
Thyroid Hormone Metabolism
Disposal of Thyroid Hormone
Summary And Future Directions
References
Chapter (85)
85. Adrenal Development and Homeostasis
Introduction and Historical Background
Anatomy
Adrenal Cortex
Embryonic Adrenal Development
Overview
Adrenal Fate Determination
Fetal Adrenal Development
Postnatal Adrenal Development
Fetal Zone Involution
Establishment of Adrenal Cortex Zonation
Homeostatic Renewal
Role of Renin-Angiotensin-Aldosterone System and Adrenocorticotropic Hormone/PKA Signaling in Regulation of Renewal
Sexual Dimorphism in Renewal
Adrenal Cortex Regeneration
Tissue Stress Progenitors and Adrenal Aging
Conclusion and Future Directions
References
Chapter (86)
86. Genetic Disorders of the Adrenal Cortex
Genetic Disorders of the Adrenal Cortex
Genetic Disorders of the Adrenal Cortex
Steroidogenesis
Hypoplasia
Familial Glucocorticoid Deficiency-Like (FGD-Like)
Other Causes Of Syndromic Adrenal Insufficiency
Conclusion
References
Chapter (87)
87. Enzymes and Pathways of Human Steroidogenesis
87
Cytochrome P450 Enzymes
Hydroxysteroid Dehydrogenases and Reductases
Acute Regulation of Steroidogenesis
Chronic Maintenance of the Steroidogenic Machinery
Human Steroidogenic Cytochrome P450 Enzymes
P450scc (Encoded by CYP11A1)
P450c17 (Encoded by CYP17A1)
P450c21 (Encoded by CYP21A2)
P450aro (Aromatase)
Redox Partner Proteins
Ferredoxin
Ferredoxin Reductase
P450 Oxidoreductase
Cytochrome b5
Steroidogenic Dehydrogenases and Reductases
3β-Hydroxysteroid Dehydrogenase/Δ5→Δ4-Isomerases
17β-Hydroxysteroid Dehydrogenases
Steroid 5α-Reductases
3α-Hydroxysteroid Dehydrogenases
11β-Hydroxysteroid Dehydrogenases
Steroid Sulfonation
Pathways
Adrenal Steroidogenic Pathways
Gonadal Steroidogenic Pathways
The Backdoor Pathway to Dihydrotestosterone
Pathways to 11-Oxygenated Androgens
Androgen Synthesis in Prostate Cancer
References
Chapter (88)
88. Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance
88
Sodium Homeostasis
Potassium Homeostasis
Specificity-Conferring Enzymes
11β-Hydroxysteroid Dehydrogenase Type 2
Mineralocorticoid Receptors
Evolution
Structure
Genomic Versus Nongenomic Aldosterone Actions
Sodium Transport
Epithelial Sodium Channels
Na+/K+-ATPase
Potassium Transport
Hydrogen Ion Transport
Nonepithelial Tissues
Cardiovascular System
Central Nervous System
Summary and Future Directions
References
Chapter (89)
89. Glucocorticoid Receptors: Mechanisms of Action in Health and Disease
Glucocorticoid Receptors: Mechanisms of Action in Health and Disease
Introduction
Glucocorticoid Receptor Polymorphisms
Glucocorticoid Receptor Physiology
Immune System
Cardiovascular System
Central Nervous System
Hepatic Carbohydrate and Lipid Metabolism
Glucocorticoid Receptor Resistance
Summary and Future Directions
References
Chapter (90)
90. Adrenal Androgens, Adrenarche, and Adrenopause
Adrenal Androgens, Adrenarche, and Adrenopause
Adrenarche
The Adrenal Glands
The Fetal Adrenal Gland
Clinical Facets of Adrenarche
Adrenal Steroidogenesis
Adrenopause
Epidemiology/Associations with Dehydroepiandrosterone Sulfate Concentrations, Aging, and Cardiovascular Risks
Conclusion
References
Chapter (91)
91. Adrenal Pathology
Accessory or Heterotopic Adrenal Tissue
Adrenal Pathology
91
Adrenal Myelolipoma
Ectopic Thyroid Tissue
Ovarian Thecal Metaplasia
Adrenal Amyloidosis
Adrenal Calcification
Adrenal Cysts
Adrenal Infection and Abscess
Congenital Adrenal Hyperplasia
Adrenal Cytomegaly
Inflammatory Adrenalitis
Functional Approach In Adrenal Pathology
Pathological Correlates of Primary Aldosteronism
Pathological Correlates of Adrenal Cushing Syndrome
Pathological Correlates of Virilism/Feminization
Pathological Correlates of Catecholamine Excess
Confirmation of Adrenocortical and Medullary Origin
Confirmation of Functional Sites in Primary Aldosteronism
Diagnostic and Predictive Biomarkers of Adrenocortical Carcinoma
Molecular Immunohistochemistry in Pheochromocytoma
References
Chapter (92)
92. Adrenal Gland Imaging
Adrenal Gland Imaging
Adrenal Gland Imaging
Introduction
Adrenal Gland Anatomy
Adrenal Gland Imaging
Adrenal Gland Imaging
Arteriography and Adrenal Vein Sampling
Clinical Utility
Adenoma or Metastases
Benign Adrenal Lesions
Neoplastic Adrenal Lesions
Functional Imaging in Primary Hyperaldosteronism
Summary
References
Chapter (93)
93. Adrenal Insufficiency
Adrenal Insufficiency
93
Epidemiology of Adrenal Insufficiency
Pathogenesis of Adrenal Insufficiency
Clinical Features of Adrenal Insufficiency
Etiology
Primary Adrenal Insufficiency
Diagnosis of Adrenal Insufficiency
Adrenocorticotropic Hormone Stimulation Testing
Overnight Metyrapone Test
Insulin Tolerance Test
Adrenal Autoantibody Tests
Serum 17-Hydroxyprogesterone
Very Long Chain Fatty Acids
Adrenal Imaging
Treatment of Adrenal Insufficiency
Replacement of Dehydroepiandrosterone
Quality of Life in Adrenal Insufficiency
Adrenal Suppression
Adrenal Crisis
Treatment of Adrenal Crisis
Prevention of Adrenal Crises
The Need for Novel Treatments for Adrenal Insufficiency
References
Chapter (94)
94. Defects of Adrenal Steroidogenesis
94
Virilizing Forms of Congenital Adrenal Hyperplasia
21-Hydroxylase Deficiency
11β-Hydroxylase Deficiency
P450 Oxidoreductase Deficiency
3β-Hydroxysteroid Dehydrogenase Type 2 Deficiency
Nonvirilizing Forms of Congenital Adrenal Hyperplasia
17α-Hydroxylase/17,20 Lyase Deficiency
Diagnosis of 17α-Hydroxylase/17,20 Lyase Deficiency
Other Conditions Affecting Adrenal Steroid Synthesis
Aldosterone Synthase Deficiency
Glucocorticoid-Remediable Aldosteronism
Smith–Lemli–Opitz Syndrome
Differential Diagnosis
Medical Therapy of Congenital Adrenal Hyperplasia
Glucocorticoid Therapy
Mineralocorticoid Therapy
Sex Steroid Replacement
Antihypertensive Therapy
Management of Atypical Genitalia
Future Directions and Novel Therapies
Conclusion
References
Chapter (95)
95. Adrenal Genomics II: Familial and Sporadic Neoplasia
Adrenal Genomics II: Familial and Sporadic Neoplasia
Genetic Causes Of Adrenal Neoplasia
Adrenocortical Tumors
Pheochromocytoma
Summary And Future Directions
References
Chapter (96)
96. Primary Aldosteronism
Pathogenesis And Genetics
Primary Aldosteronism
96
Familial Forms of Primary Aldosteronism
Somatic Mutations in Unilateral Primary Aldosteronism
Somatic Mutations in Bilateral Adrenal Hyperplasia
Prevalence
Clinical Diagnosis
Symptoms and Signs of Primary Aldosteronism
Diagnostic Workup for Primary Aldosteronism
Treatment
Anchor 472
References
Chapter (97)
97. Adrenocorticotropic Hormone–Independent Cushing Syndrome
Adrenocorticotropic Hormone–Independent Cushing Syndrome
Pathophysiology––Molecular Genetics and Animal Models
Molecular and Genetic Causes of Cortisol-Producing Adrenocortical Lesions
Animal Models
Clinical Manifestations
Hormonal Evaluation
Unilateral Tumors
Adrenocortical Adenoma
Adrenocortical Cancer
Bilateral Adrenocortical Tumors
Micronodular Adrenal Hyperplasia and Primary Pigmented Nodular Adrenal Disease
Primary Macronodular Adrenal Hyperplasia
Medical Treatment with Steroidogenesis Inhibitors (Table 97.5)
Surgery
Perspectives
References
Chapter (98)
98. Adrenocortical Carcinoma
Adrenocortical Carcinoma
Diagnosis
Hormonal Evaluation
Imaging
Histopathology
Staging
Therapy (Fig. 98.3)
Surgery
Radiation Therapy
Medical Therapy
Mitotane
Cytotoxic Chemotherapy
Targeted Therapies
Immunotherapy
Follow-Up
References
Chapter (99)
99. Pheochromocytoma
Pheochromocytoma
Catecholamine Production, Secretion, And Metabolism
Who to Test According to Presentation
Signs and Symptoms
Differential Diagnosis
Multiple Endocrine Neoplasia Syndromes
Von Hippel–Lindau Syndrome
Neurofibromatosis Type 1
Succinate Dehydrogenase Gene Family
Less Common Hereditary or Other Causes
Biochemical Diagnosis Of Pheochromocytoma
Preanalytics and Analytics
Interpretative Considerations
Follow-Up Biochemical Testing
Anatomic Imaging
Differential Diagnosis
Functional Imaging
Metaiodobenzylguanidine Scintigraphy
Positron Emission Tomography
Medical Therapy and Preparation for Surgery
Postoperative Management
Special Presentations And Therapeutic Problems
Multifocal and Metastatic Pheochromocytoma/Paraganglioma
Pheochromocytoma in Pregnancy
Future Directions
References
Chapter (100)
100. Adrenal Surgery
Adrenal Surgery
100
Preoperative Evaluation
Clinical
Familial History and Genetic Testing
Biochemical Evaluation
Imaging
Fine-Needle Aspiration
Disease-Specific Decision-Making For Surgery
Benign Nonfunctional Adrenal Tumors
Myelolipomas
Adrenal Cysts
Biochemically Functional Adrenal Disorders
Hypercortisolism
Congenital Adrenal Hyperplasia
Bilateral Macronodular Adrenal Hyperplasia
Primary Aldosteronism
Androgen- and Estrogen-Secreting Tumors
Pheochromocytoma
Adrenocortical Carcinoma
Metastatic Disease to the Adrenal Gland
Surgical Approaches To The Adrenal Gland
Open Anterior Transabdominal
Right
Left
Open Posterior (Lumbotomy)
Laparoscopic Anterior Transabdominal
Right
Left
Laparoscopic Lateral Transabdominal Adrenalectomy
Right
Left
Robot-Assisted Adrenalectomy
Bilateral Adrenalectomy
Partial Adrenalectomy
Surgical Complications
Intraoperative Complications
Postoperative Complications
Complications and Operative Approach
Bilateral Adrenalectomy
Nelson Syndrome
Additional Considerations
Conclusion
References
Chapter (101)
101. Differences of Sex Development
Introduction To Sexual Development
Embryology
Gonadal Development (Sex Determination)
Development of Internal and External Genitalia (Sex Differentiation)
Genetic Pathways Of Sex Development And Their Variations
Genetic Pathways
Differences Of Sex Development
46,XX Differences of Sex Development
46,XY Differences of Sex Development
Sex Chromosome Differences of Sex Development
Syndromic Differences of Sex Development
Tools in Differences of Sex Development Evaluation
Initial Gender Assignment
Management of Differences of Sex Development
Acknowledgements
REFERENCES
Chapter (102)
102. Endocrinology of Sexual Maturation and Puberty
Gonadarche
Endocrinology of Sexual Maturation and Puberty
102
Adrenarche
Control Of The Onset Of Puberty
Genetics of Pubertal Onset
Timing of Puberty
Physical Changes Of Puberty
Boys
Girls
Additional Changes During Sexual Maturation
Changes in Body Composition and Bone Mineral Density
Changes in Sleep and Brain Development
Other Changes at Puberty
Laboratory Testing During Puberty
Summary
References
Chapter (103)
103. Endocrinology of Pubertal Disorders
Precocious Puberty
Variants of Precocious Puberty
Endocrinology of Pubertal Disorders
103
Premature Thelarche
Premature Pubarche
Pathologic Precocious Puberty
Central Precocious Puberty
Peripheral Precocious Puberty
Abnormalities of G Proteins
Abnormalities of Gonadotropin Receptors
Tumors
Gonadal Tumors
Adrenal Tumors
Germ Cell Tumors
Exogenous Sex Steroids
Hypothyroidism
Delayed Puberty
Definition
Benign Variants of Delayed Puberty
Constitutional Delay of Growth and Puberty
Pathologic Delayed Puberty
Hypothalamic-Pituitary Hypogonadism
Primary Hypogonadism
Treatment of Delayed Puberty
Girls
Boys
Conclusion
References
Chapter (104)
104. Transgender Healthcare
Transgender Healthcare
Overview
Goal-Driven Approach to Hormone Therapy
Physical––Feminizing
Physical––Masculinizing
Sexuality Changes
Sexuality Changes––Feminizing
Sexuality Changes––Masculinizing
Sexuality and Gender-Affirming Hormone Therapy
Reproductive System Changes––Feminizing
Reproductive System Changes––Masculinizing
Risks
Laboratory Monitoring Considerations
Summary
References
Chapter (105)
105. Gonadotropin Regulation and Androgen and Estrogen Physiology
Regulation Of Gonadotropin
The Gonadotropins
Gonadotropin-Releasing Hormone
Feedback on Gonadotropin Secretion by Sex Steroids
Metabolic Regulation of Gonadotropins
Regulation of Gonadotropins by Stress
Seasonal Regulation of Gonadotropin Secretion
Androgen Physiology
Biosynthesis of Testosterone and Other Androgens
Structure Activity Relationship in Steroidal Androgens
The Regulation of Testosterone Production by LH
Transport and Bioavailability
Testosterone Metabolism
Mechanisms of Testosterone’s Action
Estrogen Physiology
Endogenous Estrogens
Regulation of Estradiol Production
Estrogen Transport and Bioavailability
Estrogen Metabolism
Mechanisms of Estrogen Action
References
Chapter (106)
106. Hypogonadism: Pathogenesis, Diagnosis, and Treatment
Introduction
106
Pathogenesis
Primary Hypogonadism
Klinefelter Syndrome
Other Sex Chromosome Disorders
Cryptorchidism and Spermatogenic Failure
Congenital Anorchia
Acquired Anorchia
Orchitis
Gonadotoxic Cancer Treatment
Noonan Syndrome
Myotonic Dystrophy
Autoimmune Polyglandular Syndrome
Sickle Cell Disease
Secondary Hypogonadism
Congenital Isolated Hypogonadotropic Hypogonadism
Congenital Hypogonadotropic Hypogonadism in Complex Genetic Syndromes
Congenital Combined Pituitary Hormone Deficiency
Pituitary and Nonpituitary Tumors
Nontumorous Hypothalamic-Pituitary Diseases/Injuries
Drug-induced
Diagnosis Of Hypogonadism
Clinical Diagnosis
History
Examination
Laboratory Diagnosis
Testosterone
Treatment Of Hypogonadism
Testosterone Replacement Therapy
Indications
Testosterone Preparations
Parenteral Testosterone Preparations
Transdermal Testosterone Preparations
Efficacy
Contraindications and Adverse Events
Monitoring of Treatment
Indications
Gonadotropin Treatment
. LH-like preparations can stimulate Leydig cells (Table 106.6). Recombinant LH is expensive, has a very short half-life, and ha...
Gonadotropin-Releasing Hormone Treatment
Future Directions
References
Chapter (107)
107. Testosterone Deficiency: Aging and Chronic Disease
Testosterone Deficiency: Aging and Chronic Disease
References
Chapter (108)
108. Gynecomastia
Physiologic Forms of Gynecomastia
Newborn Gynecomastia
Pubertal Gynecomastia
108
Adult and Senescent Gynecomastia
Pathologic Forms of Gynecomastia
Related to Elevated Estrogen
Related to Testosterone Deficiency
Related to Estradiol/Testosterone Imbalance
Related to Modulatory Hormones
Other Causes
Uncertain Causes
Treatment and Prevention
References
Chapter (109)
109. Erectile Dysfunction
Introduction
Guidelines
109
Epidemiology
Psychogenic Erectile Dysfunction
Organic Erectile Dysfunction
Penile Physiology
Anatomy of the Penis
Hemodynamics of Penile Erection
Innervation of the Penis
Neurotransmitters and Pharmacology of Erection
Testosterone and Male Sexual Function
Discussing Sexual Health with Patients
History
Physical Examination
Laboratory Testing
Specialized Testing for Erectile Dysfunction (Table 109.6)
Treatment Of Erectile Dysfunction
Adaptive Sexuality
Lifestyle Modification
Mental Health
Vacuum Erection Device
Medical Therapy For Erectile Dysfunction
Herbal Supplements and Complementary Medicine
Phosphodiesterase Type 5 Inhibitors
Intracavernosal Injection Therapy
Urethral Prostaglandin Suppositories
Malleable Penile Prostheses
Inflatable Penile Prostheses
Penile Vascular Surgery
Additional Conditions Contributing To Male Sexual Dysfunction
Disorders of Ejaculation, Emission, and Orgasm
Peyronie Disease
Priapism
New Pharmacotherapeutics
Stem Cell Therapy
Low-Intensity Extracorporeal Shock Wave Therapy
Platelet-Rich Plasma
Conclusions
References
Chapter (110)
110. Misuse and Abuse of Anabolic Hormones
Misuse and Abuse of Anabolic Hormones
AAS: Use, Misuse, and Abuse
Use
Misuse
Abuse
History and Epidemiology of Anabolic Steroid Abuse
History of Anabolic Steroid Abuse
Epidemiology of Anabolic Steroid Abuse
Ergogenic Potential of Anabolic Steroids
Types and Patterns of Androgen Doping
Direct Doping
Indirect Doping
Detection of Androgen Doping
Detection of Compounds Used in Direct Androgen Doping
Evaluation Process of AAS Abuse in Elite Athletes
Assessment of Men Abusing AAS in the Clinics
Administration of Epitestosterone
Masking Agents
Cardiovascular
Reproductive
Gynecomastia
Hepatic
Musculoskeletal
Renal
Dermatologic
Neuropsychiatric and Behavioral
Dependence
Elite Athletes
General Public
Conclusion
References
Chapter (111)
111. Regulation of Spermatogenesis
The Seminiferous Tubules
The Intertubular Tissue
111
Postnatal Growth
Cellular Development and Organization for Functional Control
The Spermatogenic Epithelium
Sertoli Cells
Spermatogonia and Spermatogonial Stem Cells
The Spermatogonial Stem Cell Niche
Spermatogonial Differentiation and Its Control
Meiosis
Spermiogenesis: Emergence of Spermatozoa
Germ Cell Associations and the Spermatogenic Cycle
Spermatogenic Efficiency and Germ Cell Loss
Genetic Defects and Spermatogenesis
Epigenetics in the Male Germline
The Intertubular Compartment
Leydig Cells
Other Interstitial Cells
The Immunologic Environment of the Testis
Endocrine and Local Control of Spermatogenesis
Interactions Between the Testis, Hypothalamus, and Pituitary Gland
Control of Sertoli Cell Function
Sertoli Cells During Fetal and Postnatal Development
Control of Spermatogenesis: Challenges Ahead
References
Chapter (112)
112. Prevalence and Causes of Male Infertility
Prevalence and Causes of Male Infertility
Common Causes Of Male Infertility
Hypothalamic-Pituitary Disorders
Testicular Disorders With Spermatogenesis Defects
Sperm Motility, Morphology, Function, or Genetic Incompetence—Spermatogenesis Qualitative Defects
Ductal Obstruction or Dysfunction
Idiopathic Azoospermia, Oligozoospermia, Asthenozoospermia, and Teratozoospermia
Other Possible Contributing Factors To Male Infertiliy
Obesity
Environmental Factors: Endocrine Disruptors
Cigarette Smoking
Cell Phone
Scrotal Hyperthermia
Epigenetics And Male Infertility
Summary And Future Directions
References
Chapter (113)
113. Clinical Management of Male Infertility
Clinical Management of Male Infertility
Introduction
Health Considerations For Infertile Men
Evaluation of Male Infertility
History and Physical Examination
Semen Analysis
Further Investigations and Complete Evaluation
Endocrine Evaluation
Low Semen Volume
Postejaculatory Urinalysis
Transrectal Ultrasound
Oligospermia/Asthenospermia/Teratospermia
Semen Leukocytes and Screening for Genital Infection
Antisperm Antibodies (ASA)
Genetic Testing (see Chapter 112)
Sperm DNA Fragmentation
Other Genetic Anomalies
Azoospermia
Management of Male Infertility
Lifestyle Modifications
Hormone Optimization
Ejaculatory Dysfunction
Ejaculatory Duct Obstruction
Pyospermia
Immunological Infertility
Varicocele Repair
Vasal Reconstruction
Assisted Reproductive Techniques (ART)
Sperm Retrieval
Conclusion
References
Chapter (114)
114. Male Contraception
The Need for Male Contraception
Current Methods
Withdrawal
Male Condom
114
Vas Occlusion
Male-Directed Contraceptive Methods in Development
Hormonal Male Contraception
Other New Hormonal Agents for Male Contraception
Nonhormonal Targets for Male Contraception
Men’s Attitude and Acceptability of Male Contraception
Summary and Future Directions
References
Chapter (115)
115. Testicular Dysgenesis Syndrome and Testicular Tumors
Testicular Dysgenesis Syndrome and Testicular Tumors
Testicular Dysgenesis Syndrome
Epidemiology
Biological Evidence
Cryptorchidism
Classification
Embryology
Etiology
Prevalence
Treatment
Long-Term Effects
Hypospadias
Embryology
Etiology
Prevalence
Treatment
Long-term Effects
Minipuberty
Minipuberty in Healthy Infants
Minipuberty in Various DSD and TDS Conditions
Testicular Tumors
Classification
Testicular Germ Cell Tumors
Fertility Preservation and Long-Term Effects of Testicular Cancer
Sex Cord-Stromal Tumors of the Testis
Other Testicular Tumors
Conclusion
References
Chapter (116)
116. Benign Prostatic Hyperplasia and Prostate Cancer
Introduction
Benign Prostatic Hyperplasia
Diagnosis
116
Management and Treatment of BPH
Summary and Future Research
Prostate Cancer
Diagnosis and Detection of Localized Prostate Cancer
Management and Treatment of Localized Prostate Cancer
Diagnosis and Detection of Advanced Prostate Cancer
Management and Treatment of Advanced Prostate Cancer
Summary and Future Challenges
References
Chapter (117)
117 - The Menstrual Cycle and Disorders of Ovulation
THE MENSTRUAL CYCLE: AN INTRODUCTION
Overview of the Hypothalamic-­Pituitary-­Ovarian Axis
The GnRH Pulse Generator
Anterior Pituitary Gonadotropes and Regulation of Gonadotropin Secretion
Ovarian Folliculogenesis and Steroidogenesis
Mechanisms of Ovarian Feedback Control of GnRH and Gonadotropin Secretion
Uterine Events During the Menstrual Cycle
Integrated Physiology of the Menstrual Cycle
Luteal-­Follicular Transition
Follicular Phase
Midcycle Gonadotropin Surge and Ovulation
Luteal Phase
Neuroendocrine Mechanisms of Anovulation
Physiological Anovulation
Selected Causes of Pathological Anovulation
Conclusion
References
The Menstrual Cycle: An Introduction
Overview of the Hypothalamic-­Pituitary-­Ovarian Axis
The GnRH Pulse Generator
Anterior Pituitary Gonadotropes and Regulation of Gonadotropin Secretion
Ovarian Folliculogenesis and Steroidogenesis
Mechanisms of Ovarian Feedback Control of GnRH and Gonadotropin Secretion
Uterine Events During the Menstrual Cycle
Integrated Physiology of the Menstrual Cycle
Luteal-­Follicular Transition
Follicular Phase
Midcycle Gonadotropin Surge and Ovulation
Luteal Phase
Neuroendocrine Mechanisms of Anovulation
Physiological Anovulation
Selected Causes of Pathological Anovulation
Conclusion
Chapter (118)
118 - Folliculogenesis, Ovulation, and Luteogenesis
Folliculogenesis
Folliculogenesis, Ovulation, and Luteogenesis
118
Preantral Follicles
Antral or Graafian Follicles
Oocyte Maturation Following the LH Surge
Luteinization
Luteolysis
Conclusion
References
Chapter (119)
119 - Ovarian Hormone Synthesis
Ovarian Hormone Synthesis
119
Acquisition of Cholesterol
The Cholesterol Side-­Chain Cleavage Reaction
Synthesis of Androgens
Synthesis of Estrogens
Steroid Hormone Metabolites
MicroRNA Regulation of Steroidogenic Enzymes
Peptide Hormones and Growth Factors Produced by the Ovary
Transforming Growth Factor-­β Superfamily
Relaxin
Gonadotropin Control of Ovarian Steroid Production
Follicle-­Stimulating Hormone and Luteinizing Hormone
Mechanisms of Gonadotropin Action
Other Hormones with Possible Actions in the Ovary
Intraovarian Regulators
Cholesterol Precursors
Role of Estrogens
Role of Androgens
Role of Progesterone
Transforming Growth Factor-­β Superfamily
Eicosanoids
Insulin-­Like Growth Factors and Their Binding Proteins
Other Intraovarian Regulators
Hormone Production During the Ovarian Life Cycle
The Fetal and Prepubertal Ovary
The Ovary During Reproductive Life
Endocrine Activity of the Postmenopausal Ovary
Conclusion
References
Chapter (120)
120. Estrogen and Progesterone Action
120
Nuclear Receptor Structure
Coactivators and Corepressors
Coactivators
Corepressors
Mechanisms of Estrogen Action
Estrogen Receptor Ligands
Estrogen Receptor Structure
Estrogen Receptor-Coactivator Interactions
Molecular Mechanisms of Estrogen Receptor Action
Mechanisms of Progesterone Action
Progesterone Receptor Isoforms
Molecular Mechanisms of Progesterone Action in the Endometrium and on Blastocyst Implantation
Conclusions
References
Chapter (121)
121. Contraception
Contraception
121
Drug Interactions
Medical Eligibility Criteria
High-Effectiveness Methods
Intrauterine Devices
Progestogen-Only Implant
Medium Effectiveness Methods
Progestogen-Only Injectable
Combined Hormonal Contraception
CHC Pill
CHC Patch and Vaginal Ring
Progestogen-Only Pill
Low Effectiveness Methods
Condoms
Diaphragm and Cap
Fertility Awareness-Based Methods
Lactational Amenorrhea
Sterilization
Emergency Contraception
Non-Contraceptive Benefits of Contraception
Summary and Future Directions
References
Chapter (122)
122. Menopause and Perimenopause
Menopause and Perimenopause
Staging Reproductive Aging
Late Reproductive Stage (Stage –3)
Menopausal Transition
Menopause
Postmenopause
Epidemiology (TABLE 122.1)
Age of Menopause
Population-Level Determinants of Menopause
Determinants of Early Menopause and Primary Ovarian Insufficiency
Pathophysiology Of Menopause
Ovarian Follicle Depletion—Current State of Knowledge
Follicular Growth Activation and Atresia
Ovarian and Pituitary Hormones
Clinical Presentation
The “Core Four” Symptoms: Hot Flashes, Vaginal Dryness, Adverse Mood, and Poor Sleep
Vasomotor Symptoms (Hot Flashes and Night Sweats)
Vaginal Dryness
Adverse Mood
Sleep Disturbances
Other Symptoms
Reproductive And Somatic Aging
Hypothalamic-Pituitary-Ovarian Axis
Somatotropic Axis
Thyroid
Adrenal
Bone
Cognition
Cardiovascular
Estrogen-Dependent Disorders
Diagnosis
Treatment
Hormone Therapy Overview
Vasomotor Symptoms
Urogenital Symptoms (GSM)
Sleep Disturbances
Adverse Mood
Other Common Issues
Complementary and Alternative Medicine in the Treatment of Menopausal Symptoms
Long-Term Use for Prevention of Chronic Conditions
Cancer Risks and Mitigation of Risk With Hormone Therapy
Summary
References
Chapter (123)
123. Female Infertility: Evaluation and Management
Female Infertility: Evaluation and Management
Introduction
History
Epidemiology
Pathogenesis And Clinical Features
Ovulatory Dysfunction
Anatomic Dysfunction
Ovarian Aging
Unexplained Infertility
Assessment of the Normal Menstrual Cycle
Assessment of Anatomic Dysfunction
Assessment of Cervical Mucus–Sperm Interaction
Assessment of Ovarian Aging
From Diagnosis to Prognosis
Ovarian Stimulation
Intrauterine Insemination
In Vitro Fertilization
Summary And Future Developments
References
Chapter (124)
124. Androgen Excess Disorders in Women
Androgen Excess Disorders in Women
Androgen Physiology and Pathophysiology
Androgen Biosynthesis
Regulation of Androgen Secretion
Blood Levels and Transport of Androgens
Mechanisms of Androgen Action
Clinical Manifestation of Androgen Excess
Cutaneous Manifestations
Polycystic Ovary and Polycystic Ovary Morphology
Metabolic Dysfunction
Common Androgen Excess Disorders
Polycystic Ovary Syndrome
Ovarian Neoplasms
Other Ovarian Hyperandrogenic Disorders
Congenital Adrenal Hyperplasia
Other Adrenal Hyperandrogenic Disorders
Idiopathic Hyperandrogenism
Diagnosis of Hyperandrogenism
Clinical Hyperandrogenism
Biochemical Hyperandrogenism
Treatment of Polycystic Ovary Syndrome and other Androgen Excess Disorders
Androgen Suppression
Ovulation Induction and Assisted Reproduction
Prevention of Long-Term Complications
Summary and Future Directions
References
Chapter (125)
125. Endometriosis
Pathogenesis
Endometriosis
125
Genetic Basis
Environment
Somatic Mutations
Pathophysiology
Cell Survival and Adhesion
Matrix Degradation And Invasion
Cell Growth and Neoangiogenesis
Altered Endocrine Activity
Immunity and Inflammation
Mechanisms Underlying Pain and Infertility
Diagnosis
Therapies
Medical
Surgical
Integrated Approach to the Patient With Endometriosis
Endometriosis-Associated Cancer Risk
Summary
References
Chapter (126)
126. Uterine Fibroids and Adenomyosis
Introduction
Epidemiology and Classification
Uterine Fibroids and Adenomyosis
126
Pathogenesis
Diagnosis
Therapies
Adenomyosis
Overview
Epidemiology and Classification
Pathogenesis
Diagnosis
Treatment
Summary
References
Chapter (127)
127. Hormonal Control of the Breast*
Hormonal Control of the Breast*
Adolescence
Anchor 466
The Mature Breast
Nulliparous Women
Parous Women
The Menopausal Breast
References
Chapter (128)
128. Sexual Dysfunction in the Female
Sexual Dysfunction in the Female
Normal Female Sexual Response
Neurotransmitters of Desire and Arousal
Endocrinology of Desire and Subjective Arousal
Endocrinology of Objective Arousal
Normal Vulvar and Vaginal Anatomy and Role of Hormones
Pathophysiology of Female Sexual Dysfunction
Hypoactive Sexual Desire Disorder
Sexual Arousal Disorders
Orgasmic Disorder
Sexual Pain Disorders
Treatments For Sexual Dysfunction
Hypoactive Sexual Desire Disorder
Nonhormonal Treatments
Hormonal Management of Hypoactive Sexual Desire Disorder
Genital Arousal and Sexual Pain
Persistent Genital Arousal Disorder
Conclusion
References
Chapter (129)
129. Hormonal Control of Human Pregnancy and Parturition
Preparation of the Endometrium
Embryo Competency
Hormonal Control of Human Pregnancy and Parturition
129
Decidualization of the Endometrium
Implantation and Placentation
Immune Tolerance of the Conceptus
Pregnancy Maintenance
Chorionic Gonadotropin
Gonadotropin-Releasing Hormone
Proopiomelanocortin-Derived Hormones
Corticotropin-Releasing Hormone
Placental Lactogen and Placental Growth Hormone
Modulation of Maternal Energy Homeostasis
Growth Factors and Cytokines
Steroid Hormones
Parturition and Birth Timing
Evolutionary Perspective
Fetal Maturation
Progesterone Withdrawal
Stress and Tissue-Level Inflammation
Progestin Therapy to Prevent Preterm Birth
Estrogens
Conclusion/Future Directions
References
Chapter (130)
130. Fetal and Neonatal Endocrinology
Fetal and Neonatal Endocrinology
Overview
Ectopic Fetal Hormone Production
The Hypothalamic-Pituitary Systems
Fetal Adrenal
Fetal Thyroid
The Fetal Autonomic Nervous System
Fetal Pancreas and Glucose Homeostasis
Fetal Parathryoid and Bone Metabolism
Parathyroid Development
Fetal Calcium and Phosphate Metabolism
Regulation of Calcium Homeostasis by PTH/PTHrP
Fetal Growth
Insulin-Like Growth Factors
Insulin
Fibroblast Growth Factor
Other Growth Factors and Signaling Pathways
Cortisol-Mediated Adaptations
Catecholamine-Mediated Adaptations
BAT Thermogenesis
Glucose Homeostasis
Calcium Homeostasis
Other Hormonal Adaptations
Future Directions
References
Chapter (131)
131. Hormonal Changes and Pregnancy Testing
Hormonal Changes and Pregnancy Testing
Introduction
Pituitary Gland
Hypopituitarism
Prolactin
Changes in Prolactin Physiology
Prolactinomas in Pregnancy
The Effect of Pregnancy on a Preexisting Prolactinoma
The Effect of Dopamine Agonists on a Developing Fetus
Prolactin and Antipsychotic Medication
Breastfeeding and Prolactin
Growth Hormone
Changes in GH Physiology During Pregnancy
GH Deficiency
GH Excess
Vasopressin
Thyroid Gland
Thyroid Hormones and Thyroid Regulation
Hyperthyroidism
Hypothyroidism
Adrenal Gland
Regulation of Glucocorticosteroid Synthesis
Parathyroid Glands
Regulation of Calcium During Pregnancy
Parathyroid Hormone–Related Protein
Vitamin D Deficiency
Hypercalcemia
Conclusion
References
Chapter (132)
132. Autoimmune Polyglandular Syndromes
Background
The Basis of Autoimmune Disease
The Normal Immune Response
Self-Tolerance
Central Tolerance
Peripheral Tolerance
Pathogenesis of Autoimmune Disease
Autoimmune Polyglandular Syndrome Type 1
Definition
Pathogenesis
Clinical Features
Chronic Mucocutaneous Candidiasis
Hypoparathyroidism
Adrenal Insufficiency
Gonadal Insufficiency
Other Endocrinopathies
Gastrointestinal Manifestations
Ectodermal Manifestations
Other Manifestations
Natural Course and Mortality
Diagnosis and Management
Autoimmune Polyglandular Syndrome Type 2
Definition
Pathogenesis
Clinical Features and Diagnosis
Management
References
Chapter (133)
133 - Multiple Endocrine Neoplasia Type 1
CLINICAL FINDINGS AND TREATMENT
Parathyroid Tumors
Pancreatic Tumors
Pituitary Tumors
Associated Tumors
Carcinoid Tumors
Other Clinical Considerations
MOLECULAR GENETICS
The MEN1 Gene
MEN1 Germline Mutations
MEN1 Germline Coding Variants
MEN1 Somatic Mutations
MEN1 Phenocopies and Mutations in Other Genes
MEN1 Mutational Analysis in Clinical Practice
SURVEILLANCE OF INDIVIDUALS AT RISK OF MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 TUMORS
FUNCTION OF THE MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 PROTEIN, MENIN, AND FUTURE TARGETED TREATMENTS
CONCLUSION
REFERENCES
Chapter (134)
134. Multiple Endocrine Neoplasia Types 2 and 3, and Medullary Thyroid Carcinoma
Multiple Endocrine Neoplasia Types 2 and 3, and Medullary Thyroid Carcinoma
Prevalence and Epidemiology
RET Structure and Function
RET Ligands
Functional Effects of RET Variants
Histology
C-Cells
C-Cell Hyperplasia
Clinical Relevance of Germline Ret Variants in Multiple Endocrine Neoplasia Type 2 and Multiple Endocrine Neoplasia Type 3
Multiple Endocrine Neoplasia Type 2 and Familial Medullary Thyroid Carcinoma
Multiple Endocrine Neoplasia Type 3
Specific Genotype-Phenotype Correlations
Clinical Presentation
Medullary Thyroid Carcinoma
Pheochromocytoma
Hyperparathyroidism
Screening in Multiple Endocrine Neoplasia Type 2 and Multiple Endocrine Neoplasia Type 3
Biochemical Screening
Genetic Screening
Management Implications of Genetic Results
Timing and Benefits of Prophylactic Thyroidectomy
Strategy and Extent of Thyroidectomy
The Role of Somatic Mutations
Sporadic Medullary Thyroid Carcinoma
Low-Penetrance Phenotypes
Hereditary Medullary Thyroid Carcinoma
Germline Screening
Sporadic Medullary Thyroid Carcinoma
Staging of Medullary Thyroid Carcinoma
Pheochromocytoma
Hyperparathyroidism
Management Of Medullary Thyroid Carcinoma
Localized Medullary Thyroid Carcinoma
Recurrent Medullary Thyroid Carcinoma
Persistent and Metastatic Medullary Thyroid Carcinoma
Management of Pheochromocytoma
Management of Hyperparathyroidism
Conclusion
References
Chapter (135)
135. Neuroendocrine Tumor Syndromes
Neuroendocrine Tumor Syndromes
Introduction
Epidemiology
Etiology
Pathologic Classification and Prognosis
Laboratory Tests and Biomarkers
Imaging
Biopsy
Clinical Features of Functional net and Their Syndromes
Insulinoma
Gastrinoma
VIPoma
Glucagonoma
Somatostatinoma
PPoma
Serotoninoma
Carcinoid Syndrome
Surgical Resection
Ablation of Primary Pancreas Neuroendocrine Tumors
Hepatic Arterial Embolization
Selective Internal Radiation Therapy
Systemic Therapies
Palliative Symptom Management
Conclusion
References
Chapter (136)
136. Ectopic Hormone Syndromes
History
Theoretical Considerations
136
Theories of the Origin of Ectopic Hormones
Hormones in Small-Cell Lung Carcinoma
Ectopic Adrenocorticotropic Hormone Syndrome
The POMC Gene
Regulation of POMC Gene Expression
Ectopic Adrenocorticotropic Hormone Syndrome
Pro-Opiomelanocortin Processing
Diagnosis
Treatment
Diagnosis
Management
Humoral Hypercalcemia of Malignancy
Parathyroid Hormone–Related Protein
Hypercalcemia in Hematologic Malignancy
Diagnosis
Treatment
Oncogenic Osteomalacia
Non–Islet Cell Tumor Hypoglycemia
Diagnosis
Management
Other Pituitary Ectopic Hormones
Gonadotropins
Ectopic Gut Hormone Syndromes
Ectopic Renin Secretion
Acknowledgments
References
Chapter (137)
137. Endocrine-Disrupting Chemicals and Human Health
Endocrine-Disrupting Chemicals and Human Health
137
Health Effects of Environmental Chemicals
The Lessons of Lead
The Lessons of PCBs
The Lessons of Diethylstilbestrol (DES)
Windows of Susceptibility
Mechanisms of EDC Actions
Dose-Response Characteristics of EDCS
Chemical Exposures in the Human Population
Disease Trends and Effects of EDCs
Cancer
Thyroid Disruption
Neurobehavior
Emerging Issues
Endocrine Disruptors, the Epigenome, and Transgenerational Effects
Increasing Exposures Worldwide
Expanded List of Diseases Related to EDCs
Mixtures
E-Waste
Green Chemistry
Conclusion and Guidance for Clinicians
References

Citation preview

DeGroot’s

ENDOCRINOLOGY

Basic Science and Clinical Practice EIGHTH EDITION

Editor-in-Chief R. Paul Robertson, MD

Emeritus Professor Pennock Chair for Diabetes Research Section Head, Endocrinology and Metabolism Medicine and Cell Biology University of Minnesota Minneapolis, Minnesota; Division Head, Clinical Pharmacology Medicine and Pharmacology University of Washington Seattle, Washington

Associate Editors Linda C. Giudice, MD, PhD

Distinguished Professor and The Robert B Jaffe MD Endowed Professor in the Reproductive Sciences Department of Obstetrics, Gynecology and Reproductive Sciences University of California, San Francisco San Francisco, California

Ashley B. Grossman, BA, BSc, MD, PhD, FRCP, FMedSci Professor of Endocrinology Green Templeton College University of Oxford Oxfordshire, United Kingdom; Professor Neuroendocrinology Barts and the London School of Medicine University of London London, United Kingdom

ELSEVIER

Gary D. Hammer, MD, PhD

Millie Schembechler Professor of Adrenal Cancer Director - Endocrine Oncology Program Professor of Internal Medicine – Metabolism, Endocrinology and Diabetes Professor of Molecular and Integrative Physiology Professor of Cell and Developmental Biology University of Michigan Ann Arbor, Michigan

Michael D. Jensen, MD

Distinguished Mayo Clinic Investigator Tomas J. Watson, Jr. Professor in Honor of Dr. Robert L. Frye Division of Endocrinology, Diabetes, Metabolism, and Nutrition Mayo Clinic Rochester, Minnesota

George J. Kahaly, MD, PhD

Professor of Medicine and Endocrinology/Metabolism Department of Medicine I Johannes Gutenberg University Medical Center Mainz, Germany

Ronald S. Swerdloff, MD, MACP

Senior Investigator The Lundquist Institute at Harbor-UCLA; Distinguished Professor of Medicine David Geffen School of Medicine at UCLA; Chief, Division of Endocrinology Harbor-UCLA Medical Center Torrance, California

Rajesh V. Thakker, MD, ScD, FRCP, FMedSci, FRS

May Professor of Medicine Academic Endocrine Unit Radcliffe Department of Medicine University of Oxford Oxfordshire, United Kingdom

Elsevier 1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-­2899 DEGROOT’S ENDOCRINOLOGY: BASIC SCIENCE AND CLINICAL PRACTICE, EIGHTH EDITION Copyright © 2023 by Elsevier Inc. All rights reserved.

ISBN: 978-­0-­323-69412-4 Volume 1: 9780443107801 Volume 2: 9780443107818

Chapter 84 by Nicole Vietor and Henry Burch, Chapter 99 by Karel Pacak, and Chapter 120 by Francesco DeMayo are in the public domain. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notice Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Previous editions copyrighted 2016, 2010, 2006, 2001, 1995, 1989, 1979.

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1 Principles of Endocrinology J. Larry Jameson

OUTLINE Definition and Scope of Endocrinology, 2 Historical Perspectives, 3 Principles of Hormone Action, 6 Hormone Biosynthesis and Secretion, 6 Feedback Regulation, 6 Paracrine and Autocrine Regulation, 7 Hormonal Rhythms and Pulsatility, 7

Hormone Transport and Degradation, 8 Hormone Action Through Receptors, 8 Membrane Receptors, 9 Nuclear Receptors, 10 Role of the Clinical Endocrinologist, 10 Major Unsolved Problems, 12

 

DEFINITION AND SCOPE OF ENDOCRINOLOGY The term endocrine was coined by Starling to contrast the actions of hormones secreted internally (endocrine) with those secreted externally (exocrine) or into a lumen, such as the gastrointestinal tract.1 This terminology continues today but makes the specialty somewhat opaque to the general public, who are more familiar with the term hormone and with particular disorders of the endocrine system. The term hormone is derived from the Greek verb hormao, which means “to set in motion.” This phrase captures the dynamic properties of hormones and their ability to elicit a cascade of physiologic responses by acting on specific target tissues. Reminiscent of Newton’s third law of motion, which states that “for every action, there is an equal and opposite reaction,” hormone action is typically counteracted by physiologic responses that restore the system to equilibrium. The major physiologic processes controlled by hormones include (1) growth and maturation, (2) intermediary metabolism, and (3) reproduction. However, the clinical specialty of endocrinology is most clearly delineated by diseases that afflict the classic glands, that is, hypothalamus, pituitary, thyroid, parathyroid, pancreatic islets, adrenal gland, testis, and ovary. In various parts of the world, additional clinical disorders, such as hypertension, nutrition, obesity, osteoporosis, and hyperlipidemia, also fall within the scope of endocrinology. The basic science of endocrinology has evolved from studies of hormone identification and structure to a greater focus on hormone action. Concepts of receptors and intracellular signaling, as well as many aspects of transcriptional regulation, remain an essential component of the field. Endocrinology is ultimately the study of intercellular communication. In some cases, communication occurs within the same tissue, as exemplified by autocrine and paracrine actions of insulin-­like growth factor-­1 (IGF-­1). More classically, hormones mediate communication between organs, as exemplified by the actions of parathyroid hormone (PTH) on bone or kidney. In this era of genomics, proteomics, and metabolomics, the traditional lines that separate endocrinology from other physiologic disciplines are becoming blurred. Erythropoietin is a classic hormone. Because it is produced by the kidney and regulates erythrocyte production, erythropoietin’s clinical

2

role is relevant primarily to the fields of nephrology and hematology. Similarly, blood cell–stimulating factors such as granulocyte colony-­ stimulating factor (G-­CSF) are studied and used by hematologists and oncologists. The receptors for colony-­stimulating growth factors such as G-­CSF and granulocyte-­macrophage colony-­stimulating factor are, however, members of a superfamily that includes the growth hormone (GH) and prolactin (PRL) receptors. These receptors share similar intracellular signaling systems, including the Janus kinase (JAK)–signal transduction and activators of transcription (STAT) pathway. Growth factors with more pleomorphic functions, such as cytokines, are being investigated and used in almost every specialty. Principles of endocrinology are readily transferable to other clinical disciplines. For example, hormones play a crucial role in blood pressure maintenance, intravascular volume regulation, and peripheral vascular resistance tone in the cardiovascular system. Angiotensin II, catecholamines, endothelins, and other vasoactive substances act via specific receptors to mediate dynamic changes in vascular tone. The heart produces hormones, such as atrial natriuretic peptide, in response to volume overload, resulting in compensatory natriuresis. The gastrointestinal tract is a remarkably rich source of peptide hormones, such as ghrelin, gastrin, glucagon-­like peptide, cholecystokinin, secretin, and vasoactive intestinal peptide, among many others. Some of these factors, such as ghrelin and cholecystokinin, modulate appetite and perform local actions in the gastrointestinal tract; others, such as gastrin and secretin, act mainly in the gastrointestinal tract to induce physiologic responses to meals. With the discovery of new hormones (e.g., PTH-­related peptide, leptin, ghrelin, activin, atrial/brain natriuretic peptide, fibroblast growth factor 21, fibroblast growth factor 23), the scope of investigative and clinical endocrinology continues to expand. In addition, many areas of traditional endocrinology have been “spun off” and transformed into other disciplines. For example, although hypothalamic regulation of the pituitary gland remains a core element of endocrinology, neuroendocrinology is rapidly becoming a distinct discipline. Similarly, calcium regulation is inextricably linked to bone metabolism. Some bone disorders, such as osteoporosis or rickets, are treated mainly by endocrinologists, whereas others, such as renal osteodystrophy or

CHAPTER 1  Principles of Endocrinology phosphate wasting disorders, are often managed by nephrologists. Reproductive endocrinology has become a subspecialty of gynecology and urology, primarily because of the skills needed to perform procedures related to the evaluation and treatment of infertility. Ovulation induction protocols and various forms of assisted reproductive technology are increasingly used to manage infertility, which affects 10% to 15% of reproductive-­age couples. Intracytoplasmic sperm injection has revolutionized the approach to male infertility. A new discipline of “oncofertility” addresses the need for fertility preservation in women, as well as men, with cancers that occur during the reproductive time frame. Common endocrine diseases, such as autoimmune thyroid disease and type 1 diabetes mellitus, are caused by abnormal regulation of immune surveillance and tolerance. Less common diseases, such as polyglandular failure, Addison disease, and lymphocytic hypophysitis, also have an immunologic basis. Although immunology is an independent discipline, the interface with endocrinology is important for understanding the pathogenesis of these disorders. Cytokines and interleukins have profound effects on the functions of the pituitary, adrenal, thyroid, and gonads. Thus, the boundaries of endocrinology change constantly, spawning new disciplines and expanding into new scientific realms.

HISTORICAL PERSPECTIVES Although concepts of fertility and reproduction can be traced to ancient times, most of our current understanding of endocrinology has evolved during the past 150 years.2 The structures of the major glands and ducts were initially captured in drawings by Renaissance anatomists and artists. The publication of De Humani Corporis Fabrica in 1543 by Vesalius provided a turning point in studies of human anatomy. Fallopio, also of the Padova School, published Observationes Anatomicae in 1561, which included a detailed description of the “slender and narrow seminal passage that arises from the horn of the uterus.” A timeline for selected advances in endocrinology is depicted in Figure 1.1. Berthold recorded the physiologic consequences of castration in 1849. He demonstrated that castration of a cock caused regression of secondary sex characteristics and mating behavior. Transplantation of the testes into the abdominal cavity restored these features, proving a role for the gonads in sexual differentiation and illustrating basic principles of hormone withdrawal and replacement. In 1855, Claude Bernard noted that the liver produced two secretions, an external secretion (bile) and an internal secretion (glucose), which passed directly into the circulation. This concept was later extended by Bayliss and Starling, who discovered that secretin, a substance extracted from duodenal mucosa, induced pancreatic exocrine secretion after intravenous injection. This observation distinguished the properties of circulating hormones from physiologic reflexes mediated by the nervous system.

KEY POINTS • Although individual hormones regulate specific physiologic processes, such as growth, metabolism, and reproduction, they typically act in a concerted or integrated manner with other hormones.

Isolation/synthesis of catecholamines, cortisol, thyroxine Rx by Iodine, gland replacement Anatomy/ histology 1900

Gene regulation by transcription factors, nuclear receptors, etc Rx GH

Rx Insulin OCPs

1925

Transsphenoidal surgery Clinical descriptions of Graves’, myxedema, Addison’s, CAH pheo, diabetes, acromegaly Secretin and hormone concept

Recombinant hormones

Radioimmunoassays

1950

3

1975 Rx RAI

Genetic disease models 2000

2025

Signaling pathways cAMP, Ca2+, etc

Proteomics

Rx ATDs

Imaging techniques

Feedback regulation

Cloning of Sms, GH, hCG, etc

Isolation peptide hormones calcitonin, PTH, insulin, GH, ACTH, TSH, LH, etc

Rx Sms A, DA agonists

New approaches to autoimmunity or neoplasia

Gene and stem cell replacement Gene profiling

Fig. 1.1  Timeline of selected advances in endocrinology. ACTH, Adrenocorticotropic hormone; ATDs, antithyroid drugs; Ca2+, calcium; CAH, congenital adrenal hyperplasia; DA, dopamine; GH, growth hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone; OCPs, oral contraceptive pills; pheo, pheochromocytoma; PTH, parathyroid hormone; RAI, radioactive iodine; Rx, treatment; Sms A, somatostatin analogs, TSH, thyroid-­stimulating hormone.

4

PART 1  Principles of Endocrinology and Hormone Signaling

In the late 1800s, the clinical manifestations of many endocrine disorders were described. The Report on Myxedema by the Clinical Society of London (1888) is a remarkable example of the power of astute clinical observation. In addition to recognition that the adult disorder of myxedema shared certain clinical features of cretinism,3 a tenuous connection to thyroid gland dysfunction was proposed. The plates shown in Figure 1.2 illustrate some of the clinical manifestations of hypothyroidism as described by William Ord, who coined the term myxedema (mucinous edema).4 Several years later, George Murray tested the role of the thyroid gland in myxedema by demonstrating that repeated subcutaneous injections of sheep thyroid extract corrected the disorder.5 This was probably the first example of successful hormone replacement and spawned parallel efforts for other glandular diseases. By the turn of the century, the clinical manifestations of Graves disease, acromegaly, Addison disease, diabetes mellitus, and

pheochromocytoma were well established. Hormone isolation and replacement strategies became a major research effort, culminating in the characterization of corticosteroids, thyroid hormones, and sex steroids. The history of endocrinology is replete with colorful renditions of hormone isolation and discovery. A recurring theme is teamwork and parallel observations by different teams working on the same problem—a testimony to the impact of scientific communication and the need for technology to drive advances. The discovery of insulin in 1921 has been chronicled extensively and is a true inflection point in endocrinology.6 The pancreatic islets are clusters of endocrine cells that are embedded within the exocrine pancreas. Early experiments in dogs by Minkowski7 showed that pancreatectomy caused diabetes, demonstrating the pancreas as the organ responsible for regulating glucose. Banting and Best set out to isolate insulin, a process that was greatly aided by the expertise of Collip, a

REPORT OF A

COMMITTEE OF THE CLIN ICAL SOCIETY OF LONDON NOMINATED DECEMBER 14, 1883 TO INVESTIGATE THE SUBJECT OF

MYXEDEMA SUPPLEMENT TO VOLUME THE TWENTY-FIRST

General Society Report on Myxedema (1888)

LONDON:

Plate 1

LONGMANS, GREEN, AND CO.

1888 A

From Photographs

Plate 2

Plate 3 Danielsson&Co., lith

B Fig. 1.2  A, Cover page from the 1888 Clinical Society of London Report on Myxoedema. B, Clinical manifestations of myxedema. Plates taken from serial photographs of a woman with untreated hypothyroidism. Plate 1: At 21 years of age, before onset of myxedema. Plate 2: At 28 years of age, showing early features of myxedema. Plate 3: At 32 years of age, illustrating overt features of myxedema. (From Clinical Society of London report on myxedema. Boston: Francis A. Countway Library of Medicine; 1888. Photographs originally published in Ord WM. On myxoedema, a term proposed to be applied to an essential condition in the “cretinoid” affection occasionally observed in middle-­aged women. Medico-­Chirurgical Trans. 1978;61:57–78.)

CHAPTER 1  Principles of Endocrinology protein chemist who isolated several other peptide hormones, including PTH.8,9 Despite erratic initial results in diabetic dogs, Banting and Best soon achieved unequivocal success using partially purified insulin. At the time of insulin isolation, children with type 1 diabetes had no treatment options aside from starvation therapy, which could not prevent their ultimate demise from hyperglycemia and ketoacidosis. The initial insulin treatment results were stunningly successful, providing immediate clinical benefits soon followed by the ability to achieve long-­term management with repeated use of insulin injections (Fig. 1.3). This dramatic treatment strategy was stymied initially by the limited supply of purified insulin, a problem that ultimately was solved by the development of recombinant human insulin. In this current era, pancreas and islet transplantation represent alternative treatment approaches. However, limited human donor tissue and ongoing challenges with immunosuppression have restricted the use of transplantation to patients with severe type 1 diabetes. There is hope, however, that stem cell biology or the ability to regenerate pancreatic islet β cells might ultimately overcome these limitations. Recognition that the hypothalamus produces a variety of pituitary regulatory factors was another major advance. In addition to establishing a link between the brain and the “master gland,” the hypothalamic-­pituitary system underscored the critical importance of anatomic proximity and vascular delivery for the regulation of hormone action. It is now appreciated that discrete pulses of hypothalamic gonadotropin-­releasing hormone (GnRH), GH-­releasing hormone, thyrotropin-­releasing hormone (TRH), and corticotropin-­releasing hormone (CRH) act locally on the pituitary gland and exert little, if any, physiologic effect at more distal sites in the body. Following the isolation of many steroid and peptide hormones during the first half of the twentieth century, a conceptual framework was outlined for mechanisms of hormone action. For peptide hormones, Sutherland established the idea of a second messenger system

5

in which a hormone binds to a membrane receptor, thereby activating intracellular second messenger pathways such as cyclic adenosine monophosphate (cAMP).10 For steroid and thyroid hormones, Tata established the concept of hormone action at the nuclear level, acting via intracellular receptors that altered gene expression, which in turn caused changes in protein levels.11 The development of the radioimmunoassay (RIA) by Berson and Yalow revolutionized endocrine physiology and diagnosis by allowing accurate measurement of minute amounts of circulating hormones.12 The impact of RIA on physiology, endocrinology, and clinical medicine cannot be overemphasized. RIA and related assays are now used routinely for almost all hormone measurements and have replaced many less-­sensitive chemical methods and bioassays. RIA was once the province of specialty endocrine laboratories but has gradually become automated and integrated into clinical pathology laboratories. Mass spectroscopy methods are being used increasingly as a means to measure steroids and peptides. These methods not only are sensitive and highly quantitative, but also do not necessarily depend upon antibodies to detect specific molecules. Important advances in therapeutic modalities have accompanied our improved understanding of endocrine diseases. Hormone replacement strategies have been refined, along with advances in surgical approaches for endocrine tumors. Many hormone excess syndromes are primarily managed surgically, including transsphenoidal surgery for pituitary tumors or excision of parathyroid, adrenal, and pancreatic tumors. Many glandular surgeries are now performed via minimally invasive techniques, such as laparoscopy or video-­assisted resection through very small incisions. In addition to hormonal replacements, important medical therapies that have been developed include the use of radioactive iodine13 and antithyroid drugs14 for hyperthyroidism, dopamine agonists for prolactinomas,15 somatostatin analogues for acromegaly, oral hypoglycemics for diabetes,16 gonadal steroids as

Fig. 1.3  Treatment of type 1 diabetes mellitus with insulin. Teddy Ryder was one of the first patients treated by Dr. Banting. After undergoing “starvation treatment” (left panel), which was the only therapy available at the time, he began insulin treatment at 5 years of age (July 10, 1922) (right panel). One year later (July 10, 1923), he is seen “cured.” Teddy Ryder lived to 76 years of age. (Modified with permission from the University of Toronto Libraries Discovery and Early Development of Insulin online collection, http://digital. library.utoronto.ca/insulin/.)

6

PART 1  Principles of Endocrinology and Hormone Signaling

contraceptives,17 and somatostatin analogues for tumors of the gastrointestinal tract.18 In recent years, the tools of molecular genetics have dramatically accelerated our understanding of endocrinology. DNA sequences encoding hormones such as somatostatin,19 GH,20 insulin,21 and chorionic gonadotropin22 were among the first human complementary DNAs (cDNAs) cloned. Recombinant DNA techniques are now used routinely to identify new hormones and receptors and to elucidate hormone function. Hormone genes have provided important models for understanding mechanisms of transcriptional regulation. Hormones typically are expressed in a cell-­specific manner (e.g., GH, thyroglobulin), providing prototypes for identifying transcription factors (e.g., Pit-­1, TTF-­1) that restrict expression to particular cells or tissues. Hormone-­regulated pathways have provided experimental variables that can be switched on or off, thereby revealing highly regulated target genes that can be used as experimental models. Thus, studies of the cAMP signaling system have unraveled the protein kinase A cascade and transcription factor targets, such as cAMP response element binding protein. Nuclear receptor pathways have been particularly illuminating. In addition to identifying target genes regulated by hormones such as estrogen, glucocorticoid, or thyroid hormone, detailed analyses of these pathways have helped to define how DNA binding specificity is encoded in promoters and how transcription factors suppress or enhance gene expression by recruiting corepressor or coactivator complexes. Transcription by nuclear receptors is arguably the best understood paradigm for how transcription factors initiate transcription, assemble a transcription complex, and renew the process to ensure multiple rounds of RNA synthesis. The genetic basis for several hundred endocrine disorders has been determined through the use of molecular biological approaches, and these tests are being used increasingly in clinical practice. In addition to technical advances such as RIAs and recombinant DNA technology, endocrinology has contributed disproportionately to pivotal conceptual advances in science and medicine. Almost every aspect of physiology is tied to rhythms. The endocrine system has provided models for rapid rhythms such as luteinizing hormone (LH) or GH pulsatility, circadian rhythms such as cortisol or vasopressin production, and longer rhythms such as the menstrual cycle or bone remodeling. Concepts of hormone–receptor interaction and second messengers established signal transduction paradigms that proliferated into innumerable signaling networks. Polypeptide precursors, such as pro-­opiomelanocortin (POMC), preproglucagon, preproparathyroid hormone, and others established pathways for protein processing, transport, and secretion. Studies of growth factors helped to refine concepts of autocrine and paracrine action, which can be viewed as an extension of classic endocrine action. Hormone replacement formed the foundation for the use of biologic agents such as factor VIII, G-­CSF, and erythropoietin. The genetic basis for cancer has been elucidated by studies of the multiple endocrine neoplasia syndromes, types I and II. KEY POINTS • The field of endocrinology has pioneered many technological advances, including protein sequencing, RIAs, ligand–receptor interactions, and recombinant DNA technology, including the large-­scale synthesis of hormones such as insulin or GH.

PRINCIPLES OF HORMONE ACTION The principles of hormone action include fundamental concepts such as hormone biosynthesis and secretion, feedback regulation,

hormone–receptor binding, and initiation of intracellular signaling. These principles are broadly applicable to other subspecialties in addition to endocrinology.

Hormone Biosynthesis and Secretion Hormones can be divided into five major classes: (1) amino acid derivatives such as dopamine, catecholamines, and thyroid hormone; (2) small neuropeptides such as GnRH, TRH, somatostatin, and vasopressin; (3) large proteins such as insulin, LH, and PTH that are produced by classic endocrine glands; (4) steroid hormones such as cortisol and estrogen that are synthesized from cholesterol-­based precursors; and (5) vitamin derivatives such as retinoids (vitamin A) and vitamin D. As a rule, amino acid derivatives and peptide hormones interact with cell-­ surface membrane receptors. Steroids, thyroid hormones, vitamin D, and retinoids are lipid-­soluble and interact with intracellular nuclear receptors. Many peptide hormones are produced from precursor polypeptides. Characteristic signal or leader sequences target these peptides for extracellular transport via secretory granules. Some precursors, such as POMC or preproglucagon, encode multiple biologically active peptides that are generated by specific processing enzymes; other precursors, such as preproinsulin and vasopressin, encode single hormones that are excised from larger proteins. The secretion of peptide hormones is tightly controlled by intracellular signals that regulate vesicle transport and fusion with the plasma membrane, resulting in hormone release into the extracellular milieu. Steroid hormones such as progesterone, cortisol, and testosterone are synthesized from cholesterol derivatives through a series of enzymatic steps. These enzymes are expressed specifically in steroidogenic tissues such as the adrenal gland and gonads. Their enzymatic activities are regulated in response to trophic hormones such as adrenocorticotropic hormone (ACTH), LH, or follicle-­stimulating hormone (FSH). Thyroid hormone is produced by modifications (iodination) of tyrosines in thyroglobulin. Vitamin D and retinoic acid are derived in part from dietary sources but can also be generated and activated by endogenous synthetic pathways. KEY POINTS • Hormones can be divided into five major classes: (1) amino acid derivatives such as dopamine, catecholamines, and thyroid hormone; (2) small neuropeptides such as GnRH, TRH, somatostatin, and vasopressin; (3) large proteins such as insulin, LH, and PTH that are produced by classic endocrine glands; (4) steroid hormones such as cortisol and estrogen that are synthesized from cholesterol-­based precursors; and (5) vitamin derivatives such as retinoids (vitamin A) and vitamin D.

Feedback Regulation The elucidation of negative feedback has had a profound impact on endocrinology. This principle holds that hormones have a particular set point that is controlled by downregulating stimulatory pathways when the set point is exceeded and upregulating stimulatory pathways when hormone levels fall below the set point. Probably every hormone is regulated in this manner, although the regulatory pathways might not be immediately evident for new hormones. These regulatory loops are well illustrated by the major hypothalamic-­pituitary-­hormone axes and include both stimulatory (e.g., TRH stimulates thyroid-­stimulating hormone [TSH]; TSH stimulates T4/T3 production) and inhibitory components (e.g., T4/T3 suppress TRH and TSH) (Fig. 1.4). Feedback regulation also occurs for endocrine systems that do not involve the pituitary gland. For example, calcium feeds back to inhibit PTH, glucose inhibits insulin secretion, and leptin acts on hypothalamic

CHAPTER 1  Principles of Endocrinology

7

CNS Factor X

Receptor X

Factor X

Receptor X

Autocrine Releasing factors Hypothalamus

Pituitary Trophic hormones

Feedback inhibition

Paracrine

Receptor Y

Gonads Thyroid Adrenal • Testis • Ovary Fig. 1.4  Feedback regulation of the hypothalamic-­ pituitary axis. CNS, Central nervous system.

pathways to suppress appetite. Although the descriptions of these feedback mechanisms oversimplify the complex physiologic pathways that regulate hormone levels, they provide useful insight into endocrine testing paradigms. For example, hypothyroidism is characterized by elevated TSH, an appropriate physiologic response to deficient thyroid hormone levels. Dexamethasone suppression of the CRH/ACTH axis is used to diagnose Cushing disease, which is characterized by impaired negative feedback regulation. A deficient adrenal response to exogenous ACTH is used to document primary adrenal insufficiency. KEY POINTS • Feedback regulation is a powerful means to achieve homeostasis. It is also the foundation for important endocrine tests, such as the ACTH stimulation test or the dexamethasone suppression test.

Factor Y

Fig. 1.5  Autocrine and paracrine regulation. Many growth factors act locally to regulate cell growth, differentiation, and function. Autocrine regulation describes the action of a factor that acts on the same cell, whereas paracrine regulation describes a circumstance in which the product of one cell acts on a different cell type.

and is neutralized by binding proteins such as follistatin. Autocrine regulation describes the action of a factor on the same cell from which it is produced. IGF-­1 acts on many cells that produce it, including chondrocytes, breast epithelium, and gonadal cells. Intracrine regulation refers to effects within a cell. The term is not commonly used but captures the important concept that many signaling and enzymatic pathways are influenced by other pathways or by substrate or product concentrations. For example, 3-­hydroxy-­3-­methylglutaryl coenzyme A reductase, the rate-­limiting enzyme in cholesterol biosynthesis, is inhibited by the end product, cholesterol. KEY POINTS • Paracrine and autocrine regulation allow growth factors and cytokines to act locally, minimizing systemic biological effects.

Hormonal Rhythms and Pulsatility Paracrine and Autocrine Regulation Whereas feedback mechanisms control many classic endocrine pathways, local regulatory systems, often involving growth factors, play critical roles in all tissues (Fig. 1.5). Paracrine regulation refers to factors released by one cell that act on an adjacent cell in the same tissue. For example, somatostatin secretion by pancreatic islet delta cells inhibits insulin secretion from nearby β cells. The oocyte produces growth and differentiation factor–9, which acts on adjacent granulosa cells to stimulate the transition of primary follicles to secondary follicles. The anatomic relationships of cells have an important influence on paracrine regulation. Seminiferous tubules are exposed to a very high testosterone concentration from the interstitial Leydig cell compartment. On the other hand, the Sertoli cell product, androgen-­ binding protein, helps to retain high local testosterone concentrations. Activin exerts paracrine effects in the pituitary gland, where it stimulates FSH production. However, activin also exerts biologic activity in many other tissues, perhaps explaining why it is regulated locally

Hormonal rhythms are used to adapt to environmental changes, such as seasons of the year, the daily light-­dark cycle, sleep, meals, and stress. In many species, reproduction is seasonal, presumably a mechanism to ensure survival of the offspring. In the extreme northern and southern hemispheres, calcium absorption and bone remodeling decline during winter, when vitamin D production is reduced. The human menstrual cycle is repeated on average every 28 days, reflecting the time required for follicular maturation and ovulation. In some species, estrus cycles are intimately linked to mating behavior induced by behavioral cues and the production of pheromones. Essentially all pituitary hormone rhythms are entrained to sleep and the circadian cycle, which in turn is dictated by sunlight exposure. The hypothalamic-­pituitary-­adrenal (HPA) axis, for example, exhibits characteristic peaks of ACTH and cortisol production before dawn and a nadir between late afternoon and midnight. Recognition of these rhythms is important for endocrine testing and treatment. Patients with Cushing syndrome exhibit inappropriately increased midnight cortisol levels. The HPA axis is more susceptible to suppression by glucocorticoids administered at night

8

PART 1  Principles of Endocrinology and Hormone Signaling

because they blunt the early morning rise of ACTH. Understanding this diurnal rhythm provides the basis for increased physiologic hormone replacement through the use of larger glucocorticoid doses in the morning than in the afternoon. Although circadian rhythms were identified initially in the context of sleep cycles and hormonal rhythms, there is mounting evidence for circadian periodicity at the level of organs and individual cells.23 For example, a number of genes expressed by hepatocytes, such as REV-­ ERB, exhibit rhythmic expression, thereby exerting epigenetic regulatory effects on metabolic pathways such as gluconeogenesis and lipid biosynthesis. Many peptide hormones are secreted in discrete pulses, often reflecting regulation by the nervous system. For example, hypothalamic GnRH induces LH pulses once every 1 to 2 hours. Intermittent hypothalamic GnRH pulses are required to maintain pituitary gonadotrope sensitivity, whereas continuous GnRH exposure causes desensitization. This feature of gonadotropin regulation serves as the basis for using long-­acting GnRH agonists to treat central precocious puberty or to decrease testosterone levels in the management of prostate cancer. The pulsatile nature of hormone secretion and the rhythmic pattern of hormone production have important implications for the measurement of circulating hormone levels, as levels can change dramatically over several hours. For some hormones, integrated markers have been developed to circumvent hormonal fluctuations. For example, a 24-­ hour collection of urinary free cortisol integrates cortisol production throughout a diurnal cycle. IGF-­1 provides a relatively stable biologic marker of GH action. Glycosylated hemoglobin is used as an index of long-­term (weeks to months) circulating blood glucose, which is linked covalently to hemoglobin in a concentration-­dependent manner. KEY POINTS • Virtually every hormonal system is linked directly or indirectly to circadian rhythms of sleep, light exposure, and feeding. These circadian rhythms not only influence physiologic responses but have practical implications for interpreting the measurements of many hormones.

Hormone Transport and Degradation The level of a hormone is determined by its rate of secretion and its circulating half-­life. After protein biosynthesis and precursor processing, peptide hormones are stored in secretory granules. These granules undergo progressive maturation and sequential translocation before arriving at the plasma membrane for imminent release into the circulation. The stimulus for hormone secretion is typically a releasing factor or neural signal that induces rapid changes in intracellular calcium concentration, which leads secretory granules to fuse with the plasma membrane and releases their contents into the extracellular environment and bloodstream. In contrast, steroid hormones usually diffuse into the circulation as they are synthesized. Thus, their secretion closely mirrors rates of synthesis. For example, ACTH and LH induce steroidogenesis by stimulating the activity of steroidogenic acute regulatory protein, which transports cholesterol into the mitochondrion. In parallel, ACTH and LH stimulate the production of other rate-­ limiting enzymes in the steroidogenic pathway, such as the cholesterol side-­chain cleavage enzyme. Hormone-­binding proteins can affect volume of distribution, level of unbound or “free” hormone, and rates of hormone clearance. Most steroid hormones and many peptide hormones circulate in association with binding proteins. T4 and T3 bind to thyroxine-­binding globulin (TBG), albumin, and thyroxine-­binding prealbumin. Similarly, cortisol binds to cortisol-­binding globulin, and androgens and estrogens

bind to sex hormone–binding globulin (SHBG). IGF-­1 and IGF-­2 bind to multiple IGF-­binding proteins. GH interacts with GH-­binding protein, a circulating fragment of the GH receptor extracellular domain. Abnormal binding proteins can significantly alter total hormone concentrations but usually have little clinical consequence, as the regulatory feedback systems respond to unbound or “free” hormone levels. For example, TBG deficiency greatly reduces total thyroid hormone levels, but the free concentrations of T4 and T3 remain normal. Liver disease and medications can also influence binding protein levels (e.g., estrogen increases TBG). Nonetheless, these abnormalities can create diagnostic confusion, and some alterations (e.g., increased SHBG) may shift ratios of hormones (e.g., testosterone, estradiol) that bind with different affinities. Knowledge of hormone half-­life is important for achieving physiologic hormone replacement, as the frequency of dosing and the time required to reach steady state are determined by rates of hormone decay. T4, for example, has a half-­life of about 7 days. Consequently, more than 1 month is required to reach a new steady state, and single daily doses are sufficient to achieve constant hormone levels. T3, in contrast, has a half-­life of about 1 day. Its administration is associated with more dynamic serum levels, and it must be administered two to three times per day to generate more constant blood levels. Synthetic glucocorticoids vary widely in their half-­lives. Analogues with a longer half-­life (e.g., dexamethasone) are associated with greater suppression of the HPA axis. Most protein hormones (e.g., ACTH, GH, PRL, PTH, LH) have relatively short half-­lives (10-­fold higher than the highest standard (Fig. 2.11)), the amount of antibody can become limiting, and the assay essentially converts to a competitive binding assay. Paradoxically, this results in a (falsely) lowered result, even though very high amounts of the analyte are present in the patient specimen. This can be resolved by serially diluting the sample to lower the analyte concentration to the point where the two antibodies are now in excess (∼1000 miU/mL in Fig. 2.11). Overall, this is a rare occurrence because commercial immunoassay manufacturers test for assay susceptibility to hook effect and alter their assay formulations accordingly. Although it is theoretically possible that the hook effect could lower the result into the reference interval, we have never observed this in the many assays we have performed.

Special Circumstances Noncompetitive immunoassays are not currently amenable to the measurement of smaller molecules like steroids and thyroid hormones, because they are too small allow for generation of two antibodies to distinct parts of the molecule. As a result, immunoassays for steroids, thyroid hormones, and small peptides (e.g., oxytocin) are still measured by competitive binding assay. The noncompetitive immunoassay design permits rapid turnaround times. The rapid PTH assay is used to intraoperatively verify parathyroid adenoma removal during minimally invasive parathyroidectomies.88 A similar modification has been made to the noncompetitive ACTH immunoassays, allowing rapid verification of intraoperative corticotropin-­secreting tumor removal.91,92

Signal

100

Not all noncompetitive immunoassays are created equally. Each vendor may use a different set of proprietary antibodies, which can lead to discordant results between methods. For example, one noncompetitive immunoassay platform has been shown to frequently give falsely increased plasma ACTH levels, leading to unnecessary testing and even unnecessary surgery.31-­33 A very interesting application of noncompetitive immunoassays is the use of combinations of antibodies to different epitopes on larger peptides and proteins to evaluate the presence of precursor peptides. An example is the evaluation of patients with the potential for high circulating levels of proopiomelanocortin and its posttranslational products (i.e., ACTH precursors). These typically arise from neuroendocrine tumors (e.g., small cell lung cancer) that can cause ectopic ACTH syndrome.93 A research group has developed antibody combinations that can quantify these ACTH precursors,87 thus enabling early detection and treatment. Unfortunately, this assay is not commercially available at this time.

FREE SERUM/PLASMA HORMONE ASSAYS KEY POINTS • Steroid and thyroid hormones circulate in the blood mainly bound to plasma proteins, with a small fraction in the free (biologically active) form. • Measurement of free serum thyroid hormones and testosterone are particularly useful for clinical care. • Methods that physically separate the free hormone from the protein bound are the gold standard. • Calculation of the free hormone from the serum concentration of total hormone and binding protein concentration may be useful but should be used with caution.

Catecholamines and most peptides and protein hormones circulate in the plasma compartment in the dissolved (free) state. Direct measurement in serum/plasma therefore accurately reflects their biological activity. Conversely, hydrophobic thyroid and steroid hormones circulate mostly bound to proteins that have varied specificity, capacity, and affinity for the hormone (Table 2.1). The unbound (free) form binds its receptor on target cells to exert its biological effects and comprises a smaller fraction of the overall concentration. The extreme example

TABLE 2.1  Specific Plasma Binding

80

Proteins for Steroid and Thyroid Hormones

60

Major Specific Binding Protein (CBG)a

40 20

00 0 00 0, 1,

00 0 10 0,

00 0 10 ,

10 00

10 0

10

0

0

25

Hormone concentration (mlU/L) Fig. 2.11  High-­dose hook effect. The response signal reaches a maximum and then decreases when the antigen concentration exceeds the limit of the noncompetitive immunoassay. (From Sluss PM, Hayes FJ. Laboratory techniques for recognition of endocrine disorders. In: Melmed SA, Goldfine, RJ, Koenig, AB, et al., eds. Williams Textbook of Endocrinology. 14th ed. Philadelphia, Elsevier; 2020:62–90.e66.)

Corticosteroid-­binding globulin CBG and sex hormone–binding globulin (SHBG) a,b CBG a SHBGb Vitamin D–binding protein Thyroid-­binding globulin (TBG) and ­transthyretin TBG

Hormone Cortisol and corticosterone Progesterone Aldosterone Testosterone and estrogen 25OHD and 1,25(OH)2D Thyroxine Triiodothyronine

Note: All of the hormones listed bind albumin to some extent (low-­ affinity/high-­capacity binding protein). aCBG binds cortisol with higher affinity than progesterone and aldosterone. bSHBG binds testosterone with higher affinity than progesterone and estrogen.

26

PART 1  Principles of Endocrinology and Hormone Signaling

is thyroxine, which is more than 99.5% bound to plasma proteins in the circulation.94,95 Overall, up to 90% to 95% of any given steroid hormone may actually be protein-­bound. Binding proteins may be specific, high-­affinity carriers (Table 2.1) or low-­affinity proteins with large binding capacity (e.g., albumin, prealbumin). Most standard assays for the measurement of steroid and thyroid hormones determine their total concentration in serum/plasma; these “total” measurements assume that the concentrations and kinetics of binding proteins are normal. There are several circumstances in which that assumption is not valid, including obesity, throughout the aging process, and during critical illness. Some additional scenarios include estrogen therapy, birth control pills containing estrogen, and pregnancy, all of which increase corticosteroid-­binding globulin (CBG) concentrations and thyroxine-­binding globulin (TBG) concentrations. Critical illness may decrease serum binding. Hyperthyroidism induces hepatic production of sex hormone–binding globulin (SHBG), which binds both testosterone and estrogen. These scenarios make total hormone measurements suspect and may affect reference intervals and dynamic testing cutoffs.48,71 For example, the cutoff for a normal baseline or ACTH1-­24–stimulated serum/plasma cortisol can be significantly increased in the context of estrogen-­induced increases in CBG.96 Serum/plasma–free hormone assays have been developed because of the issues described and because measurement of “free” hormones, at least in theory, provides the best evidence for changes in the bioactivity of the endogenous serum/plasma hormones. Some have also advocated for using salivary steroid measurements as a valid surrogate for measurement of serum free hormone concentrations.48 Free hormone assays can be technically challenging and therefore may have limitations that must be appreciated when interpreting results in the context of the clinical scenario. Their challenging nature also dictates that free hormone tests are frequently performed in reference or specialty laboratories.

Methods for Measurement of Free Hormones The most frequently used methods for estimating free hormone concentrations are physical separation of free from protein-­bound hormone and calculations based on binding constants. Additional or historical techniques include selective precipitation, calculations including ratios, and direct antibody-­based binding (immuno) assays targeted toward free hormone. Overall, these latter techniques are not recommended, and concerns regarding their use will be described later. Free hormone is typically present in very small concentrations; thus, sensitive measurement techniques are essential. An appreciation for the complexity of measuring free steroid hormones is also critical for accurate result interpretation. Protein binding kinetics, competitive binding, and equilibrium with other binding proteins all make these measurements particularly challenging. Physical Separation - Equilibrium Dialysis and Ultrafiltration: Physical separation of free from protein-­bound fractions allows for direct measurement of the biologically active hormone. Physical separation methods such as equilibrium dialysis (ED) and ultrafiltration (UF) have long been considered the gold standard for these measurements. The analytic principle of ED is based on two solution-­filled chambers separated by a semipermeable membrane; the patient specimen is located in one chamber, and an appropriate dialysis buffer is located in the other (Fig. 2.12). The membrane pores are of a sufficient diameter to allow the unbound hormone to pass through, while retaining serum-­binding proteins and bound hormone. After a period of time, the free hormone from the patient sample will come into equilibrium between the two chambers. Measuring the amount of hormone that has passed from the patient sample into the dialysis buffer chamber provides the free hormone concentration. UF similarly separates free from bound hormone via a semipermeable membrane but uses

Specimen: binding protein and steroid

Dialysis membrane or ultrafiltration filter

“Free hormone” in physiologic buffer

Fig. 2.12  Free hormone assay design using a dialysis membrane (often referred to as equilibrium dialysis). This allows separation of free hormone from hormone bound to plasma/serum protein. (From Sluss PM, Hayes FJ. Laboratory techniques for recognition of endocrine disorders. In: Melmed SA, Goldfine, RJ, Koenig, AB, et al., eds. Williams Textbook of Endocrinology. 14th ed. Philadelphia, Elsevier; 2020:62–90.e66.)

centrifugation to force the molecules through. Both techniques are routinely paired with high-­sensitivity MS-­based methods for the final measurement of free hormone. Temperature, pH, and protein adsorption to or leakage through the membrane can all have a negative impact on the results from the ED and UF methods. Importantly, the time allowed to reach equilibrium (often 12–24 hours!) and any dilution of the sample may have a significant influence on ED measurements. Calculation of Free Hormone Fraction: Testosterone provides the best example of estimated free (and/or weakly bound to albumin, or “bioavailable”) fractions by use of calculations. Formulas for the determination of free estradiol, cortisol, and others also exist97,98 but are not in routine use. The most frequently used formulas use measured values of (1) the hormone of interest, (2) its main binding protein (e.g., SHBG, CBG), and (3) albumin, along with their respective binding constants, to determine unbound versus bound concentrations. The accuracy of the calculated value is dependent on the accuracy of the individual measurements and the binding constants used. Automated methods exist for most of the individual components that would be included in these calculations; therefore, this approach is often more accessible for the typical hospital laboratory. Several free testosterone formulas have been published, and many studies report good correlation with ED methods.99,100 However, differences in measurement, formulas, and constants used can lead to significant disparities among methods, and calculated values might best be considered estimates of true free hormone concentrations.101,102 Direct (noncalculated) measurements are recommended in cases with known or suspected alternation in binding proteins.30

Additional Techniques Selective precipitation of SHBG from a patient sample using ammonium sulfate has been described for the determination of free testosterone concentrations.103 This relies on the addition of radiolabeled testosterone to the patient sample. After ammonium sulfate is added and the sample is centrifuged, the supernatant contains all non–SHBG-­ bound fractions (free and bioavailable). The nonbound radioactive testosterone is measured, and that percentage of the total testosterone concentration approximates the amount of bioavailable testosterone present in the sample. This method is not in routine use, largely because of the availability of ED and UF methods. Direct (analog) immunoassay methods have been used for the measurement of free testosterone; however, these assays are

CHAPTER 2  Principles of Endocrine Measurements

27

unacceptable because of problems with bias, reproducibility, and hormone recovery.29,30,99,100 The free androgen index (FAI) is the ratio of testosterone to SHBG multiplied by 100 to obtain a percentage. Although it is a simple calculation, caution should be exercised when interpreting the FAI. Like calculations for free testosterone, accuracy of the individually measured components is central to the overall utility of the calculated value. Although there is good correlation between the FAI and physical separation techniques in females, this relationship is not ideal and not useful in males. Yet, the performance of FAI in females has been used to identify and manage patients with polycystic ovary disease.104

salivary cortisone by LC-­MS/MS is useful for identifying contamination of the salivary sample with hydrocortisone112 and for monitoring the adequacy of cortisol replacement therapy in patients with adrenal insufficiency.113 Other salivary hormone analyses have been suggested, including testosterone and estrogen in the evaluation of gonadal function and 17-­alpha-­hydroxyprogesterone in the early evaluation of congenital adrenal hyperplasia because of 21-­hydroxylase deficiency.114-­117 Furthermore, a new, completely noninvasive nasal ACTH stimulation test using salivary cortisol as an outcome has been proposed.50 This could make the evaluation of adrenal insufficiency simpler and available to primary care physicians in any clinical setting.

Special Circumstances

PLASMA RENIN ASSAYS

Comment on Free Vitamin D Metabolites: Like other steroid hormones described earlier, 25 hydroxyvitamin D (25OHD) circulates bound to plasma proteins. Vitamin D–binding protein (DBP) carries approximately 90% of the total plasma vitamin D.105 Albumin carries approximately 10%, but with much lower affinity than DBP.105 The loose binding of 25OHD to albumin renders that portion “bioavailable,” which is similar to the fraction of testosterone that binds to albumin.30 Less than 0.05% of plasma 25OHD circulates in the free form. One may then wonder why good, direct free 25OHD or DBP assays have not been developed to allow determination of free and bioavailable serum 25OHD in a manner similar to calculated free and bioavailable testosterone.30 This is an area of significant controversy, particularly because changes in DBP were purported to account for differences in total 25OHD observed between Black Americans and White Americans.106 Subsequent studies questioned the validity of the DBP measurements used in this prior study107 and, in fact, questioned the need for measuring or calculating free 25OHD in any circumstance.108 Comment on Salivary Hormone Measurements as a Surrogate for Free Plasma/Serum Hormones: Salivary cortisol is useful as a surrogate for free plasma/serum cortisol for the diagnosis of disorders of the hypothalamic-­pituitary-­adrenal axis, particularly Cushing syndrome and adrenal insufficiency.43,109,110 Salivary cortisol may be measured by immunoassay or LC-­MS/MS. For Cushing syndrome screening, immunoassay may have a higher sensitivity because of the cross-­reactivity of most antibodies with salivary cortisol metabolites.111 Immunoassay does have poorer specificity compared to LC-­MS/MS, particularly in patients with mild cortisol excess owing to adrenal (ACTH-­ independent) Cushing syndrome.111 The measurement of

Generation step in vitro Angiotensinogen in plasma Renin

KEY POINTS • Plasma renin can be measured as plasma renin activity (PRA; measurement of angiotensin I generation in vitro) or direct renin concentration (DRC) by noncompetitive immunoassay. • The main advantage of PRA is that it assesses the actual endogenous biological activity of renin. • The main advantages of DRC are that it is simple and high-­throughput, and is not influenced by changes in plasma angiotensinogen concentrations.

Renin is not a hormone in the classic sense: it is an enzyme that catalyzes the cleavage of angiotensin I (a small peptide) from angiotensinogen (“renin substrate”). There is considerable confusion about how plasma renin is measured. Endocrinologists should take the lead in teaching the medical community the proper pronunciation of the word “renin” (i.e., REE-­nin; as in “REE-­nal” artery). The mispronunciation of the word renin as “WREN-­in” is incorrect and imprecise, and it is up to us to correct this common (annoying) error.

Plasma Renin Activity Assay and its Modifications Plasma renin activity (PRA) is measured by a hybrid assay that is part bioassay (generation of angiotensin I in vitro) and typically part immunoassay (the measurement of angiotensin I generated in vitro) (Fig. 2.13).118 The first step is the in vitro 37° C generation of angiotensin I from the plasma renin and angiotensinogen contained in the patient’s sample. Reagents are added that prevent the breakdown of angiotensin

Angiotensin I Measurement step

(Incubation at 37°C) Angiotensin I

Immunoassay or LC/MS-MS

• PRA result expressed as an enzyme activity (Ang I generated per ml per time) • To measure angiotensinogen (renin substrate), add excess renin to incubation step • To measure renin concentration, add excess angiotensinogen to incubation step Fig. 2.13  Simplified version of the plasma renin activity assay. The first step generates angiotensin I from endogenous (patient) angiotensinogen and renin. The X represents inhibitors that are added to prevent degradation of angiotensin I during the generation step. The second step is the measurement of angiotensin I concentration. LC-­MS/MS, liquid chromatography tandem—mass spectroscopy; Ang I, angiotensin 1; PRA, plasma renin activity.

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PART 1  Principles of Endocrinology and Hormone Signaling

I in vitro. A parallel sample incubation on ice (4° C) is commonly done to correct for the endogenous angiotensin I in the plasma sample. Once the angiotensin I generation step is complete and the reaction is stopped, the next step involves angiotensin I concentration measurement by immunoassay or LC-­MS/MS.119,120 The result is expressed as a biological activity (e.g., pmol of angiotensin I generated per minutes of incubation per liter of plasma). Because angiotensinogen concentration is in excess in the typical patient sample, the angiotensin I generated (renin activity) is dependent only on the concentration of renin in the sample. The PRA assay can be modified to measure plasma angiotensinogen “concentration” (Fig. 2.13). This is accomplished by adding renin in excess in the incubation step.119 Plasma renin concentration can be measured by adding excess angiotensinogen in the incubation step.119 In both cases, the measurement of angiotensin I concentration generated is the final step.

Advantages The main advantages of the PRA assay are (1) the decades of experience with the interpretation of the results and (2) assessment of the actual bioactivity of renin within the sample.

Disadvantages The PRA assay is tedious and time-­consuming. If there is an alteration in angiotensinogen concentration, it can become rate-­limiting in the generation step. This can also be considered an advantage because if angiotensinogen is rate-­limiting in vitro, it is likely to be rate limiting in vivo.

Direct Renin Concentration With the development of the two-­site immunometric (noncompetitive) assay, it is now possible to directly measure plasma renin concentration.121 This method is fast and inexpensive and correlates well with PRA.122-­124 Many reference laboratories offer both PRA and direct renin concentration because many clinicians are used to PRA in the assessment of primary aldosteronism using the plasma aldosterone to renin ratio.124,125 The main disadvantage of the method for measuring direct renin concentration is that is it not a bioassay and is unaffected by potentially rate-­limiting low concentrations of angiotensinogen in the patient’s circulation.

STRUCTURAL ASSAYS––MASS SPECTROMETRY KEY POINTS • After an initial physical separation of the compound of interest (typically by liquid chromatography), mass spectrometry is used for detection. • Mass spectrometry uses transformation of the compound into charged molecular ions, and then separation, fragmentation, and measurement of the ions according to their mass-­to-­charge ratio. • Advantages include the ability to measure only the compound of interest (high specificity) and the ability to detect low concentrations (sensitivity). • Disadvantages include instrument and maintenance costs and the technical expertise required.

Mass spectrometry instruments used in clinical laboratories include two main types: gas chromatography-­mass spectrometry (GC-­MS) and LC-­MS/MS. Both techniques rely on the physical separation of compounds on chromatography columns, the transformation of these compounds into charged molecular ions, and then separation, fragmentation, and measurement of these in the mass spectrometer.19-­23 Ions are separated according to their mass-­to-­charge ratio (m/z), which

is a dimensionless quantity calculated by dividing the mass number of an ion by its charge state.

Gas Chromatography—Mass Spectrometry GC-­MS has been widely used in steroid chemistry and is still the main technique used for the identification and characterization of inborn errors of steroid metabolism.126 Steroid profiling developed over 40 years ago, opening up the field of urinary steroid profiling, which is now sometimes referred to as urinary steroid metabolomics.127 GC-­ MS owes its success to the efficient separating capability of very long fused silica capillary columns coupled with the specificity of a mass detector. With this combination, the identification of structurally-­ related steroids can be achieved. Chromatographic separation of steroids is crucial, because there are many steroids with similar m/z values that will appear identical to the mass spectrometer. Good examples of this include 11-­deoxycortisol, corticosterone, and 21-­deoxycortisol, which share a parent ion m/z of 347 and also fragment to give the same daughter ions.128 The main problem with GC-­MS is the need to chemically derivatize steroids before analysis, because only highly volatile molecular ions will travel through the instrument. To achieve this, derivatized steroids are vaporized and are carried by inert gas through a heated silica column and interact with the chemically bonded stationary phase attached to the walls of the column. Gaseous to stationary phase interactions based on the differences in hydrophobicity of the individual steroids cause differences in retention of steroids on the column, so they arrive at the detector at different times. Derivatization also improves steroid stability and allows optimal sensitivity and chromatographic separation.129 GC-­MS has an advantage over other techniques in that it is able to measure a wide variety of steroids and their metabolites simultaneously within the same analytical run. Run times are typically 30 minutes or longer, and the sample preparation steps are long and tedious, but the data that are eventually generated are unmatched by any other technique, because novel and unknown compounds can be identified.126 It is the ability to search for previously unidentified steroids and generate metabolic fingerprints from the interrelationship of steroids that make this technique so powerful for the investigation of steroid disturbances. The main weakness of GC-­MS is the inherent complexity of sample preparation, which makes it a difficult technique to use. The introduction of electrospray ionization and improvements in liquid chromatography have propelled LC-­MS/MS to the forefront of separation techniques. Indeed, it has largely superseded GC-­MS in both routine and research laboratories. Differences between the two techniques are marked, and each have specific advantages. GC-­MS confers the ability to screen for all compounds in a sample, whereas LC-­MS/MS relies on targeted analysis of known compounds, so in the case of LC-­MS/ MS, the analyst will only detect what they expect. This is an important consideration because although LC-­MS/MS is unmatched for quantitation of targeted steroids, so far it cannot replace GC-­MS for discovery research.26 This is a situation that may change with improvements in LC-­MS/MS technology, including supercritical fluid chromatography26 and the use of accurate mass instruments.130 However, LC-­ MS/MS generally does not need derivatization, so sample preparation is simpler, and the run times are much shorter, allowing for faster throughput. Some sample preparation is still required, because neat serum, urine, or saliva samples cannot be injected directly into the LC-­MS/MS system. The main ways of preparing samples to remove proteins and other interfering substances include simple protein precipitation and liquid or solid phase extraction.131 The latter two techniques are more suitable for high-­sensitivity work, where significant sample clean-­up,

CHAPTER 2  Principles of Endocrine Measurements matrix removal, and possibly analyte concentration are needed.128 The workflow is therefore much simpler than the GC-­MS workflow, and this makes LC-­MS/MS much more amenable for use in a routine clinical laboratory. However, the high cost of instrumentation and greater technical complexity, requiring highly skilled technical staff, has impeded the wider adoption of LC-­MS/MS. As a consequence, some laboratories tend not to use LC-­MS/MS and still depend on manual or fully automated immunoassays for many analytes.

into the mass spectrometer and separated according to their m/z using quadrupoles, fragmented in a collision cell, and then detected.132 Analytical specificity in GC-­MS is determined by chromatographic separation on a very long column and measurement of molecular ions, whereas for LC-­MS/MS specificity is achieved in a three-­stage process. First, some crucial separation is achieved on the much shorter analytical column sufficient to separate compounds with the same m/z. Second, specificity is augmented by selecting the molecular ion formed in the ionization source from other potentially interfering ions using a quadrupole mass analyzer. Third, the molecular ion is selectively fragmented in the collision cell, after which molecular ion fragments are selected using the second quadrupole mass analyzer and then measured in the detector. The capacity to select a parent and a daughter ion after a chromatographic separation makes LC-­MS/MS both highly sensitive and specific. The molecular ion is often capable of forming several abundant fragment ions, and additional quality checks are now commonly incorporated by monitoring a second qualifier fragment ion to assess interference in the method. It is the fragment ions that are measured in the detector, having been produced by selectively fragmenting a molecular ion that has undergone prior separation on a chromatography column. (Fig. 2.14). This may seem complicated, but the instrument is under full software control, and each step is fully automated and highly reproducible. Once set up, the instrument can be left unattended for

Liquid Chromatography—Tandem Mass Spectrometry LC-­MS/MS uses a liquid mobile phase to achieve chromatographic separation on a short packed column. The mobile phase may contain salts or weak acids to enhance ionization in the source, and the column packing material is chemically bonded with a variety of ligands to make it hydrophobic in nature. Separation occurs by hydrophobic interactions between the analyte, the column packing material, and the mobile phase. Samples are injected as a liquid onto the column, and different analytes are separated according to their relative retention on the column and consequently reach the detector at different times. Chromatographic separation is still important to discriminate between compounds with identical mass.131 Electrospray ionization was a breakthrough, because it converts the column effluent from a liquid containing molecules into gaseous ions, thus enabling their passage through the mass spectrometer. The free ions generated are drawn

Liquid phase

Gas phase

LC column

Ion source

Analyte separation

Formation of gaseous ions

O

F

HO

HO

O HO H

H

O HO H

O H

MS2

Fragment Ion selection

Fragment Ion detection

H

O

O

O

H—O

O OH

H

OH OH

H

H O

E

Fragment Ion production

Detector

OH

O

O OH

Collision cell

Molecular Ion selection

OH

H

H H

Ion transmission

MS1

OH

H—O

H

O OH

T H H

29

H

O

Fig. 2.14  Diagram showing the measurement of cortisol using liquid chromatography tandem-mass spectroscopy. Cortisol (F), estradiol (E), and testosterone (T) are first separated using liquid chromatography. Cortisol elutes first from the column, it is ionized in the mass spectrometer source, and the cortisol ions are selectively transferred through the first quadrupole to a collision cell. Cortisol is fragmented in the collision cell, and an abundant fragment ion is selectively transferred through the second quadrupole for measurement in the detector. LC, liquid chromatographer; MS, mass spectrometer.

30

PART 1  Principles of Endocrinology and Hormone Signaling

long periods of time, including overnight and weekends, and the results downloaded to the laboratory information system for reporting. Both MS techniques are superior to immunoassay methods in terms of specificity and also because of the sample clean-­up steps. Between-­assay variability: LC-­ MS/MS is well placed for the development of niche assays that are performed less frequently, are unavailable, or perform poorly on immunoassay platforms. Some of these niche assays include the less commonly measured steroids such as 11-­ deoxycorticosterone (11-­ DOC), aldosterone, 17-­ hydroxyprogesterone (17-­ OHP), dehydroepiandrosterone, and dihydrotestosterone. There is the possibility of an immunoassay being withdrawn abruptly from the market because of quality or regulatory issues. Another important consideration is the reproducibility of immunoassays over time because of calibration or reagent changes, which can cause problems with diagnostic cutoffs in routine laboratory procedures and in longitudinal research studies.133 However, just as there are poorly performing immunoassays, there are also poorly performing LC-­MS/MS methods, and this has been shown in interlaboratory surveys for a number of steroids.134 The main problems are related to the choices of sample preparation technique, chromatography columns, and internal standards, all of which can hinder minimizing sample matrix effects. Interlaboratory variability can often be improved by standardizing sample clean-­up and chromatography conditions, because it is typically not the MS measurement technique that is at fault. Improvements in interlaboratory performance have also been achieved by the availability of commercially available calibration material.135 The steroids that are infrequently measured, such as 17-­OHP, tend to have the worst comparative performance between laboratories.136 Improvements in interlaboratory variability have been achieved with accuracy-­based initiatives such as the Centers for Disease Control and Prevention (CDC) hormone standardization program and vitamin D standardization certification program.137,138 Central to this strategy is the use of LC-­MS/MS to assign target values to single-­donor patient serum or plasma samples that can then be distributed to laboratories for analysis. Having a true assigned target concentration allows assessment of bias from the true result in a clinical sample. LC-­MS/MS assigned target values have since been adopted by many national external quality assessment schemes, but, as in the CDC program, only for a limited number of steroids.

with some drugs, particularly the estrogen receptor antagonist fulvestrant.143 Achieving the necessary sensitivity to measure E2 using LC-­MS/MS has been challenging, but improvements in instrumentation have enabled the development of assays capable of measuring concentrations as low as 0.6 pmol/L,144 which meets the required sensitivity of 1 pg/mL (3.7 pmol/L) advocated by the Endocrine Society.97 It is also important to measure E2 in postmenopausal women taking aromatase inhibitors, because treatment can reduce serum E2 by 85%; such depletion may be an efficacy or safety biomarker of adjuvant aromatase inhibitor treatment.145 Vitamin D: Automated immunoassays have proved unreliable in a number of patient groups, including pregnant women, intensive care patients, patients with liver failure, hemodialysis patients, and osteoporotic patients.146 LC-­MS/MS is the recommended method in these patients because of its superior specificity. In addition, immunoassays do not distinguish between 25(OH) D2 and 25(OH)D3 and suffer from cross-­reactivity with other vitamin D metabolites recognized by the antibody, such as 24,25(OH)2D.146 Many of the earlier LC-­MS/MS methods for vitamin D were developed for speed and sample throughput, and also suffered from some interference. Separation of 25(OH) D3 from the C3 epimer of 25(OH)D3 could not be achieved. This may be a problem in neonates, where concentrations can be high but typically return to adult levels of less than 10% of total vitamin D by 3 months of age.147 Peptides: Smaller peptides such as angiotensin 1 (Ang 1) are easily measured using LC-­MS/MS and can be used in a routine hospital laboratory setting. Larger peptides such as insulin-­like growth factor 1 and thyroglobulin offer a greater analytical challenge, because they invariably need to be digested with proteolytic enzymes to produce manageable fragments for the mass spectrometer to measure.148 Thyroglobulin measurement by LC-­MS/MS after protein digestion is the method of choice for many large contract laboratories because it is free from thyroid antibody interference.149 As mentioned previously, PRA is typically measured by immunoassay of Ang I generation but may also be measured, following a suitable incubation step, by LC-­MS/MS.120 LC-­MS/MS methods provide sensitive interference-­free measurement of Ang I and offer a good alternative to immunoassay-­based methods.150

Liquid Chromatography—Tandem Mass Spectrometry Applications

Multiplexed Liquid Chromatography—Tandem Mass Spectrometry

Cortisol: In most laboratories, cortisol is measured by immunoassay, but LC-­MS/MS is a useful alternative technology, as it is not affected by cross-­reactivity of synthetic exogenous glucocorticoids such as prednisolone/prednisone, methylprednisolone, and cortisol precursors and metabolites such as 11-­DOC. Some cortisol immunoassays are also less effective at freeing cortisol from CBG, as demonstrated by recovery in women taking the oral contraceptive pill.139 Variability between assays will affect how the cortisol result is interpreted against existing reference intervals, so the clinician should be aware of the assay characteristics of their local laboratory. From a practical standpoint, rapid LC-­MS/MS assays for the routine measurement of serum cortisol are now available, with the added benefit of identifying samples that show evidence of exogenous steroid use.140 LC-­MS/MS can also provide the added value of simultaneously measuring dexamethasone with cortisol to determine if a patient has absorbed the drug or metabolized it quickly, when interpreting dexamethasone suppression tests.141 Estradiol: Many estradiol (E2) immunoassays perform poorly for the measurement of samples from men, children, and postmenopausal women,142 because of a lack of sensitivity and the cross-­reactivity

Another advantage of LC-­MS/MS is the capability to simultaneously measure several different analytes to produce multiplexed test panels. Performing multiplexed analysis is often a minimal extra expense, because the sample preparation and LC-­MS/MS conditions are the same. Selective testing panels have been developed to investigate different clinical conditions, such as second-­tier testing for congenital adrenal hyperplasia (CAH)151 to supplement newborn screening programs and for the investigation of adrenal insufficiency.152 Commercially available kits have been developed for the measurement of steroid panels (www.Chromsystems.com), and the introduction of easy-­to-­ use kits has already simplified the introduction and service delivery of LC-­MS/MS in routine laboratory procedures. Multiplexed LC-­MS/MS panels have recently allowed a more thorough investigation of conditions such as polycystic ovary syndrome (PCOS)153 and CAH.154 There is increasing interest in the measurement of not only testosterone, but other androgens such as androstenedione and the 11-­oxygenated steroids such as 11 ketotestosterone (11KT).155 The evidence for the use of other androgens in screening for CAH is also compelling, and 11KT may prove to be an effective androgenic marker in this condition.156

CHAPTER 2  Principles of Endocrine Measurements Some have also advocated the measurement of 21-­deoxycortisol as a discriminator between 21-­hydroxylase deficiency and PCOS in women with hyperandrogenism.157 Diagnostic uncertainty often arises in these cases, because the clinical presentations of nonclassical CAH and PCOS are similar, and 17-­OHP (the traditional marker for CAH) may also be elevated in PCOS. Multiplexing tests in a single assay may confer benefits, but it can also generate large amounts of complicated data that need to be carefully interpreted. The development of urine steroid metabolomics for discriminating benign from malignant adrenal tumors has shown that this problem can be solved with the use of artificial intelligence.127,158 Urine steroid profiling, first developed using GC-­MS, has been shown to predict adrenocortical carcinoma with 95% sensitivity and specificity and can achieve an early diagnosis. The analysis has since been transferred from GC-­MS to LC-­MS/MS using fewer steroids in the panel with an increase in sample throughput but, crucially, with no detriment to the test performance. Other diagnostic areas likely to benefit from this approach include the differentiation of adrenal from pituitary Cushing syndrome, and the diagnosis of CAH.130 There is also some promising work on the use of multiplexed LC-­MS/MS assays coupled with machine learning in the investigation of Cushing syndrome159 and pheochromcytoma.160 Machine learning represents a great advance that reduces the need for expert highly subjective interpretative skills.

Special Circumstances Urine used to be the sample of choice for measuring steroids and biogenic amines, because the larger concentrations found in urine were necessary to overcome sensitivity problems in older assays. However, 24-­hour urine samples can be challenging for the patient to collect correctly and for the laboratory to process, so some urine tests are gradually being replaced by serum tests (e.g., for metanephrines161 and 5-­hydroxyindoleacetic acid162). Indeed, plasma metanephrines are now the method of choice for the investigation of paraganglioma,163 because metanephrines are continuously secreted by the tumor and are readily measured using LC-­MS/MS. Saliva offers benefits because it is easy to collect, it can be collected by the patient at any time of day and can be collected frequently. However, saliva collection is not without contamination problems, arising mainly from topical steroid use.112 Metabolism of steroids occurs in the salivary glands, allowing the use of salivary cortisone as a surrogate marker for serum free cortisol.113 Salivary cortisone is technically easier to measure than cortisol by LC-­MS/MS, because it is present in greater than 4-­fold concentrations, closely reflects free serum cortisol after adrenal stimulation, and is unaffected by CBG changes. LC-­MS/MS analysis of late-­night salivary cortisol or cortisone does not improve the already excellent sensitivity of late-­night cortisol by immunoassay, but does improve specificity of the test.111 Post dexamethasone salivary cortisone may be a better discriminatory marker between healthy subjects and patients with Cushing syndrome or with autonomous cortisol secretion than salivary cortisol.141 In addition, salivary cortisone is an excellent marker for contamination of the saliva sample by topical hydrocortisone (cortisol).112 Although technically inferior to LC-­MS/MS, cross reactivity with other corticosteroids may actually improve the diagnostic performance of immunoassays.164 Metabolomics programs are beginning to generate new candidate diagnostic markers based on multiplexed LC-­MS/MS methods.165 Fully automated clinical mass spectrometry analyzers are finally beginning to appear on the market, but currently have a limited test repertoire and are relatively expensive, a position analogous to the introduction of laboratory automation for immunoassay 30 years ago. These instruments are less technically demanding, will bring LC-­MS/

31

MS to a wider range of laboratories, and should eventually improve the availability of high-­quality tests to the clinician.

SUMMARY AND FUTURE DIRECTIONS Clinicians depend on accurate and reproducible hormone measurements for the diagnosis, treatment, and management of patients with endocrine diseases. Since the development of the RIA, many improvements have been made that increase throughput and reduce errors. However, it is incumbent on clinicians to be aware of the potential for preanalytic and analytic errors in all measurements obtained. Clinicians should be attuned to the reference intervals for the result obtained, as they can vary significantly between laboratories and methods. When a result is obtained that does not fit with the clinical scenario, clinicians should repeat the measurement and, if needed, work with their clinical laboratory partners to resolve the issue. When possible, referring a sample to another laboratory for analysis by an alternate method may be useful. All assays have strengths and weaknesses, including susceptibility to interferences such as similar hormones and metabolites and other factors such as biotin and heterophile antibodies. LC-­MS/MS is emerging as the method of choice for small molecules–– particularly steroids––although this approach is very labor-­intensive, requires sophisticated instrumentation and technical expertise, and also has analytic strengths and weaknesses. The development of methods to measure multiple steroid metabolites in a single sample by multiplexed LC-­MS/MS has promise to evaluate diseases that are difficult to diagnose and manage, such as adrenocortical cancer and PCOS. The development of accuracy-­based quality control schemes for more analytes will clearly improve agreement between laboratories and between different methods. Finally, education of clinicians as to the strengths and weaknesses of the methods for measuring hormones is a mission that will continue to improve clinical care.

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88. Lang BH, Fung MMH. Intraoperative parathyroid hormone (IOPTH) assay might be better than the second-­generation assay in parathyroidectomy for primary hyperparathyroidism. Surgery. 2021;169:109–113. 89. Kricka LJ. Human anti-­animal antibody interferences in immunological assays. Clin Chem. 1999;45:942–956. 90. Kaplan IV, Levinson SS. When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem. 1999;45:616–618. 91. Graham KE, Samuels MH, Raff H, et al. Intraoperative adrenocorticotropin levels during transsphenoidal surgery for Cushing’s disease do not predict cure. J Clin Endocrinol Metab. 1997;82:1776–1779. 92. Raff H, Shaker JL, Seifert PE, et al. Intraoperative measurement of adrenocorticotropin (ACTH) during removal of ACTH-­secreting bronchial carcinoid tumors. J Clin Endocrinol Metab. 1995;80:1036–1039. 93. Stovold R, Blackhall F, Meredith S, et al. Biomarkers for small cell lung cancer: neuroendocrine, epithelial and circulating tumour cells. Lung Cancer. 2012;76:263–268. 94. Refetoff S, Fang VS, Marshall JS. Studies on human thyroxine-­binding globulin (TBG). IX. Some physical, chemical, and biological properties of radioiodinated TBG and partially desialylated TBG. J Clin Invest. 1975;56:177–187. 95. Refetoff S, Hagen SR, Selenkow HA. Estimation of the T 4 binding capacity of serum TBG and TBPA by a single T 4 load ion exchange resin method. J Nucl Med. 1972;13:2–12. 96. Dichtel LE, Schorr M, Loures de Assis C, et al. Plasma free cortisol in states of normal and altered binding globulins: implications for adrenal insufficiency diagnosis. J Clin Endocrinol Metab. 2019;104:4827–4836. 97. Rosner W, Hankinson SE, Sluss PM, et al. Challenges to the measurement of estradiol: an Endocrine Society Position Statement. J Clin Endocrinol Metab. 2013;98:1376–1387. 98. Mazer NA. A novel spreadsheet method for calculating the free serum concentrations of testosterone, dihydrotestosterone, estradiol, estrone and cortisol: with illustrative examples from male and female populations. Steroids. 2009;74:512–519. 99. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84:3666–3672. 100. Miller KK, Rosner W, Lee H, et al. Measurement of free testosterone in normal women and women with androgen deficiency: comparison of methods. J Clin Endocrinol Metab. 2004;89:525–533. 101. Ly LP, Sartorius G, Hull L, et al. Accuracy of calculated free testosterone formulae in men. Clin Endocrinol (Oxf). 2010;73:382–388. 102. Van Uytfanghe K, Stockl D, Kaufman JM, et al. Validation of 5 routine assays for serum free testosterone with a candidate reference measurement procedure based on ultrafiltration and isotope dilution-­ gas chromatography-­mass spectrometry. Clin Biochem. 2005;38: 253–261. 103. Cumming DC, Wall SR. Non-­sex hormone-­binding globulin-­bound testosterone as a marker for hyperandrogenism. J Clin Endocrinol Metab. 1985;61:873–876. 104. Barth JH, Field HP, Yasmin E, et al. Defining hyperandrogenism in polycystic ovary syndrome: measurement of testosterone and androstenedione by liquid chromatography-­tandem mass spectrometry and analysis by receiver operator characteristic plots. Eur J Endocrinol. 2010;162:611– 615. 105. Heureux N. Vitamin D testing-­where are we and what is on the horizon? Adv Clin Chem. 2017;78:59–101. 106. Powe CE, Evans MK, Wenger J, et al. Vitamin D-­binding protein and vitamin D status of black Americans and white Americans. N Engl J Med. 2013;369:1991–2000. 107. Boers J, Oldenburg-­Ligtenberg PC, Stades AM, et al. Possible pitfalls in the workup of ectopic ACTH secretion illustrated by four rare cases. BMJ Case Rep. 2019;12. 108. Bouillon R. Free or total 25OHD as marker for vitamin D status? J Bone Miner Res. 2016;31:1124–1127. 109. Laudat MH, Cerdas S, Fournier C, et al. Salivary cortisol measurement: a practical approach to assess pituitary-­adrenal function. J Clin Endocrinol Metab. 1988;66:343–348.

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PART 1  Principles of Endocrinology and Hormone Signaling

110. Raff H. Utility of salivary cortisol measurements in Cushing’s syndrome and adrenal insufficiency. J Clin Endocrinol Metab. 2009;94:3647–3655. 111. Kannankeril J, Carroll T, Findling JW, et al. Prospective evaluation of late-­night salivary cortisol and cortisone by EIA and LC-­MS/MS in suspected Cushing syndrome. J Endocr Soc. 2020;4:bvaa107. 112. Raff H, Singh RJ. Measurement of late-­night salivary cortisol and cortisone by LC-­MS/MS to assess preanalytical sample contamination with topical hydrocortisone. Clin Chem. 2012;58:947–948. 113. Perogamvros I, Keevil BG, Ray DW, Trainer PJ. Salivary cortisone is a potential biomarker for serum free cortisol. J Clin Endocrinol Metab. 2010;95:4951–4958. 114. Otten BJ, Wellen JJ, Rijken JC, et al. Salivary and plasma androstenedione and 17-­hydroxyprogesterone levels in congenital adrenal hyperplasia. J Clin Endocrinol Metab. 1983;57:1150–1154. 115. Walker RF, Hughes IA, Riad-­Fahmy D. Salivary 17 alpha-­ hydroxyprogesterone in congenital adrenal hyperplasia. Clin Endocrinol (Oxf). 1979;11:631–637. 116. Wood P. Salivary steroids. Ann Clin Biochem. 2009;46:427. 117. Fiers T, Kaufman JM. Management of hypogonadism: is there a role for salivary testosterone. Endocrine. 2015;50:1–3. 118. Cartledge S, Lawson N. Aldosterone and renin measurements. Ann Clin Biochem. 2000;37(Pt 3):262–278. 119. Sealey JE, Gerten-­Banes J, Laragh JH. The renin system: variations in man measured by radioimmunoassay or bioassay. Kidney Int. 1972;1:240–253. 120. Carter S, Owen LJ, Kerstens MN, et al. A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solid-­ phase extraction. Ann Clin Biochem. 2012;49:570–579. 121. Menard J, Guyenne TT, Corvol P, et al. Direct immunometric assay of active renin in human plasma. J Hypertens Suppl. 1985;3:S275–278. 122. Findling JW, Raff H, Hansson JH, et al. Liddle’s syndrome: prospective genetic screening and suppressed aldosterone secretion in an extended kindred. J Clin Endocrinol Metab. 1997;82:1071–1074. 123. Hartman D, Sagnella GA, Chesters CA, et al. Direct renin assay and plasma renin activity assay compared. Clin Chem. 2004;50:2159–2161. 124. Rossi GP, Barisa M, Belfiore A, et al. The aldosterone-­renin ratio based on the plasma renin activity and the direct renin assay for diagnosing aldosterone-­producing adenoma. J Hypertens. 2010;28:1892–1899. 125. Magill SB. Pathophysiology, diagnosis, and treatment of mineralocorticoid disorders. Compr Physiol. 2014;4:1083–1119. 126. Shackleton CH, Snodgrass GH. Steroid excretion by an infant with an unusual salt-­losing syndrome: a gas chromatographic-­mass spectrometric study. Ann Clin Biochem. 1974;11:91–99. 127. Arlt W, Biehl M, Taylor AE, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors. J Clin Endocrinol Metab. 2011;96:3775–3784. 128. Keevil BG. LC-­MS/MS analysis of steroids in the clinical laboratory. Clin Biochem. 2016;49:989–997. 129. Halket JM, Waterman D, Przyborowska AM, et al. Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/ MS/MS. J Exp Bot. 2005;56:219–243. 130. Hines JM, Bancos I, Bancos C, et al. High-­resolution, accurate-­mass (HRAM) mass spectrometry urine steroid profiling in the diagnosis of adrenal disorders. Clin Chem. 2017;63:1824–1835. 131. French D. Advances in bioanalytical techniques to measure steroid hormones in serum. Bioanalysis. 2016;8:1203–1219. 132. Grebe SK, Singh RJ. LC-­MS/MS in the clinical laboratory -­where to from here? Clin Biochem Rev. 2011;32:5–31. 133. Binkley N, Dawson-­Hughes B, Durazo-­Arvizu R, et al. Vitamin D measurement standardization: the way out of the chaos. J Steroid Biochem Mol Biol. 2017;173:117–121. 134. Buttler RM, Martens F, Ackermans MT, et al. Comparison of eight routine unpublished LC-­MS/MS methods for the simultaneous measurement of testosterone and androstenedione in serum. Clin Chim Acta. 2016;454:112–118. 135. Carter GD, Jones JC. Use of a common standard improves the performance of liquid chromatography-­tandem mass spectrometry methods for serum 25-­hydroxyvitamin-­D. Ann Clin Biochem. 2009;46:79–81.

136. Greaves RF, Ho CS, Loh TP, et al. Working Group 3 “Harmonisation of Laboratory Assessment” European Cooperation in S, Technology Action BMD. Current state and recommendations for harmonization of serum/ plasma 17-­hydroxyprogesterone mass spectrometry methods. Clin Chem Lab Med. 2018;56:1685–1697. 137. Cao ZT, Botelho JC, Rej R, et al. Impact of testosterone assay standardization efforts assessed via accuracy-­based proficiency testing. Clin Biochem. 2019;68:37–43. 138. Erdman P, Palmer-­Toy DE, Horowitz G, et al. Accuracy-­based vitamin D survey: six years of quality improvement guided by proficiency testing. Arch Pathol Lab Med. 2019;143:1531–1538. 139. Hawley JM, Owen LJ, Lockhart SJ, et al. Serum cortisol: an up-­to-­date assessment of routine assay performance. Clin Chem. 2016;62:1220–1229. 140. Owen LJ, Adaway JE, Davies S, et al. Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory. Ann Clin Biochem. 2013;50:345–352. 141. Ueland GA, Methlie P, Kellmann R, et al. Simultaneous assay of cortisol and dexamethasone improved diagnostic accuracy of the dexamethasone suppression test. Eur J Endocrinol. 2017;176:705–713. 142. Middle JG, Kane JW. Oestradiol assays: fitness for purpose? Ann Clin Biochem. 2009;46:441–456. 143. Owen LJ, Monaghan PJ, Armstrong A, et al. Oestradiol measurement during fulvestrant treatment for breast cancer. Br J Cancer. 2019;120:404–406. 144. Bertelsen BE, Kellmann R, Viste K, et al. An ultrasensitive routine LC-­MS/MS method for estradiol and estrone in the clinically relevant sub-­picomolar range. J Endocr Soc. 2020;4:bvaa047. 145. Handelsman DJ, Gibson E, Davis S, et al. Ultrasensitive serum estradiol measurement by liquid chromatography-­mass spectrometry in postmenopausal women and mice. J Endocr Soc. 2020;4. 146. Dirks NF, Ackermans MT, Lips P, et al. The when, what & how of measuring vitamin D metabolism in clinical medicine. Nutrients. 2018;10:482. 147. Ooms N, van Daal H, Beijers AM, et al. Time-­course analysis of 3-­epi-­25-­hydroxyvitamin D3 shows markedly elevated levels in early life, particularly from vitamin D supplementation in preterm infants. Pediatr Res. 2016;79:647–653. 148. Rauh M. LC-­MS/MS for protein and peptide quantification in clinical chemistry. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;883– 884:59–67. 149. Kushnir MM, Rockwood AL, Roberts WL, et al. Measurement of thyroglobulin by liquid chromatography-­tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clin Chem. 2013;59:982–990. 150. Van Der Gugten JG, Holmes DT. Quantitation of plasma renin activity in plasma using liquid chromatography-­tandem mass spectrometry (LC-­ MS/MS). Methods Mol Biol. 2016;1378:243–253. 151. Schwarz E, Liu A, Randall H, et al. Use of steroid profiling by UPLC-­MS/ MS as a second tier test in newborn screening for congenital adrenal hyperplasia: the Utah experience. Pediatr Res. 2009;66:230–235. 152. Peitzsch M, Dekkers T, Haase M, et al. An LC-­MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism. J Steroid Biochem Mol Biol. 2015;145:75–84. 153. O’Reilly MW, Kempegowda P, Jenkinson C, et al. 11-­oxygenated C19 steroids are the predominant androgens in polycystic ovary syndrome. J Clin Endocrinol Metab. 2017;102:840–848. 154. Fiet J, Le Bouc Y, Guechot J, et al. A liquid chromatography/tandem mass spectometry profile of 16 serum steroids, including 21-­deoxycortisol and 21-­deoxycorticosterone, for management of congenital adrenal hyperplasia. J Endocr Soc. 2017;1:186–201. 155. Keevil B. Steroid mass spectrometry for the diagnosis of PCOS. Med Sci (Basel). 2019;7:78. 156. Turcu AF, El-­Maouche D, Zhao L, et al. Androgen excess and diagnostic steroid biomarkers for nonclassic 21-­hydroxylase deficiency without cosyntropin stimulation. Eur J Endocrinol. 2020;183:63–71. 157. Oriolo C, Fanelli F, Castelli S, et al. Steroid biomarkers for identifying non-­classic adrenal hyperplasia due to 21-­hydroxylase deficiency in a population of PCOS with suspicious levels of 17OH-­progesterone. J Endocrinol Invest. 2020;43:1499–1509.

CHAPTER 2  Principles of Endocrine Measurements 158. Wilkes EH, Rumsby G, Woodward GM. Using machine learning to aid the interpretation of urine steroid profiles. Clin Chem. 2018;64: 1586–1595. 159. Masjkur J, Gruber M, Peitzsch M, et al. Plasma steroid profiles in subclinical compared with overt adrenal Cushing syndrome. J Clin Endocrinol Metab. 2019;104:4331–4340. 160. Erlic Z, Kurlbaum M, Deutschbein T, et al. Metabolic impact of pheochromocytoma/paraganglioma: targeted metabolomics in patients before and after tumor removal. Eur J Endocrinol. 2019;181:647–657. 161. Eisenhofer G, Lenders JW, Timmers H, et al. Measurements of plasma methoxytyramine, normetanephrine, and metanephrine as discriminators of different hereditary forms of pheochromocytoma. Clin Chem. 2011;57:411–420.

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162. Miller AG, Brown H, Degg T, et al. Measurement of plasma 5-­hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—comparison with HPLC methodology. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878:695–699. 163. Darr R, Kuhn M, Bode C, et al. Accuracy of recommended sampling and assay methods for the determination of plasma-­free and urinary fractionated metanephrines in the diagnosis of pheochromocytoma and paraganglioma: a systematic review. Endocrine. 2017;56:495–503. 164. Casals G, Hanzu FA. Cortisol measurements in Cushing’s syndrome: immunoassay or mass spectrometry? Ann Lab Med. 2020;40:285–296. 165. Bancos I, Taylor AE, Chortis V, et al. Urine steroid metabolomics for the differential diagnosis of adrenal incidentalomas in the EURINE-­ACT study: a prospective test validation study. Lancet Diabetes Endocrinol. 2020;8:773–781.

3 Endocrine Rhythms, the Sleep-­Wake Cycle, and Biological Clocks Stafford L. Lightman and Thomas J. Upton

OUTLINE Introduction, 36 What are Biological Rhythms?, 36 The Sleep-­Wake Cycle as a Rhythm, 36 Clocks in the Brain and the Periphery, 36 Endocrine Rhythms, 37 Examples of Endocrine Rhythms in Humans, 37 The Hypothalamo-­Pituitary-­Adrenal Axis as a Model Endocrine Rhythm, 37 Glucocorticoid Pulsatility, 39 Rhythms in Endocrine Disease, 39 Limitations of Single Timepoint Assessments, 39

Cell Clocks, 39 The Sleep-­Wake Cycle, 40 Sleep Regulation, 40 Sleep Stages, 40 Melatonin as the Biological Maker of the Night, 40 Consequences of Rhythm Disturbance on Metabolic Health, 40 Sleep Mistiming and Human Health, 40 Jet Lag and Social Jet Lag, 41 Novel technology for the Study of Biological Rhythms, 41 Summary, 41 Conclusions, 41

 

INTRODUCTION In 2017, the Nobel Prize in Physiology or Medicine was jointly awarded for the discovery of the molecular mechanisms that control the circadian clock. In announcing the prize the Nobel Committee described how the work of Jeffrey C. Hall, Michael Rosbash, and Michael W. Young explains “how plants, animals and humans adapt their biological rhythm so that it is synchronized with the Earth’s revolutions.”1 Adaptation of life to the length of each day had proven to be literally “in our genes.”

What Are Biological Rhythms? Biological rhythms are natural oscillations that occur in physiology and behavior that respond to periodic changes in the environment. They can be described by their length and by the circumstances in which they occur. Humans and many other animals have a diurnal behavior rhythm reflecting more activity during daylight hours, and rest or sleep at night. By contrast, nocturnal species such as rodents are active at night and rest during the day. Rhythms with longer periods such as annual migration or the human menstrual cycle are infradian, whereas rhythms that occur within a period of minutes to hours are ultradian. Rhythms that are endogenous, self-­sustained, and approximate a 24-­hour day, are termed circadian. In mammals this includes secretion of many hormones, most notably cortisol and melatonin. Circadian rhythms are entrained or synchronized to external cues (Zeitgebers, from the German “time giver”), and in humans and other mammals the most potent of these are light and timing of food.2 The most important point is that circadian rhythms are intrinsic––they continue to oscillate even in the absence of a zeitgeber or synchronizing input.

36

KEY POINTS  • Measurement of melatonin in dim light conditions is considered the gold standard measure of circadian phase. Melatonin secretion is extremely sensitive to light, which may alter the phase of the rhythm or temporarily suppress secretion.

The Sleep-­Wake Cycle as a Rhythm The sleep-­wake cycle is perhaps the most obvious and visible behavioral manifestation of a circadian rhythm in humans, having evolved as a consequence of the earth’s 24-­hour rotation. Characterized by a temporary suspension of consciousness, sleep is nevertheless associated with a multitude important and dynamic processes.3 Bordered at the onset by the rise of the “sleep hormone” melatonin and at the offset by an anticipatory rise in the hormone cortisol (Fig. 3.1), sleep occurs as a series of ultradian stages, with each cycle lasting approximately 90 to 120 minutes.3 Accompanying these cycles is a complex array of physiological events, including changes in sympathetic output, fluctuations in body temperature and muscle tone, and the secretion of hormones like growth hormone and aldosterone.4,5 Disturbance of the sleep rhythm, either in time or structure, results in adverse health outcomes for the individual.6

Clocks in the Brain and the Periphery We have already noted that circadian rhythms are intrinsic, and therefore their period persists in the absence of external cues. The mechanisms by which internal time is maintained depend on a network of intracellular clocks, coordinated by a central pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN). The SCN consists of tiny bilateral nuclei of a few thousand cells each––the cells are tightly

CHAPTER 3  Endocrine Rhythms, the Sleep-­Wake Cycle, and Biological Clocks

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Clock time (hr) Fig. 3.1  Illustrative example of the daily rhythms of two circadian hormones––cortisol and melatonin. In this experiment, a healthy male volunteer was admitted to the research facility, and plasma samples were taken every 20 minutes during a 25-­hour routine in which ambient light, meals (vertical dashed lines), and sleep times (shaded areas) were controlled. Both the diurnal and ultradian rhythms of cortisol (top panel, red) can be observed. Following the nadir, pulsatile secretion begins around 3:00 am, with peak levels just after waking. In dim light conditions (

Height velocity (cm/year)

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Fig. 17.5  Height and Body Mass Index (BMI) of Indonesian Children. A, Frequency distribution of height and BMI for boys. B, Frequency distribution of height and BMI for girls. (Novina N, Hermanussen M, Scheffler C, et al. Indonesian National Growth Reference Charts Better Reflect Height and Weight of Children in West Java, Indonesia, than WHO growth standards. J Clin Res Pediatr Endocrinol. 2020; 12:410–419.)

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CHAPTER 17  Somatic Growth and Maturation: Growth Hormone and Other Growth Factors

259

TABLE 17.2  Comparison of the World Health Organization (WHO) Growth Standards

and the US Centers for Disease Control and Prevention (CDC) Growth Reference Comparison

WHO Growth Chart

CDC Growth Chart

Studied population Growth pattern Concept of growth

Breastfed infants and toddlers How healthy children SHOULD GROW in ideal conditions A STANDARD by which all children should be compared

Breastfed and formula-­fed infants and toddlers How certain groups of children HAVE GROWN in the past A REFERENCE does not imply that pattern of growth is optimal

Growth Rate The increment in height or weight between two observations provides an estimate of growth rate or velocity or tempo of growth. Measurements, however, are not always taken at prescribed dates or intervals. As such, observed increments require adjustment for the actual interval between measurements. Increments are influenced by technical errors of measurement at each observation, and also by diurnal variation (variation during the course of a day). Heights measured in the morning, shortly after arising, are generally taller than those taken later in the day. The “shrinkage” in height is attributed to the compression of the intervertebral discs associated with gravity and physical activity. Increments in height also show seasonal variation in some parts of the world. Evidence from several studies in North America and Europe indicates a greater childhood growth rate in height during the spring compared with the fall.31 Allowing for measurement variability per se and diurnal and seasonal variation, estimates of growth rates over short durations, e.g., 3 or 6 months, must be interpreted with care. Growth rates (cm/year, kg/year) are presented as velocity curves, which differ in shape from the curves of size attained at different ages described above (Fig. 17.2). The velocity curve for height (length among infants) indicates a brief acceleration in rate of growth after birth, which is followed by a constantly decelerating rate growth in stature during infancy and childhood, i.e., the child is getting taller, but at a constantly slower rate. The growth rate reaches its lowest point (“prepubertal dip”) just prior to the initiation of the adolescent spurt. The lowest point is labeled as the age at takeoff of the adolescent spurt, because this is the point at which the velocity curve begins to accelerate. In contrast, after deceleration during infancy and the second year, rate of growth in weight occurs at a slight but constantly accelerating rate until the adolescent spurt (Fig. 17.4). Rates of growth in both stature and weight accelerate during the adolescent spurt. Acceleration in height occurs prior to that in weight, and maximal velocity of growth in height (peak height velocity [PHV]) occurs earlier than that for weight (peak weight velocity). After peak velocity of growth in height is attained, the velocity of growth in height gradually declines, and growth in height eventually ceases in late adolescence. Estimated PHV occurs, on average, approximately 2 years earlier in girls than in boys. Girls stop growing in stature by approximately 16 years, on the average, whereas boys continue to grow for another 2 or 3 years. Velocity of growth in weight also declines after the peak, but often remains positive into the early twenties. Reference values for annual or semiannual height increments are available for children and youth in the Fels Longitudinal Study from birth to 18 years,32,33 and have been more recently reported for samples of Black and White American children and youth 6 to 18 years of age.34 Reference values for short-­term increments in heights of British children 7 to 10 years of age are also reported.35 In addition to the well-­defined adolescent spurt, some children show a small growth spurt in stature and weight (increase in velocity of growth) several years before the onset of the adolescent growth spurt. The midgrowth spurt usually occurs between 6.5 and 8.5 years of

age, occurs more frequently in boys than in girls, and does not appear to show a sex difference in timing.36-­38 The midgrowth spurt reflects biological variation among individuals, but the frequency of measurements in childhood is an additional factor. Children are usually measured annually during childhood “well-­child” visits, and such an interval may not be sufficiently sensitive to detect the change in velocity of growth that defines the midgrowth spurt.

Maturity Status Maturity status refers to the level or state of maturation at the time of observation. Indicators of skeletal and pubertal maturation are used most often.

Skeletal Maturity Status Skeletal maturation is generally estimated as skeletal age (SA) based on the bones of the hand and wrist viewed on a standard radiograph. Each bone goes through a series of changes from initial ossification to adult morphology. The changes provide the basis for assessing skeletal maturation based on the assumption that specific features of each bone as observed on a radiograph occur regularly and in an irreversible order, and as such provide a record of the progress of each bone towards maturity. Other parts of the skeleton, e.g., knee and foot and ankle, as well as the hemiskeleton as an “integrated” determination by the time of appearance of various centers of ossification, have also been used to derive estimates of SA.39 Methods of Assessment. Three methods are commonly used to estimate SA of the hand-­wrist. Each method calls for the hand-­wrist radiograph of a child to be compared to specific criteria; ratings are subsequently converted to an SA that is specific to the method. Indicators of maturity defined for specific bones in each method suggest discrete steps in a continuous process.1,40 The Greulich–Pyle (GP) method41 is an extension of the method initially described by Todd.42 The method was developed on upper– socioeconomic status White American children from Cleveland, Ohio. Accordingly, each individual bone of the hand-­wrist is rated relative to sex-­specific standard plates representing specific SAs from infancy through adolescence. The method often requires interpolation between the standard plates. An SA is assigned to each bone, and the median of the SAs is the estimate of SA for the child. In practice, however, the GP method is often applied by comparing the radiograph as a whole to the pictorial standards and assigning the SA of the standard to which the radiograph most closely matches. As such, variation in level of maturity among individual bones may be large and is often overlooked. By convention, the left hand and wrist are used. The Tanner–Whitehouse (TW) method43 was developed on British children in the Harpenden Study. Specific criteria or stages spanning initial appearance (ossification) to maturity were described for each of 20 bones in the hand-­wrist: the radius, ulna, and metacarpals and phalanges of the first, third, and fifth digits (long bones) and the carpals, except the pisiform. A maturity score was also assigned to each stage for each of the 20 bones. The scores assigned to each of the 20 bones are summed, with the seven carpals and 13 long bones each contributing

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50% to the skeletal maturity score; the score for the 20 bones is then converted to an SA using sex-­specific tables. The first revision of the method, TW2,44,45 did not modify the criteria for maturity indicators; however, the final stages for five carpals and the radius and ulna were eliminated. In addition to the original SA based on 20 bones (TW2 20 Bone SA), two other SAs were provided, an SA based on the seven carpals (TW2 Carpal SA) and an SA based on the radius, ulna, and short bones (TW2 RUS SA). The maturity scores assigned to the stages for each bone are sex-­specific and vary for the 20-­bone, carpal, and RUS protocols. The sum of the maturity scores for 20 bones, the seven carpals, and the 13 long bones is converted to an SA using sex-­specific tables. The most recent version, TW3,46 retained the RUS (TW3 RUS) and carpal (TW3 Carpal) SAs but eliminated the 20 Bone SA. The tables for converting carpal maturity scores were not modified; the reference for TW3 Carpal SA was the original British series. In contrast, the tables for converting the RUS maturity scores to SAs were modified. Reference values for TW3 RUS SA were based on a composite of the original British series and samples of Belgian (Flemish), Italian, Spanish, Argentine, Japanese, and American children and adolescents surveyed in the late 1960s through the mid-­1990s; the American sample was from an upper–socioeconomic status area in the Houston region (Texas). Ages at attaining skeletal maturity with the TW3 RUS protocol were also lowered from 16.0 to 15.0 years in girls and from 18.2 to 16.5 years in boys. The Fels method47 was based on participants in the Fels Longitudinal Growth Study of children from middle class families in south-­central Ohio. The method specifies criteria for the radius, ulna, carpals, and metacarpals and phalanges of the first, third, and fifth rays. Grades are assigned to each bone depending on age and sex. Ratios of linear measurements of the widths of the epiphysis and metaphysis of the long bones are also used, and the presence (ossification) or absence of the pisiform and adductor sesamoid is noted. Grades assigned to the individual bones and width measurements are entered into a program that calculates SA and its standard error; the latter provides an estimate of the error of the assigned SA, which is not available with the GP and TW methods. Skeletal Age. The SA assigned to the radiograph of an individual represents the CA at which a specific level of maturity of the hand-­wrist bones was attained by the reference sample upon which the method of assessment was developed. An individual who has attained skeletal maturity is simply noted as mature. Of relevance, the individual is skeletally mature at the time of observation; when he/she attained the skeletally mature state is not known. As such, an SA is not assigned when the hand-­wrist radiograph indicates skeletal maturity. SA is ordinarily expressed relative to CA. The difference between SA and CA (SA minus CA) is often used as to indicate maturity status, i.e., early or advanced, average or “on time,” and late or delayed. SAs derived with the different methods, though related, are not equivalent, as criteria, methods, and reference samples differ. The GP, TW 20 Bone, and Fels methods each include the carpals, while the TW2 and TW3 RUS protocols are limited to the long bones (radius, ulna, metacarpals, and phalanges). SAs based on the GP and Fels methods, and three versions of the TW method (TW2 20 Bone, TW2 RUS, TW3 RUS) in a sample of German boys 6 to 16 years of age48,49 are summarized in Table 17.2. Heights of the boys matched, on average, medians of current US reference data. The original study used the GP method, but the radiographs were made available and were assessed with the GP (bone by bone), Fels, TW 20 Bone and TW2 RUS methods.50 The TW2 RUS scores were subsequently converted to TW3 SAs (Malina, unpublished).

Standard deviations for SAs were three to four times larger than those for CA, highlighting variation in skeletal maturity status within a single-­year CA group (Table 17.3). Mean SAs with each method varied and overlapped within each CA group, except for consistently lower SAs with the most recent TW revision. Beginning at 9 to 10 years, TW3 RUS SAs were consistently lower than TW2 RUS SAs. This same trend was noted in a large series of youth soccer players.51 The trends in comparisons among methods were generally consistent with observations of GP, Fels, and TW2 SAs in a clinically normal sample of 23 boys followed from 8 to 15 years.52 As noted, TW3 RUS SAs are systematically lower than TW2 RUS SAs beginning at about 10 years of age. The rationale for assigning lower SAs for the same RUS score with TW3 compared with TW2 was to accommodate secular change.46 Although secular changes in height are evident in early childhood and continue through puberty,1,53-­54 modifications in SAs assigned to the same RUS maturity scores in boys (i.e., lower SA for the same maturity score with TW3) were only apparent beginning with SAs of about 10 years. Of relevance, secular increases in height were not necessarily accompanied by accelerated maturation between 1960 and 1980 in Belgium55,56 and between 1980 and 1997 in the Netherlands.57 Although not indicated in Table 17.3, a number of boys were skeletally mature, especially with the TW method (one each at 14 and 15 years, and five at 16 years) compared with the GP (two at 16 years) and Fels (one at 16 years). Numbers of skeletally mature boys were larger at 17 years (GP 9, Fels 11, TW 17). The discrepancy between the GP and Fels methods and the TW method regarding skeletal maturity of the hand-­wrist relates to the criterion for the final stage of the radius and ulna, respectively. With the TW method, the final stage for the radius and ulna is simply “fusion of the epiphysis and metaphysis has begun.”46 With this criterion, the interval between onset and completion of epiphyseal union of both bones is not considered. Many youth are thus classified as skeletally mature even though the process of fusion in each bone is still in progress. On the other hand, onset through complete fusion of the distal radius and ulna are used in the GP and Fels methods. SA, or more colloquially “bone age,” has been indicated for CA verification in medicolegal contexts for many years, and also in some youth sport competitions. Evidence from a review focused on male soccer players and female artistic gymnasts58 indicates the following. Among adolescent male soccer players (and likely male athletes in other sports), a significant number will be identified as older than a CA cutoff due to advanced skeletal maturity status when they in fact have a valid CA. SA assessments of soccer players were generally comparable to magnetic resonance imaging (MRI) assessments of epiphyseal-­ diaphyseal union of the distal radius, which have been recommended for age verification for international competitions among U-­17 soccer players.59,60 Both protocols indicated a relatively large number of false negatives among youth players aged 15 to 17 years. Among adolescent female artistic gymnasts, a significant number of age-­eligible gymnasts would be identified as younger than the CA cutoff due to later skeletal maturation, when in fact they have a valid CA. On the other hand, there is also the possibility of false positives––identifying athletes as younger than the CA cutoff due late skeletal maturation, when in fact their birth certificates or passports indicate a CA older than the cutoff. Given the available data on the potential for false negatives and false positives, SA is therefore not a valid indicator of CA. The situation is more complicated for CA verification in the context of immigration regulations. Given the variation in SA within single-­ year CA groups, and also variation in SAs based on currently available methods, use of SAs as an indicator of CA for the purpose of immigration has major limitations.

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CHAPTER 17  Somatic Growth and Maturation: Growth Hormone and Other Growth Factors

TABLE 17.3  Skeletal Ages With Five Different Methods of Assessment in Boys 8–16 Years

of Age

SKELETAL AGES, YEARS N 26 23 22 20 31 22 23 20 10

CA, yrs M 8.4 9.5 10.5 11.5 12.4 13.5 14.3 15.4 16.5

SD 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3

GP M 8.3 10.1 10.2 11.0 12.1 12.8 13.8 14.9 15.8

SD 0.9 1.0 1.0 0.8 1.0 1.0 1.0 0.8 0.8

Fels M 8.1 9.6 9.7 10.7 12.2 13.0 14.2 15.4 16.5

SD 0.9 1.0 1.0 1.2 1.5 1.4 1.1 0.9 0.8

TW2 20 Bone M SD 8.3 0.9 9.8 0.9 10.1 1.2 11.2 0.9 12.6 1.5 13.5 1.4 14.8 1.1 15.8 0.8 16.8 0.8

TW2 RUS M 8.0 9.8 9.9 11.3 12.6 13.5 14.9 15.9 17.0

SD 1.0 1.2 1.1 1.2 1.6 1.6 1.3 0.9 0.8

TW3 RUS M 8.0 9.4 9.5 10.5 11.6 12.3 13.8 14.9 16.0

SD 0.9 0.9 0.8 0.9 1.3 1.5 1.0 1.0 0.8

CA, Chronological age; GP, Greulich–Pyle; TW, Tanner–Whitehouse; RUS, radius, ulna, and short bones; N, number of subjects; SD, standard deviation; M, mean.

Other Protocols. Given advances in technology, several new protocols for the assessment of the skeletal maturity of the bones of the hand and wrist have been proposed. The procedures are generally based upon the GP and TW methods and are largely designed for clinical use. These include ultrasound assessment of the maturity status of the distal radius and ulna, with SA scaled relative to the GP method,61,62 although its validity has been questioned.63 Assessment of SA based on dual-­energy x-­ray absorptiometry scans of the hand-­wrist have also been proposed.64-­66 Automated methods are also available.67-­69 The BoneXpert method68 is unique in that it derives an “intrinsic” bone age based on bone borders (shapes) and wavelet texture of images of 15 bones: radius, ulna, and the five metacarpals and eight phalanges in the first, third, and fifth fingers. The “intrinsic” bone ages are subsequently calibrated to GP and TW RUS SAs. Overview of Skeletal Age. SA can be used across approximately the first two decades postnatally; in contrast, other maturity assessments are limited to puberty and adolescence (see later). Estimates of SA by each method are reasonably precise and reliable, although intra-­and interobserver variability in assessments is rarely reported. Use of SA is also criticized, because specific training is required to learn the protocol(s): this is a shallow criticism, as other protocols also need specific training. Major limitations of SA are the expense associated with the radiographs per se and radiation exposure, and also the limited availability of individuals familiar with the details of the different methods of assessment. With modern technology, exposure to radiation presents minimal risk at 0.001 millisievert, which is less than natural background radiation and radiation exposure associated with the equivalent of viewing 3 hours of television per day.70,71 The different methods for the assessment of SA are based largely on samples of European ancestry, although the TW3 RUS protocol included data from samples of Argentine and Japanese ancestry in converting maturity scores to SAs (see earlier). Nevertheless, applications of the GP and TW protocols show ethnic variation in SA.72-­78 Applications of the Fels method to youth of different ethnic groups are apparently not available, except for a study of indigenous school children in Oaxaca, southern Mexico.79 Of potential relevance, identification of the ethnicity of youth in some countries is not permitted.

Secondary Sex Characteristics Secondary sex characteristics in males include pubic hair (PH), genitalia (G; penis, scrotum, testes), testicular volume, voice change, and facial and axillary hair, while those in females include PH, breasts (B), axillary hair, and menarche.1,80 Facial hair and voice change in boys and axillary hair in both sexes are also secondary sex characteristics, but they generally develop late during puberty and are not widely used.

Pubertal Stages. The five stages of PH, G, and B described by Tanner81 are commonly the reference in assessing pubertal status (Fig. 17.6). The stages generally follow the criteria of earlier studies of Reynolds and Wines.82,83 Stages are labeled PH1 through PH5, B1 through B5, and G1 through G5. Stage 1 of each characteristic indicates the prepubertal state, an absence of overt development, although hormonal changes that trigger puberty may already be underway. Stage 2 marks the overt development of each characteristic; B2 and G2 are typically the first overt signs of the transition into puberty, but PH2 may precede B2 and G2 in some youth. Stages 3 and 4 mark progress in pubertal maturation, and the respective stages are sometimes labeled as mid-­and late-­puberty. Stage 5 indicates the mature state (Fig. 17.6). The stages are discrete categories unique to PH, B, and G. A youngster is either in a stage or not in a stage at the time of assessment; there are no intermediate stages. Moreover, stage at time of assessment provides no information on when the youngster entered the stage (timing) or how long he/she has been in the stage, related to the tempo of maturity. It is important to emphasize that the stages of PH and B are unique to girls, and the stages of PH and G are unique to boys. The stages are not equivalent, i.e., B3 ≠ PH3, G3 ≠ PH3, B3 ≠ G3, PH3 in girls ≠ PH3 in boys, and so on. The term “Tanner stages,” unfortunately, is often used without indicating the specific characteristic(s) that was (were) assessed. Direct assessments of the stage of pubertal status are made at clinical examination. Self-­assessments are often used in nonclinical settings; they require privacy, good-­quality photographs of the stages, simplified descriptions, and a mirror to assist in the process. Some self-­assessment scales include pictures or drawings of the stages, as well as questions regarding facial and axillary hair in males and axillary hair and menarcheal status in girls.1 There is a need for quality control, including intra-­and interobserver reliability in assessment of stages, and concordance between self-­ assessments and those of experienced assessors. Overall reproducibility by experienced assessors is generally good, with approximately 80% agreement in assigning stages, but lower percentages have been reported.1 Accuracy of self-­ assessments is a concern, but opinions vary depending upon the purpose of the study. Based on self-­assessments of pubertal status in three annual visits of girls between 11 and 14 years of age and assessments by trained examiners, it was concluded that “… self-­assessment can substitute for examiner evaluation only when crude estimates of maturation are needed.”84 On the other hand, agreement to within one stage was suggested as potentially useful in epidemiological surveys of youth,85 even though concordance between self-­and physician assessments indicated limited accuracy. Concordance between and among self-­assessment scales currently in use needs further evaluation.

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B Fig. 17.6  Genital and Pubic Hair Stages at Puberty in Boys and Breast and Pubic Hair Stages in Girls at Puberty. (From Tenbergen G, Wittfoth M, Frieling H, et al. The neurobiology and psychology of pedophilia: recent advances and challenges. Courtesy: Michal Komorniczak (Poland).

Assessments of pubertal status are ordinarily done in a clinical context. However, in some cases, researchers are interested in whether a youngster is pre-­or postpubertal, i.e., stages 1 and 5, respectively. Others may be interested in early-­, mid-­, or late-­puberty, i.e., stages 2, 3, and 4, respectively. Note, however, as mentioned above, that stages are specific to B and PH in girls and G and PH in boys, and are not equivalent, i.e., B2 ≠ PH2, G3 ≠P H3, etc. Analytical Concerns. Stages of PH, B, and G are variably reported. Ratings for individuals are periodically combined into a mean of B and PH or of G and PH; there is, however, no biological entity of a “mean stage.” The stages are not equivalent and must be considered separately. Stages are occasionally reported as 3+ or 4+. Note, a youngster is either in a stage or not in a stage; there are no intermediate stages. Although mean stages of PH, B, or G by CA at observation are often reported, distributions of stages within each CA group are more informative and relevant. Youth are also commonly grouped by stage of puberty independent of CA. This is problematic from at least two perspectives. First, stages of puberty vary within a CA group, and second, also vary by CA within a stage. For example, within single-­year CA groups of soccer players 11 to 14 years of age, boys in less advanced stages of PH tend to be younger, shorter, and lighter, on average, than players in more advanced stages who are older, taller, and heavier. And, among players grouped by stage of PH, younger boys tend to be, on average, shorter and lighter than older boys who are taller and heavier.86 Corresponding classifications of girls would likely yield similar results. Testicular Volume. Testicular volume provides a more direct estimate of genital maturity in boys. The method requires palpation of the testes in order to match their size with a series of models of known volume (Prader

orchidometer).87,88 The ellipsoid models have the shape of the testes and range from 1 to 25 mL; a volume above 4 mL marks the beginning of puberty. The method is used primarily in the clinical setting. Sonography can also be used to estimate testicular volume.89 In a comparison study, Joustra and colleagues noted that testicular volumes above 5 mL, when evaluated by palpation, are reasonably accurate (compared with the ultrasound method), but may differ greatly below that, when it may be most relevant.90 For clinical purposes, that may not be so important, just that the testes are prepubertal, but may take on greater importance if gonadotropin treatment is evaluated at minipuberty during infancy. Menarcheal Status. Although age at menarche is an indicator of maturity timing, whether a girl is premenarcheal or postmenarcheal is an indicator of maturity status. Classifications of girls by menarcheal status are confounded by CA per se, i.e., an 11-­year-­old premenarcheal girl is quite different physically and behaviorally from a 14-­year-­old premenarcheal girl. Overview of Secondary Sex Characteristics. Secondary sex characteristics (overt manifestations) are limited to the interval of pubertal maturation. Stages are discrete, but somewhat arbitrary. Direct assessment is often considered invasive, especially outside the clinical setting. Cultural sanctions may limit or prohibit assessment of secondary sex characteristics in some groups. Concordance of clinical and self-­ assessments is variable and needs further study. Stages of puberty are also variably reported and present analytical concerns.

Maturity Timing Maturity timing refers to the CA at which specific maturational events occur. The two most commonly used indicators of timing are age at PHV and age at menarche. Both are limited to the adolescent period.

CHAPTER 17  Somatic Growth and Maturation: Growth Hormone and Other Growth Factors Age at Peak Height Velocity. Age at PHV is the estimated CA at the maximum rate of growth in height during the adolescent spurt. Age at PHV is estimated from serial height measurements of individuals taken annually or semiannually from late childhood through adolescence. Historically, determinations from individual height records were graphically plotted to identify takeoff, peak, and eventual cessation of growth. Mathematical modeling or fitting of individual height records is currently used, and a variety of methods are available.91 Estimated ages at PHV vary somewhat among methods, but are generally more uniform than estimated peak velocity of growth in height (cm/yr). Mean ages at PHV are reasonably similar among longitudinal samples of European and North American youth.1,92 On the other hand, variation in ages at PHV among individuals is considerable. In longitudinal samples of British, Swiss, Polish, Belgian, Canadian, and American youth, estimated ages at PHV ranged from 9.0 to 15.0 years in individual girls and 11.1 to 17.3 years in individual boys.1,93-­96 Age at Menarche. Menarche refers to the first menstrual flow; age at menarche is an indicator of the timing of this pubertal event. In longitudinal studies, girls and/or their mothers are interviewed at each regularly scheduled visit/observation regarding whether or not menarche has occurred. If menarche occurred between visits, further questions pinpoint the specific date/age of the first menstrual flow occurred. This is labeled the prospective method. Prospectively recorded ages at menarche in two longitudinal studies, one of American94 and one of Polish96 girls, ranged from 10.77 to 15.25 years and 10.49 to 16.30 years, respectively. Longitudinal studies generally follow subjects across adolescence so that early-­and late-­maturing girls are included. Depending on ages at which short-­term longitudinal studies start and conclude, there is potential risk that early-­and late-­maturing girls may be excluded. Ages at menarche based on the prospective method are sometimes confused with estimates based on the status quo method. The status quo method requires two pieces of information in a cross-­sectional sample spanning 9 through 17 years: CA and whether or not menarche has occurred (yes/no). The data are subsequently analyzed with probits or logits to derive a median age at menarche and associated variance statistics for the sample. The status quo method is generally used in surveys. In contrast to the prospective and status quo methods, ages at menarche are commonly obtained with the retrospective method, with late adolescents and/or adults asked to recall when they experienced their first menstruation. The method relies on memory, i.e., recall of the age when first menstrual flow occurred. In addition to potential errors with memory per se, reported ages are influenced by recall bias (the shorter the recall interval, the more accurate the recall, and vice versa) and a tendency to report whole years, typically age at the birthday before menarche.1 Estimates of age at menarche based on the retrospective method with samples of young adolescents are biased. Girls who have not yet attained menarche are excluded from the estimates. Some late-­ maturing girls may not attain menarche until 15 or 16 years, or perhaps later. In a nationally representative sample of US girls, 90% attained menarche by 13.75 years,97 but 10% of girls attain menarche after this age. Other Indicators of Timing and Interrelationships. Assuming longitudinal data are available, other potential maturity indicators can be estimated, e.g., age at takeoff of the growth spurt in height, ages at peak velocity for other body dimensions, and ages at attaining specific SAs, stages of pubertal maturation or specific percentages of adult height (see later). Analyses of ages at attaining several different maturity indicators in two longitudinal series highlight interrelationships among indicators

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during adolescence.98-­100 Common indicators in the two longitudinal series included ages at PHV and menarche, ages at attaining stages of pubertal development, and ages at attaining specific SAs and percentages of adult height. The analyses indicated a general maturity factor in both sexes underlying the timing of maturity indicators during the interval of the adolescent spurt. The analyses for boys suggested a second factor that loaded on ages at attaining SAs of 11 and 12 years, 80% of adult height, and early stages of PH and genital development, which are characteristic of early puberty. The results also suggested a degree of independence of ages at attaining several maturity markers characteristic of the transition into puberty, i.e., late prepuberty or early puberty.100 Longitudinal data for 30 boys also indicated considerable variation in SA at the time of pubertal onset (serum testosterone ≥30 ng/dL).101

Tempo of Maturation Tempo refers to the rate at which maturation progresses. Evidence from the Zurich Longitudinal Study indicated the following trends. Intervals (means ± standard deviations) between B2 and B3 and between PH2 and PH3 in Swiss girls were, respectively, 1.4 ± 0.8 years and 1.8 ± 1.0 years, while intervals between G2 and G3 and between PH2 and PH3 in Swiss boys were, respectively, 1.7 ± 1.0 years and 1.3 ± 0.9 years.102,103 The intervals between the transition into puberty (B2, G2, PH2) and the mature state (B5, G5, PH5) were, on average, 2.2 ± 1.1 years for B and 2.7 ± 1.1 years for PH development in girls, and 3.5 ± 1.1 years for genital and 2.7 ± 1.0 years for PH development in boys. The standard deviations for the transition through puberty for each characteristic approximated 1 year and highlight the variation in tempo of maturation of secondary sex characteristics within and among individuals. Although estimated increments in GP SAs in a longitudinal sample of American children approximated 1 year, variation was considerable and was associated in part with maturity status, i.e., early versus late.104,105 In a mixed longitudinal sample of White and Black American girls aged 6 to 12 years, mean single-­year velocities for TW2 20 Bone SAs varied between 0.66 and 1.14 years/year, and standard deviations varied between 0.33 to 0.52; corresponding mean single-­year velocities for boys varied between 0.75 and 1.27 years/year, and standard deviations ranged from 0.32 to 0.60.73 Single-­year rates of skeletal maturation expressed as maturity points per year of the American children72 overlapped mean rates and ranges for British children.106 Observations in a longitudinal series of 34 boys suggested that annual increments (years/year) in TW2 SAs (presumably 20 Bone) increased during the interval of puberty and the growth spurt, and that the increments appeared to reach a peak near PHV.105 Allowing for limited data, it is legitimate to inquire whether a skeletal year equals a chronological year.

Evaluation of Children for Disorders of Growth One of the most common reasons for referral to a pediatric endocrine clinic is to evaluate an infant/child/adolescent for short stature for his/ her CA relative to a reference for the general population. In contrast, relatively few children are referred for tall stature, although the paradigm for evaluation is, in general, similar to that for short children. Although the most common causes of short stature for calendar age are variations on the theme of normal growth, there are a number of conditions for which evaluation will yield a proper diagnosis and suggest an appropriate therapy; the latter, however, is often a matter of reassurance. Inextricably entwined in evaluations of short stature are nutritional status and the psychosocial concomitants of being short or maturing late. Nevertheless, evaluation of short stature must be viewed in the context of expected age and gender interactions that influence developmental, behavioral, metabolic, biochemical, and hormonal factors, in addition to genetic and environmental factors, including socioeconomic status. Interactions among the preceding are often

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brought into sharper focus at the time of puberty, especially when peers have begun pubertal maturation. Socialization during puberty, including sports participation, often tracks with biological maturation to a larger degree than CA. Evaluation of children for short stature and/or delayed maturation begins somewhat differently for boys and girls, although the differential diagnosis may extend beyond the variation in normal growth and maturation. Perceptions and observations of children and parents suggest that the psychosocial aspects of being short affect boys to a greater extent than girls; as such, boys are more often presented to a pediatric endocrinologist for earlier evaluation than girls. Normal linear growth in stature and weight is generally accepted as a sign of good health. The process of growth is continuous, but not linear, and shows a wide range of variation at different stages of a child’s life. Three distinct periods have been formally noted: infantile, when the rapid rate of in utero growth in length declines during the first year of life; childhood, when growth rate is largely steady but declines somewhat just before the pubertal growth spurt begins; and pubertal, as the growth spurt occurs.107,108 Estimated rates of growth in length/height decline from more than 25 cm/year shortly after birth to approximately 12 cm/year at the end of the first year and to approximately 7 cm/year at the end of the second year. Subsequently, growth rate in height varies between approximately 5 and 7 cm/year and declines, on average, slightly until the initiation of the pubertal growth spurt (age at takeoff), when the rate of growth in height accelerates and reaches approximately 9 cm/year in girls and 10.5 cm/year in boys at PHV. After PHV, growth rate declines until growth in height terminates in the late teens or early twenties. Individual children will often cross major percentiles on the growth chart during the first 24 months as they are no longer bound by the constraints of the intrauterine environment and move toward their genetic potential. Of interest, the correlation between birth length and adult height is approximately 0.1, while that between length (height) at 2 years and adult height is approximately 0.7. Growth disorders may be divided into at least three categories: primary or intrinsic to the growth plate; secondary or due to changes in the milieu of the growth plate, as in hormonal deficiencies; and idiopathic. Most systemic diseases or their treatments will slow linear growth (at least transiently) such that velocity of growth in height is sufficiently reduced that height begins to fall from a previously defined percentile. Allowing for normal variability, the pediatrician becomes concerned when two major percentile lines on the standard growth chart for height (size attained) are crossed (Fig. 17.4). The preceding is more apparent using height velocity charts with due care in estimating growth velocities from sequential measurements (e.g., measurement variability per se, adjustment for the interval between observations) (Fig 17.4). The hallmark of this transient slowing of growth is the phenomenon of catch-­up growth, i.e., growth in height at a higher than normal rate that allows a child to move upward on the growth chart, usually to the percentile along which he/she was previously growing, after the growth inhibiting factor(s) has (have) been removed. Full evaluation includes noting chronological and biological age, measurements (and charting) of height (length) and weight, estimates of height velocity, as well as parental heights, personal and family history, physical examination, state of pubertal maturation, and review of different systems. Subsequent laboratory and imaging studies are based on these initial observations.

Growth Charts Multiple growth charts for height, height velocity, and weight are available. The WHO charts are recommended for international use, while other charts are based on specific countries, regions or ethnic groups

(Fig. 17.4). Growth charts can be integrated into the electronic health record and automatically plotted to permit immediate comparison with earlier observations. A secondary benefit of immediate comparison is a reduced risk of inaccurate measurements, as the child can be remeasured if necessary. Nevertheless, appropriately calibrated instruments and care in the measurement process are essential. In addition, a child’s record can be evaluated by a growth “algorithm,” which can also take into consideration deflection from the midparental target height and perhaps accelerate referral to specialists for children who may require such attention. There are at least two types of charts: reference charts that provide a snapshot of how children are growing relative to the population, and standards that provide an estimate of how children should grow given optimal nutrition and care. These were noted in more detail earlier. KEY POINTS  • Maturity status may be determined by physical examination of the secondary sex characteristics, often using the Tanner criteria or by assessing skeletal (bone) age by one of several standardized methods. The timing of certain events and the tempo of maturation are important milestones.

EVALUATION OF CHILDREN WITH SHORT STATURE Short stature, defined as a height below –2 standard deviations (SD) for age, sex, and genetic background, including midparental height, is a statistical definition encompassing 2.3% of all children. It may, however, be the first presenting sign for an underlying condition. Short stature is not itself a disease, and the majority of children with short stature have a physiologic variant, i.e., familial short stature, constitutional delay of growth and then of puberty, or idiopathic short stature. The last category, which is also not a diagnosis, is ever-­shrinking as genetic variants that affect the growth plate are identified.109-­111 Large-­ scale genome-­wide association studies have noted that height is highly polygenic (hundreds of genes), with thousands of genetic variants distributed across the entire genome. These height-­associated regions are enriched for genes in multiple metabolic pathways associated with growth.111 Guidelines (guidance) for the referral of a short child to a pediatric subspecialist, usually a pediatric endocrinologist, and for their detailed evaluation may vary widely by country or among regions of a country.112,113 The goals of the evaluation are several. The evaluation should initially differentiate variants of normal from pathological (and often treatable) causes of short stature. The former may account for 80% of referred children in many pediatric endocrine clinics.114 Accurate height measurements following standardized procedures are essential, although children are often referred with few accurate measurements. Proper technique includes an appropriate device and a standardized protocol, but differs for infants and children. Infants are measured horizontally (recumbent length) on an examining table device with a flat board at the top and a moveable foot piece. The measurement is a two-­person procedure, with one at the head being sure that it is firmly against the end, with the nose straight up. The second person applies pressure to the knees so that they are flat against the table and moves the foot piece to the soles, with the toes pointing vertically. The length is read from the scale along the table (Fig. 17.1). A stadiometer or wall-­mounted device (e.g., steel tape) is used for children 2 years of age and older. Standing height is measured with the child (shoes removed) positioned with heels, back, and shoulders against the device or wall, and the head in the Frankfurt horizontal

CHAPTER 17  Somatic Growth and Maturation: Growth Hormone and Other Growth Factors plane with the eyes looking straight ahead. A head piece (or right-­angle triangle) is then moved to the top of the head. The height of the child is read either from the scale on the device or from the steel tape (Fig. 17.1). In some cases, the head piece may have to be applied with pressure to compress the hair. This procedure should be repeated after the child steps away from the device. The two measurements can be averaged if they agree within 0.4 cm, or the process repeated if they do not. Sitting height (upper segment) is measured with a similar device, except that the child sits on the table with the head in the same plane as noted above and the head piece lowered until it reaches the top of the head. Sitting height is then read from the attached scale. Leg length (lower segment) is estimated as the difference between standing height and sitting height, and the ratio between the two (sitting height/leg length) is the upper-­to-­lower ratio (Fig. 17.1).

History The history should start with the pregnancy with the child to determine if there were difficulties (including drugs the mother may have taken) that might have led to a small-­for-­date child. Birth weight and length are important to determine if the child was small-­for-­date. Other early historical notes should include feeding problems, medical diagnoses and treatments, motor and mental development, and behavioral issues in addition to height and weight records. Others in the family with a similar condition, as well as consanguinity, may offer clues for diagnosis. The heights of the parents, preferably measured, will provide an indication of the expected, sex-­specific target height and range. A child’s target height is based on the sex-­adjusted midparental height. It is a projected adult height (and range) based on the heights of the biological parents. The sex adjustment is important, because men are on average 13 cm (∼5 inches) taller than women. The simplest way to predict the genetic potential for adult height is to add the parental heights (cm or in) and then add 13 cm for boys or subtract 13 cm for girls and then take the average. That places the other-­sex parent on the same centile on the opposite sex chart. This is an estimate the genetic potential for all children of these parents. For the individual child, adult height is predicted from his/her present height and bone age using, for example, the Bayley and Pinneau tables in the Greulich and Pyle atlas.41 This may be done because bone age denotes approximately the percentage of adult height that one is at the time of the radiograph. It should be noted that many conditions that present as short (or tall) stature do not begin at birth. As such, information that may identify a secondary growth disorder (due to nutrition, hormones, inflammatory cytokines, or extracellular fluid, as in renal tubular acidosis) should be sought. With emphasis on the growth curves for weight and height, a significant weight loss or gain, or a slowing of growth in height and when it began should be noted. Issues of relevance include growth faltering (in addition to short stature) or acceleration. Diseases of the gastrointestinal tract are often associated with both decreased weight and growth faltering, whereas several endocrine disorders may present a weight percentile greater than the height percentile (increased weight-­for-­height) and a growth trajectory that may be negative for height but positive for weight (as may be noted with Cushing syndrome or severe hypothyroidism). Child complaints should also be considered; for example, abdominal pain/discomfort and diarrhea may suggest inflammatory bowel disease, or severe headaches may suggest an intracranial mass. The history of medication is critical, even if certain medications have been stopped by the time the patient presents for evaluation, e.g., drugs for attention deficit disorder or glucocorticoids for inflammatory conditions. The family history may again be helpful concerning others with a similar condition and/or psychosocial, intellectual, or behavioral issues.

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Physical Examination The physical examination is likely more helpful in those with primary growth disorders (clinically defined syndromes and skeletal dysplasias caused by variants of genes associated with the growth plate), e.g., dysmorphic features, abnormal body proportions, and cardiac and other organ system abnormalities (see Fig. 17.7 for growth plate physiology). Nevertheless, most children with acquired growth faltering present a relatively normal physical examination and normal body proportions. The degree of short stature may also offer clues to diagnosis. Children whose heights are slightly below the –2 SD cutoff are more likely to have a variant of normal growth than children with heights below –3 or –4 SD. Among more than 785 subjects with severe short stature (heights below –3 SD) after 3 years of age in the Helsinki University Hospital district, a pathologic cause for short stature, whether primary or secondary, was noted in approximately 70% of the children.115 The conditions included various syndromes, disorders of organ systems, GH deficiency (GHD), small-­for-­gestational age without catch-­up growth, and skeletal dysplasias. Normal variant, idiopathic short stature was far more prevalent at heights –3.0 to –3.5 SD than at heights more than –4.0 or –5.0 SD. More severe growth disorders (–4.0 SD and greater) were noted in children with syndromes and skeletal dysplasias (both primary growth disorders). Thus, the degree of short stature, especially at the extremes, separates the likelihood of a variant of normal short stature from the diagnosis of a pathological form.

Preliminary Laboratory Evaluation The information in the preceding paragraphs focuses on the appropriate evaluation for children with asymptomatic short stature, i.e., those children who are short but growing at the lower end of the normal range for age. Many will have heights in the –2.0 to –3.0 SD range and a height velocity that follows along one of those trajectories. If they are truly asymptomatic after a careful history and a normal physical exam, it is overwhelmingly likely that they have a variant of normal growth and can be followed by the primary care physician or endocrinologist without laboratory evaluation, with the exception of a bone age determination. Evidence suggests a low incidence of pathology among children with asymptomatic short stature. Among 235 such patients, at most only three new pathologic diagnoses were noted; nevertheless, laboratory screening following the guidance of several Pediatric Endocrine Societies cost more than $100,000 per diagnosis.116 The authors concluded that “… a clinician should use history, review of systems, and physical examination to guide the evaluation of individuals with short stature” [p 1050]. This information is quite helpful from several perspectives. Certain children with short stature do not require evaluation by an endocrinologist, although parents often request one. Perhaps more importantly, the history and physical examination, along with minimal laboratory testing, may lead the referring physician to a more appropriate pediatric subspecialist, for example, gastroenterologist, nephrologist, or pulmonologist. KEY POINTS • Is the child short? • What is the height velocity? • Detailed personal and family history • Physical examination • Bone age • Parsimonious laboratory evaluation (at first)

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Resting zone Proliferative zone Hypertrophic zone

A

Metaphysis

Hormonal signaling (GH1, GHR, IGF1, IGF1R, STAT5B, IGFALS)

Fundamental cellular processes (SOX9, SHOX, LARP7, BRF1, CREBBP, PTPN11, SPRED1, PRKAR1A, PIK3R1, ATR, DNA2, TRAIP, SMARCAL1, LIG4, XRCC4, MCM9, RECQL4, ERCC6, NIPBL, LMNA, TRIM37, CDH7, SCARP, DHCR7, PCNT, CRIPT, POC1, CUL7, OBSL1, ORC1, CDKN1C, GNAS1 locus)

Growth plate RZ PZ HZ

Paracrine signaling (FGFR3, PTHLP, PTH1R, IHH, BMPR1B, GDF5, NPR2, WNT3, ROR2, WNT5A, DVL1, IGF2)

Extracellular matrix defects

B

(Col2a1, Col9a, Col10a1, Col11a, ACAN, COMP, MATN3, FBN1)

Endocrine signals

Intracellular mechanisms

C

Nutrition

Paracrine signals

Chondrocyte

Extracellular matrix

Inflammatory cytokines

Extracellular fluid

Growth plate

Fig. 17.7  Growth Plate Physiology. A, Histology of the growth plate. The growth plate is a thin cartilage structure situated in the ends of tubular bones. It is commonly subdivided into three distinct zones; the resting, proliferative, and hypertrophic zones. B, Molecular mechanisms involved in longitudinal growth. C, Human growth plate histology from an 11-­year-­old boy. The growth plate comprises three histologically and functionally distinct zones; the resting, proliferative, and hypertrophic zones. Bar represents 100 μm. (A from Nilsson O, Marino R, De Luca F, et al. Endocrine regulation of the growth plate. Horm Res. 2005;64:157–165; B from Andrade AC, Jee YH, Nilsson O. New genetic diagnoses of short stature provide insights into local regulation of childhood growth. Horm Res Pediatr. 2017;88:22–37; C from Baron J, Sävendahl L, De Luca F, et al. Short and tall stature: a new paradigm emerges. Nat Rev Endocrinol. 2015;11:735–746.)

Evaluation for Endocrine Disorders of Growth The hallmarks of endocrine disorders of growth are greater deflections in height velocity and height standard deviation score (SDS) than in weight velocity or weight status. An upward trajectory in weight may be noted as height SDS is declining. The most prevalent endocrine system–related conditions include hypothyroidism, Cushing syndrome, and GHD, and perhaps GH insensitivity. If poor nutrition, either malabsorption or insufficient intake, is prominent as in celiac

disease, inflammatory bowel disease, or chronic caloric deficit, the loss of weight is generally in excess of that in height. Hypothyroidism and Cushing syndrome are described in specific chapters. The current discussion focuses on disorders of the GH/IGF-­1 axis.

Growth Hormone/Insulin Growth Factor-­1 Axis The GH/IGF-­1 system is the main regulator of postnatal human growth (Figs. 17.8 and 17.9). It regulates this complex process by integrating

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GHRH Adipose tissue Stimulate lipolysis Decrease de novo fatty acid synthesis Increase FFA use

Ghrelin Exercise Stress Somatostatin Pituitary gland

GH

Stimulate hepatocyte proliferation Stimulate liver regeneration Increase glucose production through gluconeogenesis and glycogenolysis from the liver Stimulate hepatic fatty acid oxidation

Skeletal muscle Increase amino-acid uptake Increase RNA synthesis Increase protein synthesis Induce FFA uptake into skeletal muscle Increase muscle growth

Heart and Cardiovascular system Have inotropic effect Increase cardiac output Increase myocardial mass Induce NO endothelial production

Liver

IGF-1

Bone and Cartilage Stimulate proliferation and differentiation of osteoblasts Stimulate osteoclast differentiation and activation of mature osteoclasts Increase bone turnover and bone mass Stimulate the colony formation of young prechondrocytes (GH) and cells at a later stage of maturation (IGF-1) Increase endochondral ossification and linear growth

Fig. 17.8  Growth Hormone/Insulin Growth Factor-­1 Main Actions on Liver, Adipose Tissue, Skeletal Muscle, Heart and Cardiovascular System, Bone, and Cartilage. FFA, Free fatty acids; NO, nitric oxide. (Sbardella E, Pozza C, Isidori AM, et al. Dealing with transition in young patients with pituitary disorders. Eur J Endocrinol. 2019;181:R155–R171.)

genetic and nutritional factors, along with signals from thyroid hormone, insulin, and glucocorticoids. Psychosocial aspects may also be important.117 Human GH is a heterogeneous protein with several molecular isoforms. This heterogeneity is expressed at the level of the GH gene, mRNA splicing, posttranslational processing, and GH metabolism, and poses a challenge to complete understanding of GH bioactivity, accurate measurement, and assay standardization.118,119 Two GH genes, GH1 and GH2, are on the long arm of chromosome 17q23.3: the former encodes the predominant isoform, 22 kDa (191 amino acids), which contains two intramolecular disulfide bonds.

Growth Hormone Release and Action The release of GH from the anterior pituitary is mediated by two peptides secreted by the hypothalamus, GH-­releasing hormone (GHRH) and the inhibitory hormone somatostatin (somatotropin release– inhibiting hormone [SRIH]) (Fig 17.8). A pulse of GH is generated by the simultaneous rise in GHRH and decline in SRIH. The amount of GHRH released likely determines the amplitude of the GH peak, and the frequency and duration of the GH secretory event is primarily under SRIH control. The pulse is also stimulated by ghrelin, which is produced mainly in the stomach, but also in the hypothalamus. IGF-­1 and GH itself exert negative-­feedback control at the hypothalamus. GH is transported in the serum by binding proteins: the major one, GHBP, is identical to the receptor.118

GH acts by binding to the GH receptor (GHR), which is expressed in most tissues. The GHR is a transmembrane receptor belonging to the class 1 cytokine receptor family. Once this binding takes place, the GHR undergoes conformational changes that result in activation of Janus kinases (JAK2), followed by recruitment and phosphorylation of signal transducers and activators of transcription (STATs) that, among other actions, induce the synthesis of IGF-­1, IGFBP3, and the acid-­ labile subunit (ALS) encoded by the IGFALS gene (Fig. 17.9).120 This ternary complex circulates assembled as a 150-­kDa protein, then IGF-­1 (a 7.6-­kDa protein, 70 amino acids), whose gene is located at chromosome 12q23, is liberated so it may bind with its receptor (IGF-­IR) to promote growth. Circulating IGF-­1, stimulated by GH action on the liver, is the main mediator of generalized GH actions; however, it is not the only route through which GH can exert its effects, for example, at the growth plate.121,122 IGF-­1 levels are age-­and sex-­dependent. Serum levels are high in the fetus, but drop shortly after birth and then increase slowly until late prepuberty or very early puberty. The levels subsequently increase approximately 3-­fold, concomitant with the rise in the quantity of GH secreted, peaking near the time of PHV.123 Peak values occur earlier in girls as their pubertal maturation increases. Once free IGF-­1 binds to its receptor, signaling cascades are triggered intracellularly, with two essential ones being the mitogen-­activated protein kinase (MAPK) and the phosphatidylinositol 3-­kinase (PI3K) cascades. The MAPK cascade is primarily responsible for the proliferation of muscle cells, and the PI3K cascade for cell differentiation.

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PART 2  Neuroendocrinology and Pituitary Disease Hypothalamus GHRH

Ghrelin

SST

The somatotropic axis

Pituitary

GH IGF-I

Regulation of Hepatic IGF secretion

Regulation of IGF bioavailability

Vasculature Liver ALS IGFBP3/5

IGFBP IGFBP IGF-I

IGF-I

IGF-II

IGF-I IGF-II

Intestine

IGFBP protease

Ghrelin

IGF-I IGF-II

GHR

IGF-II

IGF-I

IGF-II

Insulin

IGF-IR

IGF-IR

Hybrid R

IR

Target cell Fig. 17.9  The Somatotropic Axis. Hypothalamic growth hormone (GH)-­releasing hormone and somatostatin, as well as intestine-­secreted ghrelin, regulate pituitary secretion of GH. Once released to the circulation, GH stimulates liver production of insulin growth factors (IGFs) and a few of the IGF binding proteins (IGFBPs). In the vasculature, IGFs are found in binary and ternary complexes, which increase their half-­lives. Serum and tissue proteases act upon the IGFBPs to liberate IGFs and increase their bioavailability. Target cells expressing the IGF-­IR, insulin receptor, or hybrid receptors bind the IGFs and initiate phosphorylation cascades to enhance cellular proliferation, differentiation, and function. GH receptor is found on almost all cells. Upon binding to its receptor, GH initiates signaling cascades to promote cellular function that may be IGF-­dependent or -­independent. (Yakar, S, Werner H, Rosen CJ. Insulin-­like growth factors: actions on the skeleton. J Mol Endocrinol. 2018;61:T115–T137.)

GH and IGF-­1 (and other growth factors) work in concert to promote cartilage and bone growth. GH has a dual effect on the growth of epiphyseal cartilage and differentiation of cartilage cells, as well as the generation of IGF-­1. The proximal zone of cartilage, which is close to the bony segment of the epiphysis, consists of a narrow band of germinal or stem cell chondrocytes. GH preferentially stimulates differentiation of these prechondrocytes, while IGF-­1 stimulates the clonal expansion of the more differentiated cells in the distal proliferative zone. GH, but not IGF-­1, stimulates lipolysis. GHD may be congenital or acquired. It may also be isolated or in combination with other anterior hormone deficiencies (multiple pituitary hormone deficiencies [MPHD]) and dysmorphic findings or with involvement of other organ systems (see also Chapter 18). Children with congenital severe GHD have only a slightly reduced birth length and may not immediately show growth failure, which becomes prominent in the second half of the first year. The children also show a higher frequency of breech presentation and perinatal asphyxia. Neonatal morbidity may include hypoglycemia and prolonged jaundice. When GHD is combined with deficiency of adrenocorticotropic hormone (ACTH), hypoglycemia may be severe. The combination of GHD with gonadotropin deficiency can cause microphallus, cryptorchidism, and hypoplasia of the scrotum. Children with acquired GHD present with severe growth failure, delayed bone age, and increased weight:height ratios. Causes of

acquired GHD include intracranial tumors involving the hypothalamic-­ pituitary region (e.g., craniopharyngioma), cranial irradiation, and head trauma (Table 17.4). Another cause of hypopituitarism in children more than 2 years of age is psychosocial short stature, also known as deprivational short stature. Children exposed to a severely traumatic home environment are characterized by bizarre behaviors including gorging and vomiting (hyperphagia); sleep disturbance, night wandering often in search of food; pain agnosia; abnormal and disturbed relationship with primary caregiver; temper tantrums; and poor peer relationships. Objectively, the children have subnormal height velocity, but often an appropriate weight for height. Catch-­up growth may be marked with removal from the disturbed environment, as may be amelioration of many of the signs and symptoms noted previously. The relationship to the GH/IGF-­1 axis is that, if tested early after removal from the environment, whether measured by the spontaneous release of GH or by the GH response to a pharmacologic stimulus, the children respond as if GH-­deficient. Within a few days, responses to these tests generally revert to normal. Growth rates become remarkably high within weeks following removal from the home environment, even during hospitalization. Given this high rate of growth following an extended period of subnormal growth and the reversion of the testing to normal, this condition may be considered a form of reversible hypopituitarism, at least for the GH/IGF-­1 axis, and often for the corticotropin-­releasing hormone/ACTH/adrenal axis.117

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TABLE 17.4  Genetic Disorders of Growth Hormone (GH) GENETIC DISORDERS OF GROWTH HORMONE (GH) Gene

Phenotype

Inheritance

OMIM #

Isolated GH deficiency GH1

Isolated GH deficiency

AR, AD

GHRHR

Isolated GH deficiency

AR

139250, 612781, 173100, 262400 139197

Combined pituitary hormone deficiency POU1F1 GH, TSH, prolactin deficiencies GH, TSH, LH, FSH, prolactin and evolving ACTH deficiencies PROP1

AR, AD AR

173110 262600

Specific syndromes HESX1 LHX3 LHX4 SOX3 GLI2 SOX2 GLI3 PITX2

AR, AD AR AD X-­linked AD AD AD AD

182230 600577 262700 312000 610829 206900 146510 180500

Septooptic dysplasia GH, TSH, LH, FSH, prolactin deficiencies; limited neck rotation GH, TSH, ACTH deficiencies with cerebellar abnormalities Hypopituitarism and mental retardation Holoprosencephaly and multiple midline defects Anophthalmia, hypopituitarism, esophageal atresia Pallister–Hall syndrome Rieger syndrome

AD, autosomal dominant; AR, autosomal recessive; TSH, thyroid-­stimulating hormone; LH, luteinizing hormone; FSH, follicle-­stimulating hormone; ACTH, adrenocorticotropic hormone.

A number of conditions (syndromes) that involve linear growth and body composition have become of interest to internists because the children/adolescents who present with them survive into young adulthood. The largest group is likely the survivors of childhood cancer, many of whom have endocrine system–related morbidities, especially those related to growth and puberty.124-­126 New to many internists are those adolescents with Prader–Willi syndrome or cystic fibrosis. Prader–Willi syndrome (OMIM 176270) is a multisystem, genetically heterogeneous condition caused by a lack of paternal gene expression in the chromosome 15q11-­q13 PWS locus. Although the clinical phenotype may change over time, many adolescents and emerging adults have had the benefit of GH therapy, with increased adult height and a more normal body composition, indicated by an increased lean body mass and decreasing fat mass; although both remain abnormal in most subjects. The most common metabolic issue, in addition to hypogonadism, either secondary or mixed primary and secondary, is insulin resistance and disordered glucose metabolism. This often occurs in concert with the unresolved issue of adult treatment with recombinant human GH (rhGH), with effects on body composition and the regional distribution of body fat, as well as quality of life.127 Three and four decades ago it was uncommon for children and adolescents with the usual mutation causing cystic fibrosis to survive to emerging adulthood; many now survive into their fourth and fifth decades. Nutritional support is the cornerstone for metabolic function, but virtually half have a “new variety” of diabetes mellitus or cystic fibrosis–related diabetes, whose treatment is radically different from that for either type 1 or type 2 diabetes mellitus. The key deficiency is insulin itself and its strong anabolic action in muscle tissue. As this disease complication unfold during adolescence, there is a diminution of the anabolic action on muscle, and often a worsening of the pulmonary disease.128 Patients with these and similar conditions now require an endocrinologist for part of their ongoing medical and psychological care.

Diagnostic Approach GHD should be considered in the context of auxology, i.e., growth faltering and usually an increased weight:height ratio, but it is confirmed by

biochemical and stimulation testing. The first step is to evaluate for other potential causes of growth failure, including chronic systemic disease, e.g., hypothyroidism, Turner syndrome (in girls), and skeletal disorders. This is accomplished through a thorough medical history, physical examination, and bone age determination. Additional laboratory evaluation should be performed when appropriate, including screens for systemic disease, undernutrition, inflammation, and thyroid function, and a karyotype in girls to rule out Turner syndrome. GHD is effectively excluded in children with normal height velocity and bone age. Specific to GHD, in addition to the bone age radiograph obtained for most evaluations of short stature, an IGF-­1 level should be measured; in children below age 3 years, an IGFBP-­3 level should be measured. Although individual values will not permit the specific diagnosis of GHD, children with levels above 0 SDS for age and sex may be excluded. These findings are generally sufficient to exclude GHD without stimulation tests (see later). When GHD is congenital and near-­ complete, the diagnosis is straightforward, as affected children present with severe growth failure, delayed bone age, and very low serum concentrations of GH, IGF-­1, and IGFBP-­3. For patients with these clinical characteristics, it is reasonable to make the diagnosis of GHD without performing GH stimulation testing (see later). Lesser degrees of growth faltering and decreased IGF-­1 and IGFBP-­3 levels are consistent with GHD, but are also consistent with a number of other causes of growth failure, including poor nutrition. If not explicable on the basis of undernutrition, low IGF-­1 and/or IGFBP-­3 levels are strongly suggestive of a diagnosis of GHD, but this must be confirmed by provocative GH testing (see later). MRI of the hypothalamo-­pituitary region (with and without contrast) is recommended for children with suspected GHD and may be specifically useful in infants, where it is more likely that structural abnormalities will be found.129 A significant minority of children (3%–30%, depending on the study) have an affected parent or sibling, while several genetic causes, for example, transcription factor mutations or mutations of GH itself, the GHRH receptor Gsα or the GH secretagogue receptor, have been described in detail.130,131

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Growth Hormone Stimulation Tests

Indications. Provocative (stimulation) GH testing is indicated for most patients to confirm a diagnosis of GHD. Because these tests have limitations, the results should not be used as the sole diagnostic criterion and should be interpreted in the context of auxologic findings, bone age, and IGF-­1 and IGFBP-­3 concentrations.113 Provocative testing is not necessary for patients in whom other clinical criteria are sufficient to make the diagnosis of GHD: • Pituitary abnormality (secondary to a congenital anomaly, tumor, or irradiation) and a known deficiency of at least one other pituitary hormone, in addition to auxologic criteria.113,132 • Newborn with a congenital pituitary abnormality (ectopic posterior pituitary and pituitary hypoplasia with abnormal stalk) or known deficiency of a pituitary hormone, along with hypoglycemia, at which time a simultaneous serum GH concentration is less than 5 mcg/L.132 • Infant or young child with extreme short stature (height T, p.(Q87X), in three siblings from a Caribbean pedigree. All three affected children manifested a phenotype characterized by CPHD (GH, TSH, and PRL deficiencies), as well as learning difficulties and congenital cataracts.77

Structural Abnormalities. In addition to the structural abnormalities associated with the genetic problems described above, GHD can occur in the setting of other cranial or midline abnormalities such as holoprosencephaly, nasal encephalocele, single central incisor, and cleft lip and palate. As methods of radiologic evaluation of the CNS have improved, an increasing percentage of patients with idiopathic GHD have been identified to have structural abnormalities.78 Many of these are associated with some of the genetic abnormalities described above, but the findings are worthy of separate consideration. In particular, earlier studies showed that the finding on MRI of an undescended (frequently called “ectopic”) posterior pituitary (posterior pituitary ectopia [PPE]) was commoner in males than females (3:1 when PPE present vs. 1:1 if normal anatomy), in patients with CPHD as compared with IGHD (49% vs. 12%), breech delivery (32% vs. 7%), and associated congenital brain anomalies (12% vs. 7%).79 These findings appear to be best explained by a defect in induction of the mediobasal structure of the brain in the early embryo rather than the product of birth trauma, as previously suggested. Whether pituitary insufficiency is the result of hypothalamic or pituitary dysgenesis, or the product of hypoplasia or sectioning of the pituitary stalk, is not always clear. Perinatal problems, however, including breech presentation, may prove to be the consequence rather than the cause of underlying CNS abnormality. The concept that PPE, stalk section or hypoplasia, and pituitary hypoplasia may represent abnormal embryonic development rather than the consequences of birth trauma is supported by the finding of similar anatomic abnormalities in patients with SOD, type I Arnold-­Chiari syndrome, and holoprosencephaly, and increasingly in patients with mutations in the genes controlling pituitary development. In the empty sella syndrome, abnormalities of the sellar diaphragm allow herniation of the suprasellar subarachnoid space into the region of the sella turcica.80 This may result in damage to the sella, including the pituitary. Empty sella syndrome may be the consequence of surgery or irradiation or may be idiopathic. It is often found in patients with mutations in PROP1, when it may have been preceded by a pituitary mass.

Acquired Defects A wide range of destructive lesions involving the hypothalamus or pituitary may present with isolated GHD or CPHD. Birth trauma, associated with abrupt delivery, prolonged labor, or extensive use of forceps, has been associated frequently with subsequent hypothalamic or pituitary dysfunction.81

Destructive Lesions of the Hypothalamus and Pituitary. An increased incidence of GHD has been reported in breech deliveries, although it is still unclear whether such deliveries lead to the acquisition of pituitary dysfunction or, on the other hand, whether preexisting CNS abnormalities result in higher rates of abnormal birth presentations.

Tumors. CNS tumors are an important cause of isolated GHD and CPHD and must be excluded in every child with GHD who does not have an obvious alternative explanation for growth failure. Midline brain tumors include germinomas, meningiomas, gliomas, colloid cysts of the third ventricle, ependymomas, and optic nerve gliomas. GHD or CPHD may also occur from local extension of tumors affecting the head or neck, such as craniopharyngeal carcinomas and lymphomas. The major pediatric tumor involving the pituitary is the craniopharyngioma, which is probably an evolving congenital malformation that develops from remnants of Rathke’s pouch.82 It accounts for 5% to 15% of intracranial tumors in childhood and 80% of tumors in the HP region. Arising from rests of squamous cells at the embryonic junction of the adenohypophysis and neurohypophysis, it forms an enlarging cyst filled with degenerating cells, leading to cyst fluid or calcification but never to malignant degeneration (Fig. 18.5). These calcifications may be seen at times on skull films and constitute an important diagnostic sign. Although craniopharyngiomas represent the consequences of a congenital malformation, they may present clinically at any age. Significant growth failure may be observed in 30% to 50% of children at the time of diagnosis,83 but patients most commonly present with complaints of increased intracranial pressure, such as headaches, vomiting, and oculomotor disturbances; visual field defects are frequently noted at the time of diagnosis. Deficiency of at least one pituitary hormone, most commonly GH or gonadotropin, is present in 50% to 80% of patients. DI is reported in 25% to 50% of patients at diagnosis.82,83 The variation in the reported incidence of DI may be due to the fact that incidence of DI is either underestimated or may be masked by the simultaneous presence of ACTH deficiency. The commonest type of craniopharyngiomas in childhood is adamantinomatous craniopharyngioma (ACP). Activating mutations in the gene encoding β-­catenin (CTNNB1), a component of the Wnt signaling pathway, have long been identified in ACPs.84 A murine model that expresses a degradation-­resistant mutant form of β-­catenin in early progenitors of Rathke’s pouch has furthered our understanding.85 Most mutant mice die perinatally, by 4 weeks of age, and those who survive exhibit pituitary hyperplasia and marked hypopituitarism with severe disruption of the differentiation of the POU1F1 lineage and extreme growth retardation. Ultimately, all animals develop lethal pituitary tumors that closely resemble human ACPs. The tumorigenic effect of the activated mutant β-­catenin is observed only when it is expressed in undifferentiated progenitors, demonstrating that mutated β-­catenin in pituitary progenitor/stem cells has a causative role in the etiology of murine tumors resembling human ACPs.85 Pituitary adenomas, dermoid or epidermoid cysts, lipomas ,and teratomas are less frequent causes of GHD in childhood.86 Langerhans cell histiocytosis (LCH) may also present at any age. LCH is characterized by clonal proliferation and accumulation of

CHAPTER 18  Growth Hormone Deficiency in Children

A

287

B

Fig. 18.5  Magnetic resonance imaging (MRI) of cystic craniopharyngioma. Saggital MRI scan (A) revealing a large, multicystic craniopharyngioma arising from the pituitary fossa and extending up to hypothalamus. Coronal section (B) of the same lesion delineating upward and lateral spread. Both images are T1-­weighted, gadolinium-­enhanced scans.

abnormal dendritic cells that can affect either a single site or many systems, causing multiorgan dysfunction. In children, the median age of diagnosis ranges between 1.8 and 3.4 years. LCH infiltrates the HP area in 15% to 35% of patients, with subsequent development of at least one pituitary hormone deficiency.87 In a multicenter French national study of 589 pediatric patients with LCH, 145 patients (25%) had pituitary dysfunction. In 60 patients, pituitary involvement was already present at the time of diagnosis, and in 20 of them it was the first manifestation of the disease. Patients at high risk of pituitary involvement seem to be those with multisystem disease involving the skull and facial bones, mastoid, sinuses, and mucous membranes (i.e., gums, ear, nose, and throat region). Furthermore, compared with patients without pituitary involvement, patients with pituitary involvement have a higher rate of relapse (10% at 5 years vs. 4.8% at 5 years) and a higher incidence of neurodegenerative LCH.88 DI is the most frequently reported permanent consequence of LCH and the commonest endocrinopathy; almost all patients with pituitary involvement have DI. The second commonest endocrinopathy is GHD, which occurs in 14% of all patients with LCH and in more than 40% of patients who have pituitary involvement. In the vast majority of patients, GHD is associated with DI, with a median interval of 2.9 to 3.5 years between the diagnosis of DI and development of GHD. Isolated GHD, or the association of GHD with other anterior pituitary hormone deficiencies, occurs less commonly. Pituitary MRI findings in patients with LCH include thickening of the pituitary stalk, suggestive of the infiltrative process enhancing changes in the pituitary gland and hypothalamus, and absence of the bright signal of the posterior pituitary in T1-­weighted images, caused by the loss of the phospholipid-­rich antidiuretic hormone secretory granules. The latter is an invariable feature of patients who develop DI.89 Although at the time of diagnosis of DI 75% show a thickened pituitary stalk, only 24% have persistent stalk thickening after 5 years. These changes are variable and do not correlate with treatment or with clinical recovery; DI persists in all cases. Long-­term follow-­up of patients with LCH has shown that the already established hormone deficiencies cannot be reversed by

treatment.89 Recently, however, isolated case reports have suggested that treatment with the purine analogue 2-­chlorodeoxyadenosine (2-­ CDA) may reverse established DI. Subsequent studies of this form of therapy, used in refractory cases of LCH involving the CNS, showed that 2-­CDA may result in partial or complete radiologic improvement of the mass lesion, but the endocrine consequences of the disease, including DI and panhypopituitarism, do not reverse.90 Patients treated with the JLSG-­96 protocol who have been followed up for 5 years developed DI with an incidence of 3.1% to 8.9%, depending on the extension of the disease (single system multisite vs. multisystem).91 Radiotherapy used for the treatment of LCH is within the dose range of 10 to 15 Gy, which is known to be unlikely to cause GH insensitivity. However, radiotherapy has been associated with an increased risk of GHD despite the use of a dose less than 15 Gy, a finding that may reflect the severity and extent of the disease rather than the direct effect of radiotherapy.

Irradiation of the Central Nervous System. Cranial irradiation used for the therapy of solid brain tumors and as prophylaxis for leukemia can lead to abnormal HP function. The sensitivity of the HP axis to radiation depends upon the dose, the fractionation, the tissue location, and the age of the patient.92 Such damage is typically difficult to assess precisely, because the hypothalamus and pituitary may differ in the extent of involvement, and the loss of function may evolve with time. Sensitivity to CNS radiation may differ among patients, although the majority of children will experience some degree of hypothalamic or pituitary dysfunction within 5 years of receiving 30 Gy. GHD also occurs with doses of 18 to 24 Gy,93 and subtle dysfunction may be observed at even lower doses. GH secretion generally appears to be the most sensitive to irradiation, followed by TSH, gonadotropins, and finally ACTH. This may relate to the unique position of the GHRH neurons on the surface of the hypothalamus and not deep within the structure, as previously thought.94 Pituitary dysfunction evolves over several years following irradiation, so such children should be monitored for growth deceleration. Provocative GH testing may be within normal limits, but measures of spontaneous GH secretion frequently demonstrate abnormalities.

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Serum concentrations of IGF1 or insulin-­like growth factor-­binding protein-­3 (IGFBP-­3) may not be reduced in the early years following cranial irradiation.95 Cranial irradiation may also result in precocious puberty, leading to an early pubertal growth spurt, advanced skeletal maturation, and ultimately reduced stature. This may be superimposed upon any growth restriction that results from the spinal irradiation for the primary problem.96 Low-­dose irradiation is frequently associated with a precocious onset of puberty; higher doses may result in gonadotropin deficiency and pubertal delay. In the irradiated child with early puberty, therapy with gonadotropin-­releasing hormone analogs (GnRHas) should be considered, with or without GH treatment, to delay epiphyseal fusion. Lower doses of radiation (24 Gy) are also associated with GHD in approximately 30% to 60% of cases. Craniospinal irradiation used in the treatment of posterior fossa tumors and total-­body irradiation used in conditioning regimens for bone marrow transplant are also associated with damage to the epiphyses, with subsequent disproportionate short stature.

Traumatic Brain Injury. Traumatic brain injury (TBI) has been recognized as a cause of acquired hypopituitarism in a number of adult studies. Data on pediatric patients are sporadic, but TBI is probably underdiagnosed.97 These effects may be significant, considering the scale of the problem. In the United Kingdom, 180 children per 100,000 population per year sustain a head injury, with 5.6 per 100,000 requiring intensive care and almost one third of those admitted to the intensive care unit undergoing neurosurgery. Although the pituitary gland is protected within the bony cavity of the sella turcica, the rich vascular network of the hypothalamus and pituitary and the structure of the pituitary stalk make it vulnerable to the effects of TBI. The hypothalamus and pituitary have a complex vascular supply consisting of an arterial supply via the superior and inferior hypophyseal arteries from the internal carotid artery, as well as long hypophyseal vessels and a rich network of portal capillaries that surround the pituitary and infundibulum. The pathophysiology of hypopituitarism related to TBI is not clearly defined, but it is thought that it is the result of direct trauma or of vascular injury resulting in ischemia and infarction,98 an observation supported by the anatomical findings of autopsies following head trauma, which include anterior lobe necrosis, pituitary fibrosis, hemorrhage, infarction, or necrosis of the pituitary stalk.99 Hormone deficiencies may be identified in the first days to weeks posttrauma (acute phase) or may develop over time (late effect). Because there is overlap between the symptoms and signs of hypopituitarism and those of neurologic/psychologic sequelae of TBI, it is possible that late-­evolving or partial deficiencies can remain undiagnosed for extended periods. In the acute phase, alterations in the endocrine function may reflect an adaptive response to acute illness. The clinically significant alterations involve mainly the regulation of fluid and electrolyte balance (DI, syndrome of inappropriate secretion of antidiuretic hormone, cerebral salt wasting) and the hypothalamo-­pituitary-­adrenal axis. Most of the pituitary hormone changes observed in the acute phase are transient, and their development cannot predict the development of permanent hypopituitarism.100 Pituitary hormone deficiencies present in the acute phase are usually transient, but they may persist or appear and evolve over time. In adults, the incidence of permanent hypopituitarism ranges between 23% to 69%, depending on the study. The GH axis is the most frequently affected (10% to 33%), followed by the gonadal (8% to 23%), adrenal (5% to 23%), and thyroid (2% to 22%) axes. The prevalence

of permanent DI varies between 0% and 6%. In children with a TBI diagnosis there was a 3.22 times higher risk of a subsequent central endocrine diagnosis compared with the general population (± 0.28).101 Until recently, there were only sporadic reports of hypopituitarism following TBI in children, but prospective studies designed to address the problem in the pediatric and adolescent population are in progress. The incidence of hypopituitarism is reported to range from 10% to 60%, and although this is lower in children as compared with adults, it is not uncommon.102 In general, the long-­term outcome of TBI seems to be more favorable in children, although quality-­of-­life issues and minor disability may persist. The extent to which endocrine dysfunction contributes to these outcomes has yet to be defined. GHD appears to be the main endocrine manifestation, followed by gonadotropin deficiency. GHD can present as growth failure, whereas delayed or arrested puberty and secondary amenorrhea may present in adolescents and in patients in the transition phase. In a number of case reports, central precocious puberty has also been described in association with head injury, presenting 0.4 to 1.6 years after the event.103 Patients with hypopituitarism after head injury may have no clinical signs and symptoms suggestive of this disorder; its correct identification requires a high degree of suspicion. An international consensus guideline on the screening of adult patients post-­TBI suggests that all patients who had TBI, regardless of its severity, should undergo baseline endocrine evaluation 3 and 12 months after the event or discharge from the intensive treatment unit.104 With reference to children, there are still not enough data from prospective studies to recommend a systematic approach for screening patients after TBI.105 Even more controversial is the issue of recommending treatment with rhGH post-­TBI to improve cognitive outcome and quality of life106 or in patients with evidence of abnormal GH secretion on provocation testing who have normal growth.105

Infiltrative and Inflammatory Disorders. Infiltrative diseases are uncommon causes of GHD in the pediatric population, but pituitary insufficiency may be observed secondary to CNS involvement in tuberculosis, sarcoidosis, or toxoplasmosis. Inflammation associated with bacterial, viral, fungal, or parasitic disease may also result in HP dysfunction.107 Lymphocytic hypophysitis has also been reported.108 A recently reported rare cause of acquired hypopituitarism in adults with GH, TSH, and PRL deficiency results from insults specific to the somatotrope cell lineage, associated with autoimmunity to Pit-­1 (POU1F1), positive serum anti-­Pit1 antibodies, and histologically a marked reduction of pituitary somatotropes with infiltration by lymphocytes and plasma cells.109 It is as yet unknown if this may also be a cause of acquired GHD in children and adolescents with autoimmune disorders. Thalassemia is a hereditary disorder characterized by quantitative defects in synthesis of globin chains that result in ineffective erythropoiesis and, in its more severe forms, transfusion dependence. The majority of complications are the consequence of the toxic effects of iron, which is deposited in organs of the reticuloendothelial system, the heart, and all target organs of the endocrine system, including the pituitary. The anterior pituitary is very sensitive to iron overload, resulting in defective GH secretion, reduced responsiveness of GH to GHRH, and hypogonadotropic hypogonadism. The gonadotrope cells seem to be particularly vulnerable to the toxic effects of iron deposition, which may be related to the way iron is transported in cells. Failure of pubertal development and growth impairment are the most prominent endocrine complications, and may occur despite early initiation of chelation therapy. It is estimated that 56% of thalassemic patients have at least one endocrinopathy; almost half have hypogonadism (40% to 59%), and 33% to 36% manifest growth failure.110 Of

CHAPTER 18  Growth Hormone Deficiency in Children 202 thalassemic patients below the age of 18 years, the reported endocrine complications included GHD in 4.5%, latent hypocortisolism in 4.4%, and central hypothyrodisim in 0.5%.111 The growth impairment is the result of a number of factors that include chronic anemia and tissue hypoxia, overchelation due to the toxic effects of desferrio-­xamine on spinal cartilage, GH insufficiency, and possible GH insensitivity.

Vascular Lesions. Aneurysms may behave as space-­ occupying lesions and cause hypothalamic or pituitary destruction.112

Psychosocial Dwarfism. Psychosocial dwarfism is a form of poor growth associated with bizarre eating and drinking behavior, social withdrawal, delayed speech, and on occasion other evidence of developmental delay. Periodic hyperphagia is associated with decreased GH responsiveness to standard provocative stimuli but also with subnormal responses to exogenous GH therapy. Removal from the stressful environment, which usually involves removal from the home, is accompanied by a restoration of normal GH secretion, typically within weeks, and a period of catch-­up growth.113 The mechanisms for this reversible form of GHD are unclear, but it is of note that a variety of psychiatric conditions in adults may be associated with decreased spontaneous and provocative GH secretion. Establishing the diagnosis of psychosocial dwarfism requires documentation of catch-­up growth and restoration of normal GH secretion following correction of the environmental situation.

The Hypothalamo-­Pituitary-­Somatotroph Axis GH is secreted by somatotropes in the anterior pituitary gland. The secretory pattern is pulsatile, with discrete pulses of GH every 3 to 4 hours and virtually undetectable GH concentrations in between. Secretion of GH varies considerably with age and shows a sexually dimorphic pattern, with a greater average daily GH output in women. This pattern is the result of an interaction between the hypothalamic peptides GHRH and somatostatin (SS). The amplitude of the GH peak is determined by GHRH, which stimulates the pituitary somatotrophs to increase both the secretion of stored GH and GH gene transcription. SS determines trough levels of GH by inhibition of GHRH release from the hypothalamus and GH release from the pituitary. Withdrawal of SS, on the other hand, determines the timing of a GH pulse. More recently, the use of synthetic GH-­releasing peptides (GHRPs) has led to the identification of a GH secretagogue (GHS) receptor (GHS-­R type 1a). The receptor is strongly expressed in the hypothalamus, but specific binding sites for GHRP have also been identified in other regions of the CNS and peripheral endocrine and nonendocrine tissues in both humans and other organisms.114 The endogenous ligand for the GHS receptor, ghrelin, was isolated from the stomach and is an octynylated peptide consisting of 28 amino acids. It is expressed predominantly in the stomach, but lower amounts are present within the bowel, pancreas, kidney, immune system, placenta, pituitary, testis, ovary, and hypothalamus. Ghrelin not only leads to the secretion of GH but also stimulates PRL and ACTH secretion. Additionally, it influences endocrine pancreatic function and glucose metabolism, gonadal function, appetite, and behavior.114 It can also control gastric motility and acid secretion and has cardiovascular and antiproliferative effects.114 The role of endogenous ghrelin in normal growth during childhood remains unclear. Both ghrelin and GHRPs release GH synergistically with GHRH.114 The expression of the human GH1 gene is regulated not only by a proximal promoter, but also by a locus control region (LCR) 15 to 32 kb upstream of the GH1 gene. The LCR confers pituitary-­specific, high-­level expression of GH.115 The full-­length transcript from the

289

GH1 gene encodes a 191–amino acid, 22-­kD protein that accounts for 85% to 90% of circulating GH. Alternative splicing of the mRNA transcript generates a 20-­kD form of GH that accounts for the remaining 10% to 15%. Within both the proximal promoter and the LCR are located binding sites for the pituitary-­specific transcription factor Pit1. Therefore, the LCR region may be considered as initial “entry” point for the recruitment of the tissue-­specific Pou1f1 (Pit-­1), which in turn triggers chromatin remodeling and recruitment of coactivators.116 Additional binding sites for the transcription factor Zn15 are also located within the proximal promoter. In the circulation, GH binds to two binding proteins, high-­affinity GHBP and low-­affinity GHBP.117 Little is known about the low-­affinity GHBP, which accounts for approximately 10% to 15% of GH binding, with a preference for binding to 20-­kD hGH. The high-­affinity GHBP is a 61-­ kD, glycosylated protein that represents a soluble form of the extracellular domain of the GHR that can bind to both 20-­and 22-­kD hGH and thereby prolong the half-­life of GH. The GHR is present in a number of tissues. GH binding induces a conformation change of constitutively dimerized GHRs by rotation, activating a receptor-­associated tyrosine kinase JAK2 that in turn is autophosphorylated and also phosphorylates the GHR. This then leads to signal transduction using the MAPK, STAT, and PI3 kinase pathways. The end result is activation of a number of genes that mediate the effects of GH. These include early-­response genes encoding transcription factors such as c-­Jun, c-­Fos, and c-­Myc— implicated in cell growth, proliferation, and differentiation—and IGF1, which mediates the growth-­ promoting effects of GH.118 IGF1 and IGF2 are single-­chain polypeptide hormones that are widely expressed. Together with a family of specific binding proteins, they are believed to mediate most of the actions of GH.119 KEY POINTS  • The development of the pituitary is a highly complex process dependent upon a number of genetic factors encoding transcription factors as well as signalling molecules. Mutations in several of these genes are associated with various congenital forms of GHD including syndromic and non-syndromic forms of hypopituitarism as well as isolated GHD. The inheritance, penetrance and phenotypes associated with these mutations are highly variable. Acquired forms of GHD are caused by tumours and their treatments, inflammation, infiltration, and rarely, trauma.

CLINICAL FEATURES Neonatal Presentation Recent studies in humans and in animal models have demonstrated marked similarities, but also critical differences, between the clinical features of GHD and various forms of IGF deficiency.4,120 In GHD, prenatal growth is near normal, although mild reductions in birth length and weight have been observed. GHD does not cause severe intrauterine growth restriction, whereas loss of placental GH does. However, loss of IGF1 in utero results in severe intrauterine growth restriction in both humans and mice, suggesting that IGF1 and the IGF1 receptor are critically involved in intrauterine growth.121 IGF1 synthesis and secretion in utero are not regulated primarily by pituitary GH. IGF1 production comes under GH regulation either in the last few months of fetal life or shortly after birth, and is well-­established by 6 months of age. Growth failure is greater for skeletal growth than for body weight, so infants and young children have an appearance of relative adiposity. Neonates may present with hypoglycemia, and this suggests the possibility of other pituitary hormone deficiencies, especially ACTH. Normoglycemia is only maintained when cortisol replacement therapy is commenced, suggesting that ACTH (and consequently cortisol) secretion is critical for glucose homeostasis. However, the

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GH-­IGF1 axis also plays a role in maintaining glucose homeostasis, although IGHD is rarely associated with neonatal hypoglycemia. A diagnostic fast may be required to dissect CPHD from other causes of hypoglycemia, although the distinguishing feature from hyperinsulinism is the absence of ketone body formation in the latter. The presence of concomitant gonadotropin deficiency is suggested by the presence of microphallus, cryptorchidism, and scrotal hypoplasia. Genital ambiguity would not be expected, owing to placental production of hCG. Prolonged jaundice with conjugated hyperbilirubinemia and cholestasis may also be observed, typically in patients with CPHD. The relative contributions of GH, ACTH, and TSH deficiency to this presentation are unclear. It is imperative that the diagnosis of pituitary insufficiency be considered in any infant (especially term) with hypoglycemia, cryptorchidism, and microphallus, or conjugated hyperbilirubinemia. Associated features that might indicate more widespread problems (midline defects of the face, a single central incisor, nystagmus, and/or optic nerve hypoplasia) should be looked for, and MRI undertaken.22

Infant And Childhood Years After the perinatal period, the defining feature of GHD is growth failure. Reduced skeletal growth may be observed during the first 6 months of life in congenital GHD, but by 6 to 12 months of age early growth failure is almost inevitable.3 Height velocity is usually between –2 and –5 standard deviations (SDs) from the mean, leading to progressive height centile crossing. In patients with acquired GHD, the critical feature is a change in growth rate. Between the age of 2 years and the onset of puberty, children maintain their height percentile with remarkable integrity. Deviation from this channel (either acceleration or deceleration) needs investigation. Thus, a child who has been growing along the 75th percentile but moves across to the 25th percentile warrants evaluation, even though his/her height may still be within the normal range. Bone age is often delayed in patients with GHD, but this may not be so in acquired GHD. The close proximity of time to the growth failure or acquired GHD accompanied by accelerated puberty is occasionally seen in patients with intracranial tumors, when bone age may be accelerated. Delayed dentition may be observed, but in the absence of midline craniofacial abnormalities is otherwise normal. Other skeletal appearances include hypoplasia of facial bones, hypoplastic nasal bridge, frontal bossing, and delayed closure of sutures. Head circumference is usually at the lower limits of normal, indicating normal brain growth.3,123 An increase in adiposity, particularly central adiposity, can be detected by careful measurement of skinfold thickness. Genital growth prior to the onset of puberty is usually proportional to body size. Puberty may be delayed, but in the absence of other endocrine deficiencies is otherwise normal. Limited data are available on the adult height of untreated GHD patients. These results are often difficult to interpret because of (1) heterogeneity in the timing of GHD, (2) heterogeneity in the severity of GHD, (3) the presence or absence of other pituitary deficiencies, and (4) delay in puberty, resulting in late epiphyseal fusion. Wit et al.124 summarized the results from studies of 22 men and 14 women with untreated isolated GHD who underwent spontaneous puberty and reached a mean final height of 4.7 SDs (range 3.9–6.0) below the population mean.

DIAGNOSIS OF GROWTH HORMONE DEFICIENCY IN CHILDHOOD The diagnostic evaluation of children with growth failure is complex, because there are multiple causes for short stature (Table 18.4). In the pursuit of the diagnosis of GHD, other causes for short stature need

TABLE 18.4  Causes of Short Stature Nonpathogenic Constitutional delay of growth and puberty Familial short stature Nutritional Low Birth Weight Systemic Disorders Cardiovascular disease (e.g., congenital heart disease) Renal (e.g., chronic renal failure, renal tubular disease) Respiratory (e.g., cystic fibrosis, asthma) Gastrointestinal disease (e.g., Crohn disease) Neurologic (e.g., brain tumor) Psychologic (e.g., anorexia nervosa, child abuse) Endocrine Causes GH (growth hormone)-­related causes GH deficiency: isolated or combined with other hormone deficiencies Resistance to GH Insulin-­like growth factor-­1 deficiency Hypothyroidism Pseudohypoparathyroidism Glucocorticoid excess Cushing syndrome Congenital adrenal hyperplasia Exogenous administration Genetic Causes Turner syndrome Noonan syndrome Down syndrome Skeletal dysplasias: hypochondroplasia, achondroplasia, spondylo-­epiphyseal dysplasia Russell–Silver syndrome Seckel syndrome Prader–Willi syndrome Miscellaneous other syndromes (e.g., Rothmund–Thompson syndrome, ­ Leri–Weill syndrome, progeria, mucopolysaccharidoses)

to be considered and excluded. This is because the diagnosis of GHD is a multistep process that requires meticulous evaluation of the clinical history, auxology, and biochemical data, with increasing contribution from pituitary MRI and results of genetic studies.125 GH is the final common pathway for postnatal growth, and many causes of poor growth may secondarily affect GH secretion. There are a number of tests available for assessing GH status.126 Considerable attention has been paid to the underlying mechanisms assessed by the tests, how the samples should be collected, and what type of measurement should be performed. Less attention has been paid to the statistical assumptions underlying the performance of diagnostic tests. The statistical theory behind many tests is complex, because the results do not follow an all-­or-­none law. Rather than being left with a clear-­cut answer to the initial diagnostic question, the clinician is more likely to be left with a series of probabilities as to whether or not the patient is likely to have GHD.

Guidance Derived From Clinical Assessment

Neonatal Period. Several pointers to the diagnosis of GHD have already been considered in this discussion, but in the neonatal period GHD may be isolated or associated with other pituitary hormone deficiencies. Small genitalia may point to associated gonadotropin deficiency. Hypoglycemia in the newborn period is often a feature

CHAPTER 18  Growth Hormone Deficiency in Children of ACTH deficiency, although, on an arbitrary basis, a serum GH of less than 10 ng/mL is considered consistent with a diagnosis of GHD under these circumstances. This is not universal, however, and caution needs to be exercised in interpreting the GH response to hypoglycemia under different circumstances.127 Recently, Binder et al. reported that, using a highly sensitive hGH enzyme-­linked immunosorbent assay, the median GH concentration in dry blood spot samples of healthy neonates was 16.4 μg/L, while the median serum GH concentration of nine newborns diagnosed with hypopituitarism was 2.1 μg/L (maximum 5.5 μg/L), with no significant overlap between the groups. Based on these groups, the authors concluded that a single cutoff value of 7 μg/L, when measured with the same assay, would have high sensitivity (100%) and specificity (98%) for the diagnosis of neonatal GHD.128 Prolonged neonatal jaundice raises the question of thyroxine (unconjugated) or cortisol (conjugated hyperbilirubinemia) deficiency. Given these features, it might be possible on the basis of pattern recognition to ascribe the diagnosis of GHD to a patient with a high degree of certainty. MRI of the brain should be obtained to look for an undescended posterior pituitary, anterior pituitary hypoplasia, hypoplasia or absence of the pituitary stalk, hypoplasia of the optic chiasm, and absence or hypoplasia of the corpus callosum and septum pellucidum.129

Infancy and Childhood. Diagnostic evaluation in children must be based upon auxology. Although there are a number of clinical features of GHD that are said to be classic, none is specific. For example, obesity is listed as a clinical feature of GHD, but if we simply restricted biochemical evaluation to patients with obesity as the main feature, testing the GH axis would yield a large number of individuals with a poor GH response, because obesity per se is associated with blunted GH responses to various stimuli. Individuals who exhibit GHD are often obese, but the converse is clearly not the case. Little is known of the sensitivity and specificity of many of the clinical observations, either alone or in combination. The prevalence of many of the clinical features within the general population is unknown, which heightens the problem. Even the presence of specific features or a combination of features will only slightly increase the likelihood of disease if they are relatively insensitive. The manifestation of GHD as a result of a GH gene deletion is early, and poor growth can be detected as early as the sixth month of postnatal life. With advancing age, more GH has to be secreted to maintain concentrations of GH sufficient for growth, so idiopathic isolated pituitary GHD may present at any time. It is the degree of deficiency that dictates when the individual comes to medical attention. Table 18.5 provides general clinical rules that are a useful aid when selecting patients for further study of the GH axis.

Principles of Testing The aim of any diagnostic test is to progress the clinical history and examination to the point where patient care is altered. No test will ever benefit a patient. It is only when subsequent treatment has to differ depending on the test result that patients will be better off. There is a vast and bewildering body of literature on GH testing, but the clinician can be guided by asking the questions detailed in Table 18.6. It is important to remember that a diagnostic test is not just about whether a disease is present or not; it may also be important in determining severity and prognosis and responsiveness to and monitoring of therapy, and as a screening tool. As such, how the test performs under one circumstance may not be the same in another. Measuring serum IGF1 concentrations may be unhelpful in screening for GHD but may be excellent as a marker of response to therapy.

291

TABLE 18.5  Clinical Indicators for Further

Evaluation of the Growth Hormone Axis204

1. Height SDS below –2. Although WHO growth charts can be utilized for children up to 2 years of age, local growth charts, when available, are more appropriate for older children. 2. Height that clearly deviates from the familial background. 3. A significant decrease in height SDS (i.e., a deflection of at least 0.3 SDS/ year that is not explained by the normal channeling in infancy to adjust linear growth to target height trajectory, by the prepubertal growth dip or by pubertal delay). 4. Predisposing condition (tumor, radiation, etc.) or features suggestive of an underlying syndrome. 5. Neonatal signs consistent with pituitary hormone deficiencies. SDS, Standard deviation score; WHO, World Health Organization.

TABLE 18.6  Underlying Principles of

Assessing Tests

1. Has there been an independent blind comparison with the diagnostic “gold standard”? 2. Was the test conducted in a wide range of patients with and without the condition? 3. Is the test reproducible? 4. What was the definition of “normal” in the test situation? 5. How might the test interact with others in a diagnostic sequence? 6. Does the test entail risk or reduce risk for the patient?

Two points deserve special mention. First, it is unusual in endocrinology for there to be a diagnostic “gold standard.” The anterior pituitary is not accessible, and molecular biology is not sufficiently advanced to give definitive answers. Second, care needs to be taken in ascribing the role of a gold standard. It may change with time, and the test must be well validated by application to large numbers of individuals with and without the condition. The temptation is to use the extremes, but this may lead to a considerable overestimate of sensitivity and specificity, which may not be borne out in field studies.130 Two principles operate when using diagnostic tests.131 First, probability is a useful marker of diagnostic uncertainty. This is when the sensitivity (ability to detect a target disorder when present or true positive rate) and specificity (ability to identify correctly the absence of the disorder or true negative rate) become important. If both were 85%, 15% of patients with disease would have a negative result (false negative), and 15% without disease would have a positive result (false positive). Abnormal results would occur in patients with and without disease. Whatever the result, new information has been generated that may or may not influence decision-­making. Second, diagnostic tests should be obtained only when they can alter the management of the case—that is, if the test result alters the probability of the disease.

Pre-­and Posttest Probability. The relationship between the probability of disease after the results of diagnostic tests are known (the posttest probability), and pretest probability of disease depends on the sensitivity and specificity of the test, as shown in Fig. 18.6. There are two important points to note: the first is that the more certain the clinician is of the diagnosis before the test is performed, the less effect the confirmatory test has on the probability of disease. The obverse is also true. The second point is that tests will have major effects on probability of disease in the intermediate zone. Testing is not likely to be beneficial if the pretest probability is very high or low. This is one reason why screening for GH problems in short children on the basis

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TABLE 18.7  Effect on Posttest Probability

Post-test probability

1.0

of Differing Pretest Certainty, Assuming Constant Sensitivity and Specificity

0.8

POSTTEST PROBABILITY

0.6 0.4 0.2

0

0.2

A

0.4

0.6

0.8

Test Negative

90 50 5

98 (+8) 87 (+37) 25 (+20)

67 (–23) 19 (–31) 1 (–4)

1.0

Pre-test probability 1.0

Post-test probability

Test Positive

Change from pretest probability in parenthesis.

0

0.8 0.6 0.4 0.2 0 0

B

Pretest Probability (%)

0.2

0.4

0.6

0.8

1.0

Pre-test probability

Fig. 18.6  The relation between pretest and posttest probability of disease. The data were constructed by using Bayes’ theorem with a test sensitivity and specificity of either 70% (solid line) or 90% (dashed line). A, The posttest probability if the test were positive. B, The posttest probability if the test were negative. If the posttest probability were the same as the pretest probability, then the relation would be given by the line of identity. (Reproduced from Brook CGD, Hindmarsh PC, Jacobs HS, eds. Clinical Pediatric Endocrinology. 4th ed. London: Blackwell Publishers, 2001.)

of biochemical tests is unhelpful; the pretest probability is 1 in 3000, or 0.03%. Clinicians are often faced with the situation where they feel really sure the patient has the condition, but the test does not confirm this. Table 18.7 analyzes this concept. Here, specificity and sensitivity have been fixed, and the effects on posttest probability are considered. In the situation where there is a 90% pretest probability that the patient has GHD, then even if the test is negative in the individual, there is still a 67% probability (reduced by 23%) that they have the condition, so treatment would still be justified. When the pretest probability was 5% (very certain that the patient does not have GHD) and the test is positive, all the result says is that the patient has a 1 in 4 chance of having the condition, so we would probably not treat. In the middle ground, certainty in either direction is dramatically improved.

Multiple Tests. Table 18.7 could have been made much larger by introducing any number of pretreatment probabilities. There comes a point, however, when posttest probability changes to a level where a decision has to be made to stop and either accept or reject the proposal that the condition is present. The decision to stop investigation and to treat or not depends on how convinced the clinician is of the diagnosis, the benefits and risks of the therapy, and the potential yield and risks of further tests. There are two ways to assist this situation: conduct

another test or use a more sophisticated analysis rather than a simple positive or negative. This is problematic in the GH field, because the methodology assumes that the results of the two tests are independent. In normal individuals undergoing repeat GHRH tests, dependence cannot be assumed.132 Where repeat tests have been performed in children, concordance was observed 50% of the time, a value close to that calculated for independent events using a test with 70% to 85% efficiency. If all tests are treated as independent, there is a risk of over-­or underestimating the presence of the condition. Another important issue is whether the test may change in individuals as they age. There is evidence that the clonidine test is less effective in releasing GH in young adults compared with children. Whether the magnitude of the response to other stimuli can be assumed to remain unchanged is unknown. Assuming that the two tests are performed (on different days) and that they are dependent, then if both tests need to be positive for diagnosis, this maximizes specificity and avoids falsely labeling normal children, but it misses many treatable individuals. Insisting that both tests are negative maximizes sensitivity and minimizes misdiagnoses, but falsely labels many more normal child. These challenges in the diagnosis of childhood GHD are highlighted by studies showing that up to 85% (28/33) of short prepubertal children who were diagnosed with GHD based on a peak GH of less than 10 μg/L in two provocation tests had a normal GH response when retested 1 to 6 months later.133 In other series, almost 60% to 85% of patients diagnosed with GHD in childhood had an adequate GH secretion when retested in late adolescence or adulthood.134

Diagnosis of Growth Hormone Deficiency Assessment of GH secretion is problematic, in part because of the pulsatile nature of GH secretion. The most consistent GH surges accompany slow-­wave electroencephalographic rhythms during phases 3 and 4 of sleep. Although this rhythmicity is characteristic of GH secretion at all ages, the size of the amplitudes and the total integrated GH secretion varies with sex, age, pubertal status, and nutrition.135 Between pulses, serum GH concentrations are extremely low, often less than 0.1 ng/mL. Consequently, measurement of random serum GH concentrations is of no value in the diagnosis of GHD. Measurement of spontaneous GH secretion requires multiple sampling, typically every 15 minutes over a 12-­to 24-­hour period. Such methodologies are inconvenient and expensive, and, while they allow identification of the patient with severe GHD, it is not clear that they can discriminate between partial GHD and normal secretory variation.136 However, even the reproducibility of GH secretory patterns in children from day to day is uncertain. Rose and colleagues136 reported that measurement of spontaneous GH secretion identified only 57% of children diagnosed as GHD by provocative testing. Lanes137 reported that approximately 25% of normally growing children have low overnight GH concentrations. A longitudinal study of GH secretion in normal

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TABLE 18.8  Growth Hormone Stimulation Tests Stimulus

Dose

Sampling Protocol (min)

Notes

Exercise Levodopa Clonidine Arginine HCl

15 min for 90 min 15 min for 90 min 30 min for 90 min 15 min for 90 min

Variable response, highly dependent on degree of exercise Nausea Tiredness; postural hypotension May cause insulin release

Insulin

Cycle for 10–15 min 30 kg, 500 mg 0.l5 mg/m2 0.5 g/kg (max, 30 g) IV given as 10% arginine HCl in 0.9% NaCl over 30 min 0.05–0.1 units/kg IV

15 min for 120 min

Glucagon GHRH

0.1 mg/kg IM (max, 1 mg) 1 μg/kg IV

30 min for 180 min 15 min for 120 min

Hypoglycemia; requires supervision. Can also measure cortisol reliably Nausea Flushing. Only assesses pituitary reserve, not whole HP axis Needs further work to assess value in pediatrics

GHRH-­arginine Tests should be performed after an overnight fast. Patients should be documented to be euthyroid. Prepubertal children should be primed with gonadal steroids. GHRH, Growth hormone-­releasing hormone; IM, intramuscular; IV, intravenous.

boys during puberty indicated wide intersubject variation, and day-­to-­ day variation has been noted among normal subjects. An alternative approach has been the measurement of urinary GH concentrations.138 This methodology requires a timed urinary collection and a GH assay of high sensitivity, because urinary GH concentrations are low. The theoretical advantages of this approach include its relative ease of performance and noninvasive nature, as well as the requirement for only a single GH measurement. This must be balanced, however, by the need to assess the effects of renal function, the wide interindividual variation, and the lack of adequate age-­and sex-­related reference ranges. Obesity is another aspect that complicates primary GHD diagnosis, as visceral obesity per se results in a secondary reduction in serum GH concentrations.139 The reasons for the hyposomatotropinism in obesity and its mechanisms have yet to be clarified. Reductions in spontaneous GH secretion (as much as 6% for each unit increase in body mass index [BMI]) and in the half-­life of circulating GH have been reported. The GH response to pharmacological and physiological stimuli is impaired in obesity.140 Some of the theories on the cause of altered GH physiology in obesity involve the increased concentrations of leptin, insulin, free fatty acids, and IGF1. As a result of these difficulties, the standard for the diagnosis of GHD has been provocative testing of GH “secretory reserve” (Table 18.8). Physiologic stimuli for such tests have included sleep and exercise, and pharmacologic stimuli have included a wide variety of agents.141 None of these tests truly mimics normal GH secretory physiology, and none has been evaluated adequately in normal children and normal short children. The limitations of provocative GH testing in the diagnosis of GHD are described below and need to be considered in the light of statistical theory (see earlier): 1. Provocative testing by its nature is nonphysiologic. None of the commonly used stimuli truly mimics normal regulation of GH secretion. 2. The definition of a “normal” response to stimulation is arbitrary. Normal values are difficult to obtain in pediatric practice, and reference ranges would be needed for tall, normal, and short children, because their GH secretion differs. In addition, both age and pubertal stage influence GH secretion, as does body composition.142 Values for these would also have to be included. 3. The classic approach of defining normal data in terms of a Gaussian distribution does not come without hazard. Endocrine testing rarely fits this distribution; even if it did, it would imply that the

lowest and highest 2.5% of values are abnormal, and that all diseases have the same frequency—clearly unlikely. Creating upper and lower limits does not help either. It is more appropriate to identify a range of diagnostic test results, beyond which GHD is likely. 4. Most decisions on placing the value have been empirical rather than statistical. In practice, cutoff values could be chosen at an absolute extreme. If 100 short children were studied, and GH sufficiency or deficiency was defined by a peak response of less than 3 ng/mL, only 3% to 5% might have a response at this level. When testing the next 100 children, one or two normal individuals might have such a response. They will be outliers, but they are important, because the more patients studied, the greater the chance of finding outliers. 5.  Moving the cutoff to more extreme values to exclude these patients restricts the population of treatable individuals. Relaxing the criteria interposes normal individuals into the diagnosis zone. Placing the cutoff is based partly on clinical judgment. Because there is no perceived disadvantage apart from financial cost in falsely labeling someone with GHD and treating him or her, a relaxed cutoff would be acceptable. However, this could lead to some children receiving unnecessary treatment with GH, which is not completely without side effects. 6. The question of cutoff points for use in tests becomes more important as individuals with differing severities of the disorder are considered. In constructing normal ranges, it is clearly best if large sample sizes are chosen. Choosing populations of disease-­ positive and disease-­negative is unwise, either for this task or assessing test performance, because it is unlikely that the test will perform as well in the less severe cases.143 Some useful points in the assessment of studies are outlined in Table 18.9. Referral bias remains a major issue in many studies. In studies emanating from referral centers, the strength of a factor such as short stature may appear to be less important, in that patients are already selected for this in the referral. Changes in the prevalence of a condition do not change test properties, whereas changes in the spectrum of the disorder do.144 7. The dependence of GH secretion on other factors needs to be taken into account. Marin and colleagues145 demonstrated that, when exercise and arginine stimulation tests were performed on normal-­stature children without sex steroid priming, the lower-­ limits-­ of-­ normal (–2 SD) peak serum GH concentration for

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TABLE 18.9  Considerations to Give to Test

and Control Populations

1. What is the population from which patients are drawn? 2. How have patients been filtered before joining the study? 3. Test properties may differ in subpopulations within the sample. 4. Recruit consecutive patients with the problem. 5. Can the findings be generalized?

prepubertal children was as low as 1.9 ng/mL and rose to 7.2 ng/ mL on estrogen priming. Thyroxine and cortisol, which directly alter gene transcription, influence the results obtained, and these need to be controlled before undertaking a diagnostic study. Similarly, the presence of high concentrations of glucose or free fatty acids may influence the response obtained. 8. GH assays may measure a variety of immunoreactive molecular forms of GH, and individual standards need to be established for each laboratory. These should take into account factors intrinsic to the assay used, the heterogeneity of the GH molecule (different isoforms circulating in hetero-­and homodimers), circulating GHBP, and the standard preparation used for calibration. At the time of the 2000 Consensus of the GH Research Society on the diagnosis of GHD in children,125 the international standard (IS) available was the IS 88/624 (recombinant 22-­kDa GH). Since then, it has been replaced by the IS 98/574, the implementation of which has been recommended by an international consensus statement on the standardization and evaluation of GH and IGF assays.146 These recommendations and the current use of monoclonal assays make it imperative to review the accepted GH cutoff used for diagnosis of GHD in children. Assay precision, accuracy, and sensitivity play an important role in determining the success or failure of the diagnostic test. For example, assays for IGF1— despite having good standardization from the technical aspect (elimination of interference of IGFBPs and the use of high-­affinity, high-­specificity antisera)—may have different performance characteristics when concentrations are either above or below the normal range.147 9. Most endocrine tests are conducted over short periods of time, and results are extrapolated to longer time frames. GH provocation tests take 2 hours to perform, and the results are then compared with height velocity measurements obtained over a longer period of time, often 1 year. That there is a relationship is perhaps surprising; that there are high false-­positive and false-­negative rates is probably not. 10. Hormone pulsatility may also influence diagnostic tests if the test itself is influenced by oscillations (e.g., the stimulus applied) within the system under study. The GH response at any point in time is going to be heavily dependent on the interplay between the hypothalamic regulatory peptides involved in GH release, namely GHRH and SS. SS, in particular, is a key determinant of the amount of GH released as a result of GHRH stimulation. Attempts have been made to take control of this variable by the use of GHRH combined with arginine.148 An alternative approach is the use of ghrelin.149 However, in patients with congenital hypopituitarism the GH response to ghrelin depends on the degree of the anatomical abnormalities, as the main action of the peptide is at the hypothalamic level and requires the integrity of hypothalamic–pituitary connections.150 11. Endocrine systems are also subject to feedback from target tissues, and this is an issue not only in the interpretation of single provocation tests, but also where second tests are performed in

rapid succession to the first. A diminished response to GHRH can be observed if the second stimulus is applied 1, 2, or 3 hours after the first. The implication of doing two tests on the same day, often following each other, are immense; the cutoff that might be implied to determine normality or not may not be the same for the second test as for the first, especially if the second stimulus is different from the first. 12. Provocative testing fails to give any consideration to the effect of negative feedback by serum IGF1. It probably makes more sense to interpret serum GH concentrations in the light of serum IGF concentrations, much as TSH concentrations are best assessed with a knowledge of circulating thyroxine concentrations. 13. In assessing the results of endocrine evaluations, it is generally assumed that the single or multiple samples measured are relatively stable, at least over short periods. When important changes are postulated to be taking place, for example in a disease process, some knowledge of the inherent variability within the measurement system is required. In the short term, a number of studies have demonstrated variability within and between individuals in terms of GH tests. Group data are usually reproducible, but problems can arise if it is assumed that individual oscillatory profiles are consistent from day to day. 14. In considering provocative tests, the situation may arise where no response is observed. A possible explanation is that the strength of the stimulus was insufficient to provoke hormone release. In such a situation, it is valuable to have an independent marker of stimulus application. In the insulin-­induced hypoglycemia test, this marker is glucose and the attainment of adequate hypoglycemia. In the glucagon test, it may be the release of glucose. In other tests, there may be no independent markers, so doubt may be cast on the reliability of the nonresponse. 15. Careful consideration needs to be given to the age of the child under study. Not only may the cutoff point criteria differ at different ages, but the likelihood of disease presence will change with age. It is highly unlikely that GHD will manifest itself during puberty. It is possible, but it is more likely that any growth or GH secretory problems at this age relate more to delayed puberty rather than an abnormality in the GH axis. Even in childhood, the return in terms of diagnosis of GHD is not high if height screening is undertaken at school entry, so that with increasing age there is a diminishing diagnostic return. 16. Provocative GH testing is expensive and uncomfortable, and has risk. Insulin-­induced hypoglycemia should only be performed in a supervised setting. Deaths have been documented in patients rendered hypoglycemic and corrected in an overly vigorous manner. 17. Of the provocative tests listed in Table 18.8, it should be noted that stimulation with GHRH is not designed to document whether a patient has GHD, but rather whether GHD, established by other methodologies, is the result of pituitary or hypothalamic dysfunction. Failure to respond to GHRH suggests that the abnormality is at the pituitary level. This test may be enhanced by the addition of arginine or pyridostigmine.

A Practical Approach to Diagnostic Evaluation A practical approach to the diagnosis of a child with GHD is grounded on clinical assessment with allocation of pretest probability of disease presence. In the neonate and first few months of life, Binder and colleagues151 proposed a GH cutoff of 7 μg/L, while most recent guidelines have suggested that a cutoff of 5 μg/L in a newborn with additional pituitary hormone deficiencies and/or structural abnormalities of the pituitary is more appropriate.152 In the prepubertal child with abnormal growth, serum concentrations of IGF1, IGFBP3, and/or ALS

CHAPTER 18  Growth Hormone Deficiency in Children

295

TREATMENT

randomized to rhGH at a dosage of 0.175, 0.35, or 0.7 mg/kg/week for the first 2 years of treatment. Significantly greater height velocities and gains in height SD resulted from the 0.35 mg/kg/week versus 0.175 mg/ kg/week, but no further significant improvement was observed with the 0.7-­mg/kg/week dosage. Ultimately, the issues that should determine dosage in the child with GHD are (1) how best to return the GHD child to the normal growth curve, (2) how best to ensure that the child attains his or her genetic height potential, (3) risks, and (4) cost. However, instead of fixed dose regimens, titration of the dose of rhGH according to a target IGF1 SDS near the age-­and gender-­ adjusted mean (IGF1 SDS of −0.5 to +0.5) seems to be beneficial in terms of growth, at least in the short term, with great variability in the dose required.178 Although dose titration to achieve higher IGF1 targets (IGF1 SDS of +1.5 to +2.5) resulted in a higher growth response over a 2-­year term, it is not possible to conclude that this dosing strategy will result in improved adult height outcome.158 Response to GH therapy varies even when the diagnosis is homogeneous. This probably reflects differences in tissue responsivity, which may relate in part to the function of the GHR. A polymorphism in the GHR gene leading to retention (full-­length) or deletion of exon 3 (d3), which encodes a 22–amino acid sequence in the extracellular domain,159 has been associated with a greater height increase in response to GH replacement in children born small for gestational age (SGA), those with idiopathic short stature (ISS),160 and in a GHD population.161 Patients with at least one d3 allele had a significantly better first-­year response leading to an improved adult height on GH treatment than patients with homozygosity for full-­length GHR. However, not all reported studies are consistent, which may reflect differing populations and conditions.162 In the current context, the use of the GHR polymorphism, alone or in association with other genetic variants within the GH-­IGF1 axis, is of little value in prediction models to estimate the response to rhGH treatment in short children.163 False-­positive findings are more likely with small sample sizes, and for quantitative trait loci, phenotypic variations tend to be overestimated with small sample sizes.

Growth Hormone

Frequency of Administration. Several studies have compared the

The first successful treatment of human GHD with hGH was in the 1950s, while rhGH became available in 1985. This allowed for potentially unlimited supplies, obviating the need for low-­dose usage and interrupted therapeutic regimens. The initial rhGH preparation was an N-­terminal methionine, met-­rhGH, which was fully active biologically but was ultimately replaced by the mature 191–amino acid protein. Since then, information on the safety of rhGH treatment has mainly been obtained from large postmarketing databases including patients with GHD of variable etiology and non-­GHD subjects, confirming an overall safe profile.153,154

short-­term effects of administering hGH either daily or thrice weekly. Generally, daily injections are more effective, but increasing the frequency more than this makes little difference. Multiple long-­acting growth hormone (LAGH) preparations are currently at various stages of development, allowing for decreased GH injection frequency from daily to weekly, biweekly, or monthly, and thus improved adherence.164,165 Longer-­acting agents (LAGHs) can be divided broadly into depot formulations, PEGylated formulations, prodrug formulations, noncovalent albumin-­binding GH, and GH fusion proteins. Following administration of LAGH, the serum peak and trough GH and IGF1 concentrations vary depending upon the mechanism used to prolong GH action. Randomized, controlled clinical trials have not reported significant LAGH-­related adverse events during short-­term therapy. In order for LAGH to replace daily rhGH in the treatment of GHD, further studies are needed to clarify methods of dose adjustment, timing of monitoring of IGF1, safety, efficacy, and cost-­effectiveness. Furthermore, long-­ term surveillance registries to evaluate efficacy and safety will be essential for understanding the impact of prolonged exposure to these compounds.166

provide a means for excluding a diagnosis of GHD. Provocative GH testing with appropriate sex steroid priming will provide information on GH secretory capability, and GHRH stimulation, with or without arginine or pyridostigmine, will allow determination of whether the defect is at the hypothalamic or pituitary level. All data need to be interpreted together with known test performances and integrated with the pretest probability to generate a posttest probability that would then lead to a decision as to whether intervention is required. The interpretation of second tests of GH secretion may be problematic from both the physiologic and statistical standpoints, and should be analyzed with extreme caution. Documentation of GHD also requires that other pituitary functions be assessed periodically, including TSH, ACTH, and gonadotropin status. Other pituitary deficiencies may not be evident upon initial assessment but may develop over time. MRI of the hypothalamus and pituitary should be performed initially to determine if there is evidence of intracranial tumors, pituitary hypoplasia or PSIS, or midline defects. Even if the baseline MRI is normal, in the absence of an alternative explanation for GHD or CPHD, the possibility of tumors or structural defects should not be dismissed permanently. With increasing knowledge of the genetics of pituitary disorders, these should be looked for, because they have an impact on the likelihood of other pituitary hormone deficiencies evolving with time and allow genetic counseling to be undertaken. KEY POINTS  • The diagnosis of GHD is by no means simple, and often involves careful evaluation of the clinical history, examination, basal biochemistry, GH response to stimulation, radiology including a bone age and MRI of the brain and pituitary, and molecular analysis. Confounding factors in the interpretation of GH responses to provocation include obesity.

Dose Studies. Investigations of optimal dosing of rhGH have been complicated by the use of heterogeneous study populations, as studies frequently include patients with unequivocal and complete GHD together with patients with partial GHD. Therefore, it is not surprising that, despite long experience in the use of rhGH, there still remains uncertainty regarding the best dosing regimen, its calculation based either on weight or surface area, and a satisfactory way to quantify and predict responsiveness to treatment. It is generally accepted that a daily dose of 0.025 to 0.035mg/kg/day is sufficient to increase growth velocity to more than 10 cm/year in children with severe GHD and the adult height in treated patients ranges from –1.5 to –0.8 SDS, depending on the study.155,156 Several studies have demonstrated a dose-­response relationship for hGH, but the slope of the response is relatively shallow. Cohen et al.157 compared the growth responses of prepubertal, naive patients

Prediction Models. A series of models have been derived167 that describe factors that may influence response, but none has gone on to be tested in formal randomized controlled trials. Further problems arise when large multicontributor databases are used, because of the

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TABLE 18.10  Specific Issues with

Prediction Models of Response to Growth Hormone Treatment Development 1. Regression to the mean 2. Prediction gives average effect; how to apply to individuals for prognosis 3. Predictors may not be independent of each other; assumptions made in linear and multiple regression modeling 4. Assumptions about uniformity of response 5. Spurious correlations involving time

accuracy of the data entered; when prognosis is considered, factors that may be important may not have been recorded. It has been reported that, in children with GHD, auxologic parameters such as chronologic age (the younger the patient, the better the response) and the difference between target height and actual height (the smaller the patient, the better the response) are better predictors of growth response than the cumulative weekly GH dosage. There are several problems with these types of models: 1. Although prediction models are useful to give an average effect,168 they are not individualizable. These models explain only approximately 40% to 60% of the observed variability and are limited by their short-­term endpoints, the lack of validation in long-­term randomized trials, and the fact that they do not take into account the genetic makeup of the individual. 2. They often only focus on one outcome, usually short-­term growth, whereas interest may be more centered on final height. The two need not necessarily be related, and the factors that influence response in the first year of treatment may differ totally from those that lead to prediction of the individual’s final height. 3. Very few prediction models have been constructed from an a priori hypothesis, and care needs to be taken that there has been no interference from other factors accompanying the disease that might affect prognosis. 4. Rules derived from one data set may reflect associations that have occurred by chance and often result from overfitting of the data. 5. There is always the possibility that the predictors are idiosyncratic to the population, to the setting, to the clinicians, or to other aspects of the original study. Specific issues associated with growth-­ response models are summarized in Table 18.10.

Height and Other Outcomes. Early initiation of therapy, combined with careful attention to dosage adjustments and compliance, is the best predictor of cumulative growth response in patients with GHD. Final height correlates with height at the initiation of puberty, so it is important to maximize growth during the prepubertal period, within the limits of safety and economy. Analysis of data on final heights of rhGH-­treated GHD is complicated by the heterogeneity of patient groups and dosage. In earlier reports a common observation was a general failure of children to reach their full genetic height potential, especially in the case of IGHD and particularly in females. Price and Ranke69 reported final heights of –1.26 SD and –1.45 SD from the mean in males and females, respectively, with IGHD, and –0.22 SD and –0.52 SD from the mean in males and females, respectively, with CPHD. In patients with longer durations of treatment and higher dosages of rhGH, adult heights tended to be greater, although they still failed to achieve full genetic height potential. Recent reports of the final height of GHD children treated with rhGH show that, as a group, they achieve a normal final height within their genetic potential,155 although there is still considerable variability in the response depending on the population, sex, and underlying diagnosis (IGHD compared

with CPHD).154,170 Males of European/North American and Japanese origins with IGHD achieved a near-­adult height of –0.8 SDS, while females achieved a height of –1.0 SDS; differences between near-­adult height and midparental height ranged between –0.6 and +0.2 SDS.155 The timing of GH treatment initiation increases the chance to achieve near-­adult height. The NordiNet International Outcome Study, a noninterventional, multicenter study, identified that near-­ adult height SDS achieved by patients starting treatment early (n = 40 [boys, 70.0%)]; least squares mean [standard error] −0.76 [0.14]) exceeded that achieved by those starting later (intermediate, n = 42 [boys, 57.1%]; −1.14 (0.15); late, n = 90 [boys, 68.9%]; −1.21 [0.10]). The study concluded that early initiation of GH treatment in children with isolated GHD improves their chance of achieving their genetic height potential.171 It is clear that the timing of puberty has a significant impact on adult height of individuals with rhGH-­treated GHD. The duration of rhGH treatment and the height gained prepubertally are typically greater when puberty is induced rather than spontaneous. Final heights were greater after induced puberty compared with spontaneous puberty in boys (171.3 vs. 166.0 cm) and in girls (157.0 vs. 155.0 cm). Therapy designed to delay the onset of puberty (both normal and precocious) may augment the cumulative growth response to rhGH, but this is not accepted practice, and should only be considered in exceptional cases. Final height gain can be particularly variable in children who have had treatment for malignancies. GHD is often complicated by skeletal damage following TBI or craniospinal irradiation, early puberty, hypothyroidism, gonadotropin deficiency, malnutrition, and concomitant chemotherapy. GnRHa therapy to arrest early puberty has been used in conjunction with GH treatment in this group of patients, with encouraging results. The use of GnRHa reduces the concentration of sex steroid, with a consequent delay in epiphyseal fusion. However, GH and GnRHa combination therapy in children with GHD is not widely used at present, and the long-­term beneficial effects in terms of adult height are not well established.172 It may be beneficial under certain circumstances—for example, where the diagnosis of GHD has been delayed. The effects of GnRHa in the long term are unknown, particularly on bone health; and the cost of this combination therapy would need to be weighed against the presumed benefit.172 Although considerable attention has been paid to growth, there is now recognition that the beneficial effects of rhGH in children extend beyond height. GH treatment in childhood can also normalize body composition, with a reduction in body fat, although effects on lean body mass are less evident.173 It is also associated with reversible insulin insensitivity154 and an increase in the ratio of high-­density lipoprotein (HDL) to total cholesterol, improved cardiac function, and reduction of proinflammatory cytokines, along with an increase in glomerular filtration rate and acceleration of bone remodeling, with an increase in bone mineral mass.174 An increasing number of studies have focused on the role of GH in memory and neurocognition.175 On the other hand, functional MRI has shown that, although children with IGHD do not have a global reduction in brain volume, specific structures are affected, with significantly lower volumes of the splenium of the corpus callosum (P < 0.02), right pallidum (P < 0.007), right hippocampus (P > γ

α >> ACTH, β, γ

MC1R MC2R MC3R MC4R MC5R Fig. 21.3  Proopiomelanocortin (POMC)-­derived peptides and melanocortin receptors. POMC is processed into adrenocorticotropic hormone (ACTH) and α, β, and γ-­melanocyte stimulating hormone (MSH) peptides, which act on the melanocortin 1–5 receptors (MC1R–MC5R).

enzyme PCSK2, which acts on the products of the prior PCSK1 cleavage to produce the smaller melanocortin peptides. A further enzyme, carboxpeptidase E, is needed to produce the final mature peptides. Impaired processing of POMC contributes to the hyperphagic severe early-­onset obesity and ACTH deficiency in people lacking PCSK1.24 Hypogonadotropic hypogonadism due to the impaired processing of prepro–gonadotropin-­releasing hormone and postprandial hypoglycemia due to impaired processing of proinsulin to insulin are additional clinical features. Where these assays are available, the finding of high proinsulin levels in the context of a low plasma insulin can point to this diagnosis. Impaired processing of gut-­derived peptides in the

enteroendocrine cells that express PCSK1 throughout the gut25 may contribute to neonatal enteropathy; indeed, patients may first present to gastroenterologists with these symptoms. A single family with a homozygous mutation in carboxypeptidase E with overlapping clinical features has also been reported.26 Setmelanotide has been shown to be effective in POMC and PCSK1 deficiency27,28 and has been licensed for chronic weight management in these disorders in the United States.

MC4R Deficiency POMC-­derived peptides act as agonists at the MC4R. Targeted disruption of Mc4r in mice leads to increased food intake, weight gain, increased lean mass, and linear growth.29 Heterozygous loss-­ of-­ function mutations in MC4R are found in 5% to 6% of patients with severe early-­onset obesity30,31 and at a frequency of approximately 1/330 in the general UK population, making this the commonest gene in which variants contribute to obesity. Most naturally occurring disease-­ causing MC4R mutations disrupt the expression and trafficking of the receptor to the cell surface.32 The mechanism of G protein–coupled receptor (GPCR) dysfunction has potential interest, as pharmacological chaperones can increase the cell surface expression and signaling of mutant GPCRs, which represents a rational therapeutic approach for this condition.33 Additionally, mutations can affect the production of cyclic adenosine monophosphate, the homodimerization of MC4R, the endocytosis of MC4R, and the recruitment of β-­arrestins.34 MC4R mutations are inherited in a codominant manner, with variable penetrance and expression in heterozygous carriers30; less commonly, homozygous and double heterozygous (different mutations on both alleles) mutations have been reported in patients with severe obesity. The features of MC4R deficiency include hyperphagia, hyperinsulinaemia, increased lean mass, and increased linear growth.30,35

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CHAPTER 21  Genetic Syndrome Associated with Obesity Complete loss-­of-­function mutations have a larger impact on phenotype than partial loss-­of-­function mutations.36 MC4R-­/-­ mice maintain a normal blood pressure despite severe obesity.37 Similarly, loss-­ of-­ function MC4R mutations in humans are associated with a reduced prevalence of hypertension, low systolic blood pressure, lower urinary noradrenaline excretion, and reduced peripheral nerve sympathetic activation compared with similarly obese people without MC4R mutations.38,39 As MC4R neurons synapse with preganglionic sympathetic neurons, which regulate vascular tone, increased signaling through MC4R can lead to an increase in blood pressure, as seen with first-­generation melanocortin receptor agonists given to obese volunteers. Setmelanotide, a second-­generation MC4R agonist that causes weight loss without increasing blood pressure, has been shown to be very effective in POMC and PCSK1 deficiency and is currently being trialed in heterozygous MC4R deficiency. Liraglutide, the GLP-­1 receptor agonist, is effective in some patients with MC4R deficiency,40 and some patients with heterozygous (but not homozygous) mutations benefit from Roux-­en-­Y-­bypass surgery.41

LEPR

SH2B1

STAT3

STAT3

STAT3

PCSK1

SIM1 Deficiency SIM1 is a transcription factor that plays a key role in the development and function of the paraventricular nucleus of the hypothalamus, where MC4R is highly expressed. Dominantly inherited or de novo deletions of chromosome 6q14-­21 that encompass the SIM1 gene, as well as heterozygous loss-­of-­function SIM1 mutations,42,43 cause hyperphagia, severe obesity, and relatively low systolic blood pressure, features that closely overlap with those seen in MC4R deficiency. Many SIM1 mutation carriers have speech and language delay in childhood and exhibit neurobehavioral abnormalities, including autistic-­ type behaviors. These features show some overlap with the behavioral phenotypes seen in Prader–Willi syndrome (PWS). Expression of the neuropeptide oxytocin is reduced in mouse models of Sim1 deficiency, and oxytocin administration reduces food intake in Sim1-­haploinsufficient animals.44 As oxytocin is involved in energy homeostasis, emotional responses, and social interaction, reduced levels of oxytocin may contribute to the behavioral phenotype seen in SIM1 deficiency. A reduced number of oxytocin neurons and reduced oxytocin mRNA and protein levels have also been reported in PWS.45,46 For this reason, some reports have referred to SIM1 deficiency as being PWS-­like. A small number of mutations in OTP, another transcription factor involved in paraventricular nucleus development, have been reported in patients with obesity and autistic behaviors.47

BDNF, TRKB, and SH2B1 Deficiency Chromosome 11p.12 deletions encompassing the brain-­derived neurotrophic factor (BDNF) gene48 or missense mutations that disrupt BDNF or its tyrosine kinase receptor, tropomycin-­related kinase B (TrkB), are associated with hyperphagia and obesity.49 Mutations often arise de novo, and as such should be considered where both parents are of normal weight and IQ. The neurotrophin BDNF is widely expressed in the brain and signals via TrkB to regulate neuronal differentiation and survival, synapse formation, and activity-­ dependent changes in synapse structure and function. BDNF and TrkB are expressed in areas of the brain involved in learning and memory, including the hippocampus. Obesity-­associated mutations that impair TrkB signaling affect dendritic spine structure and function, which forms the neural substrate for learning and memory in hippocampal neurons.49 In addition to severe obesity, carriers of these functional TrkB mutations have learning difficulties, impaired short-­term memory, hyperactivity, repetitive behaviors often considered to be autistic-­like, fearlessness, and in some cases aggression. Some of these neurobehavioral phenotypes, as well as increased

1 CSR

POMC α/β MSH

Fig. 21.4  Genes that modulate leptin signaling to cause obesity. Leptin signaling through its receptor (LEPR) is enhanced by SH2B1 and results in the activation of STAT3, which dimerizes and translocates into the nucleus to activate proopiomelanocortin (POMC) transcription. SRC-­1 modulates POMC transcription by interacting with activated STAT3. POMC is processed by PCSK1 to yield the melanocortin peptides (α and β-MSH).

locomotor activity, have been observed in animal models of Bdnf/ Trkb disruption.50 Src-­homology-­2 (SH2) B-­adaptor protein-­1 (SH2B1) is an intracellular adaptor protein that mediates signaling through a number of receptor tyrosine kinases (including TrkB) and cytokine receptors (including the leptin receptor) (Fig. 21.4); mutations impair signaling and neurite differentiation. Chromosomal deletions on 16p11.2 that include SH2B151 and heterozygous mutations in the gene itself52 are associated with hyperphagia, dominantly inherited severe early-­ onset obesity, and disproportionate insulin resistance. Male mutation carriers often have behavioral problems, including social isolation and aggressive behavior from childhood.52,53 Intriguingly, mice with brain-­specific deletion of Sh2b1 gain weight and develop reactive aggression54; brain-­specific restoration of Sh2b1 completely reverses intermale aggression. Neural SH2B1 promotes brain development and growth, at least in part by enhancing the actions of BDNF and related neurotrophic factors.

PLEIOTROPIC OBESITY SYNDROMES In a number of syndromes, obesity is one of a number of features (pleiotropic) alongside developmental/learning difficulties and dysmorphic features (Table 21.2). A number of these disorders arise from chromosomal abnormalities.

Prader–Willi Syndrome PWS is relatively common, with an estimated prevalence of 1 in 25,000 births,3 and is characterized by diminished fetal activity, hypotonia,

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TABLE 21.2  Pleiotropic Genetic Obesity Syndromes Syndrome

Inheritance

Additional Clinical Features

Prader–Willi

Autosomal dominant

Albright’s hereditary osteodystrophy

Autosomal dominant

Bardet–Beidl

Autosomal recessive

Cohen Carpenter

Autosomal recessive Autosomal recessive

Alstrom

Autosomal recessive

Tubby

Autosomal recessive

Hypotonia, failure to thrive in infancy, developmental delay, short stature, hypogonadotropic hypogonadism, sleep disturbance, obsessive behavior Short stature in some, skeletal defects, developmental delay, shortened metacarpals; hormone resistance when mutation is on maternally inherited allele Syndactyly/brachydactyly/polydactyly, developmental delay, retinal dystrophy or pigmentary retinopathy, hypogonadism, renal abnormalities Facial dysmorphism, microcephaly, hypotonia, developmental delay, retinopathy Acrocephaly, brachydactyly, developmental delay, congenital heart defects; growth retardation, hypogonadism Progressive cone-­rod dystrophy, sensorineural hearing loss, hyperinsulinemia, early type 2 diabetes mellitus, dilated cardiomyopathy, pulmonary, hepatic and renal fibrosis Progressive cone-­rod dystrophy, hearing loss

Chromosome 15q11-13 SNURF-SNRPN Centromere MAGEL2 NECDIN IC

Telomere HBII-85

HBII-52

Fig. 21.5  Chromosomal deletion in Prader–Willi syndrome (PWS). Paternal chromosome 15q11-­13 deletions cause PWS. A number of genes (MAGEL2, NECDIN), small nuclear ribonucleoprotein polypeptide N (SNRPN), and the imprinting center (IC) are deleted in most patients, but the minimal critical region involved in the phenotype involves the HBII-­85 snoRNAs (noncoding small nucleolar RNAs).

developmental delay, short stature, hypogonadotropic hypogonadism, and obesity. PWS is caused by deletion of an imprinted region on the paternal chromosome 15q11-­q13. Loss of the entire paternal chromosome 15 with the presence of two maternal homologs (uniparental maternal disomy) occurs in approximately 20% of patients. While a number of genes within the imprinted region have been studied, small deletions restricted to a family of noncoding RNAs (HBII-­85 snoRNAs) recapitulate the classical features,55,56 providing strong evidence that these noncoding RNAs play a causal role in PWS (Fig. 21.5). These noncoding RNAs affect the alternative splicing of a large number of target genes, with evidence of intron retention,46 as is also seen in a number of neurodegenerative diseases. RNA sequencing of brain tissue from PWS patients has shown that expression of BDNF and its receptor TrkB is reduced, and downregulated genes affect neuronal differentiation, maintenance, and synaptic plasticity.46 Neuroimaging studies in PWS have identified reduced gray matter volume in cortical areas and abnormal gyrification.57 As such, it is likely that the complex clinical phenotype arises from the reduced expression of large number of genes in the hypothalamus and other brain regions. Patients often have low birth weight at term (in keeping with other imprinted disorders) and poor feeding as neonates due to diminished swallowing and sucking reflexes, which may require assisted feeding for 3 to 4 months. Feeding difficulties generally improve by the age of 6 months. From 12 to 18 months, hyperphagia is a dominant feature in children with PWS, often associated with pica behavior. One suggested mediator of the obesity phenotype in PWS patients is the enteric hormone ghrelin, which is implicated in the regulation of mealtime hunger and also stimulates growth hormone (GH) secretion via the GH secretagogue receptor. Fasting plasma ghrelin levels are 4.5-­fold higher in PWS subjects than in equally obese controls and patients with other obesity syndromes58; whether this finding is relevant in the pathogenesis of hyperphagia in these patients remain unclear. The mainstay of management has centered on low-­calorie diet with regular exercise, rigorous supervision, restriction of food, and behavioral counseling for

the patient and family, often in the context of group homes for PWS adolescents and adults. Children with PWS have reduced linear growth, reduced muscle mass, and increased fat mass, body composition abnormalities that are consistent with GH deficiency. Treatment with GH significantly improves growth and final height, and GH is licensed for use in PWS. GH treatment also decreases body fat and increases muscle mass.59 Gonadal maturation is delayed or incomplete as the result of hypogonadotropic hypogonadism.

Albright’s Hereditary Osteodystrophy Albright’s hereditary osteodystrophy (AHO) is an autosomal dominant disorder caused by heterozygous loss-­of-­function mutations in GNAS, the gene encoding Gαs (stimulatory G-­protein alpha subunit), which mediates GPCR signaling. Patients present with developmental delay, brachydactyly (shortened metacarpals and/or metatarsals), subcutaneous ossifications, childhood-­onset obesity with short adult stature (height below the 3rd percentile for chronological age), and in some cases hormone resistance syndromes.60-­62 Imprinting at the GNAS locus results in selective silencing of the paternally-­inherited GNAS allele in some tissues. Patients with mutations on maternally-­ inherited alleles, which are preferentially expressed in the thyroid, pituitary, and renal proximal tubule, develop resistance to parathyroid hormone and other hormones that signal through Gαs-­coupled receptors (pseudohypoparathyroidism type 1A),61 while patients with mutations on paternally-­inherited alleles have AHO without hormone resistance (pseudopseudohypoparathyroidism [PPHP]). Obesity is a well-­recognized feature of PHP when mutations occur on the maternally inherited GNAS allele. Studies in patients presenting with severe childhood-­onset obesity have revealed an unexpectedly high prevalence of GNAS mutations, indicating that screening for mutations in GNAS should be incorporated into the diagnostic workup of severe childhood-­onset obesity.63 Early diagnosis guides monitoring for hormone resistance syndromes, recognition of hypocalcemia as a

CHAPTER 21  Genetic Syndrome Associated with Obesity cause of seizures, and treatment with thyroxine and with recombinant human GH before fusion of the growth plate.62,64 Brain-­specific deletion of the maternal Gαs allele impaired the ability of an MC4R agonist to reduce body weight, suggesting a role for centrally expressed MC4Rs in mediating obesity in GNAS deficiency.65 Recent studies have shown that obesity-­associated missense mutations in GNAS impair MC4R signaling.

Bardet–Biedl Syndrome Bardet–Biedl syndrome (BBS) is an autosomal recessive syndrome characterized by obesity, developmental delay, syndactyly, brachydactyly or polydactyly, retinal dystrophy or pigmentary retinopathy, hypogonadism, and renal abnormalities. BBS is genetically very heterogeneous, with mutations in at least 20 genes reported to date and evidence of triallelic inheritance in some families (two mutations in one BBS gene plus an additional mutation in a second, unlinked BBS gene).66 Most of the genes involved in BBS affect the structure and/or function of the basal body, a modified centriole that is essential for the function of nonmotile cilia.67 There is some evidence that BBS proteins affect leptin signaling, and as such the MC4R agonist setmelanotide (which targets the MC4R pathway downstream of leptin) has been trialed in these patients, with some success.68

Alstrom Syndrome Alstrom syndrome shows some overlap with BBS (retinitis pigmentosa, deafness, obesity, diabetes mellitus with recessive inheritance); however, classically, developmental delay, polydactyly, and hypogonadism are not features. Patients present with a severe visual defect in infancy, photophobia, nystagmus, and loss of central vision. Some affected individuals present with dilated cardiomyopathy (often diagnosed in infancy), hepatic dysfunction, renal dysfunction, hypothyroidism, male hypogonadism, short stature, and mild to moderate developmental delay.69 Although obesity is common in this syndrome, it is rarely severe. In contrast, insulin resistance due to impaired adipose tissue expandability70 is frequently severe, and once diabetes develops, it may be very difficult to control. Hypertriglyceridemia may be severe and may result in acute pancreatitis. Mutations in ALMS1, which is localized to the centrosome and the ciliary basal bodies in vitro, suggest a role in the structure of the basal body or in the transport of proteins between the cytoplasm and the ciliary axoneme.

Cohen Syndrome Cohen syndrome is an autosomal recessive disorder characterized by nonprogressive mild to severe psychomotor retardation, motor clumsiness, microcephaly, characteristic facial features, childhood hypotonia, intermittent isolated neutropenia, and a cheerful disposition. Progressive, often high-­grade myopia and retinochoroidal dystrophy resembling retinitis pigmentosa are essential features in Cohen syndrome.71 Vision starts to deteriorate early but generally is preserved until adulthood, and by the age of 40 years many patients are severely visually impaired. Mutations in COH1 found in some Finnish patients with Cohen syndrome suggest a role in intracellular vesicle–mediated sorting and transport, although the link to obesity has not been established. KEY POINTS  • Monogenic obesity syndromes are characterized by hyperphagia (increased drive to eat) and weight gain that begins before the age of 5 years. Disorders affect hypothalamic pathways involved in the regulation of hormone secretion, and as such patients sometimes have endocrine features such as hypogonadism. Genetic obesity syndromes that affect neural development can be associated with developmental delay and behavioral difficulties.

339

RARE VARIANTS ASSOCIATED WITH OBESITY In addition to these dominant or recessively inherited monogenic obesity syndromes, there is increasing recognition that variants in a broader set of genes contribute to severe obesity. As these variants are rare, statistical evidence of enrichment in obese cases versus controls is often not achieved (power calculations predict that studies involving hundreds of thousands of patients would be needed72). While patients in whom these variants are described often share a set of clinical features, severe obesity is often not inherited in a classical Mendelian manner, and there can be incomplete penetrance and variable expressivity within and across families. Nonetheless, where variants cause a loss of function in cells, affect a mechanism known to regulate weight, and/ or cause obesity when modeled in mice, there is sufficient evidence to indicate that rare obesity-­associated variants contribute to the missing heritability in obesity, as seen for other complex traits.73 Where the molecules encoded by these genes converge on pathways known to be involved in weight regulation, there is sometimes sufficient evidence to treat subsets of patients. The semaphorin 3s are a large family of secreted proteins that have both repulsive and attractive effects on axons during development.74 Recent evidence in mice has shown that SEMA3A guides vomeronasal neurons, which migrate into the hypothalamus to develop into gonadotropin-­releasing hormone–expressing cells, and loss-­of-­ function variants contribute to hypogonadotropic hypogonadism in a non-­Mendelian manner. Semaphorin 3s acting via the neuropilin-­2 receptor direct the development of the melanocortin circuit formed by POMC projections extending from the arcuate to the paraventricular nucleus of the hypothalamus.75 Rare loss-­of-­function variants in semaphorin 3 ligands, receptors, and coreceptors are enriched in severely obese individuals compared with controls.75 Some individuals had learning difficulties, behavioral abnormalities, and neurological disorders including epilepsy, as well as medication-­resistant constipation in childhood. SEMA3 signaling is known to regulate the development of the enteric nervous system, and rare heterozygous loss-­of-­function variants in SEMA3C and SEMA3D have been associated with Hirschsprung disease,76 a disorder characterized by failure of development of parasympathetic ganglion cells in the large intestine. In the arcuate nucleus of the hypothalamus, leptin signaling leads to the phosphorylation of STAT3, which interacts with the transcription factor steroid receptor coactivator (SRC)-­1 to modulate POMC transcription (Fig. 21.4). Disruption of the SRC-­1-­pSTAT3 interaction causes obesity in mice, and rare heterozygous variants in SRC-­1 identified in severely obese people decrease leptin-­pSTAT3 mediated signaling and POMC expression in cells.77 A mouse model of a human loss-­of-­function SRC-­1 variant gains weight on a high-­fat diet. As a result of this evidence, trials are ongoing to investigate whether patients with SRC-­1 deficiency may lose weight with setmelanotide. Nuclear pleckstrin homology domain–interacting protein (PHIP) directly affects the transcription of POMC, and obesity-­associated PHIP mutants have been shown to decrease POMC transcription.78 Heterozygous PHIP deletions and frameshift mutations have been reported in patients with developmental delay, intellectual disability, dysmorphic features, and in some cases obesity.79 The absence of severe obesity in some family members carrying PHIP loss-­of-­function variants, as well as the presence of missense variants in control participants without obesity, suggests variable penetrance. In keeping with the growth phenotype seen in null mice, some, but not all, PHIP variant carriers exhibit low birth weight, reduced linear growth in childhood, hyperphagia, insulin resistance, and early type 2 diabetes. Rare variants in the gene encoding MRAP2, an accessory protein that interacts with MC4R and leads to obesity when disrupted in mice,

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have been associated with severe obesity, although the detailed molecular mechanisms underlying this association are not known.80 To date, most of the genetic obesity syndromes are characterized by hyperphagia as the major driver of the obesity. Loss-­of-­function mutations in kinase suppressor of Ras2 (KSR2) increase food intake but are predominantly associated with reduced basal metabolic rate in the presence of normal thyroid function.81 Some KSR2 mutation carriers experience marked weight loss in childhood when prescribed the antidiabetic drug metformin (for severe insulin resistance). Further work will be needed to see if these observations can be replicated and to investigate the cellular mechanisms underlying these effects. KEY POINTS  • Rare variants that disrupt the function of key genes involved in weight regulation can contribute to severe obesity. Patterns of inheritance may not be clearly Mendelian (e.g., dominant or recessive), with variable penetrance within and between families. Nonetheless, some of these disorders are treatable.

CONCLUSIONS Cumulatively, up to 20% of children with severe obesity have rare chromosomal abnormalities and/or highly penetrant genetic mutations that drive their obesity.78 This figure is likely to increase with wider accessibility to genetic testing and as new genes are identified from exome and genome sequencing. A genetic diagnosis can inform management (many such patients are relatively refractory to weight loss through changes in diet and exercise) and can inform clinical decision-­ making regarding the use of bariatric surgery (feasible in some; high-­ risk in others). Importantly, some genetic obesity syndromes are treatable. Recombinant leptin is dramatically effective at treating the severe obesity of congenital leptin deficiency. Setmelanotide, an MC4R agonist, has been used effectively in phase II/III clinical trials of POMC and LEPR deficiencies and is being explored for the treatment of other genetic obesity syndromes affecting the melanocortin pathway. Ultimately, understanding how these pathways are disrupted in people with weight problems may inform strategies to target these pathways for prevention and treatment.

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81. Pearce LR, Atanassova N, Banton MC, et al. KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation. Cell. 2013;155:765–777.

22 Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise David E. Kelley and Bret H. Goodpaster

OUTLINE Postabsorptive Intermediary Metabolism, 343 Introduction, 343 Resting Energy Expenditure and Postabsorptive Respiratory Quotient, 344 Postabsorptive Glucose Utilization, 345 Postabsorptive Fatty Acid Utilization, 345 Lipolysis and Control of Fatty Acid Availability During ­Postabsorptive Conditions, 347 Fatty Acid Reesterification as a Component of Flux, 348 Postabsorptive Glucose Production, 348 Hormonal Governance of Glucose Production, 349 Glucose Counterregulation, 350 Prandial and Postprandial Intermediary Metabolism, 351 Introduction, 351 Digestion, Absorption, and Enteroendocrine Cells, 351 Insulin and Glucagon Responses, 353

Effects on Energy Expenditure and Respiratory Quotient, 353 Hepatic Glucose Metabolism, 353 Postprandial Peripheral and Central Nervous System Glucose Utilization, 354 Postprandial Adipose Tissue Metabolism, 355 Hepatic Metabolism of Fatty Acids and Lipoproteins, 356 Methodologic Approaches to Investigating Postprandial ­Conditions, 356 Protein and Amino Acid Metabolism, 357 Exercise Metabolism, 358 Exercise Dramatically Increases Demand For Energy, 358 Energy Metabolism During Exercise, 358 Glucose Metabolism During Exercise, 358 Fatty Acid Metabolism During Exercise, 360 Effects of Exercise Training on Intermediary Metabolism, 360 Summary and Concluding Remarks, 360

 

POSTABSORPTIVE INTERMEDIARY METABOLISM Introduction All major body processes require energy, which is provided by intermediary metabolism, the conversion of nutrient-­derived substances into forms that can be combusted to yield energy immediately or after a period of storage, and that collaterally can generate substrate for use in anabolic processes. A useful definition of postabsorptive metabolism is a time when energy needs are being met through mobilization of endogenous substrates, after digestion, absorption, tissue distribution, oxidation, and storage of ingested food has been completed (postprandial metabolism). A typical duration of postprandial metabolism is at least a few hours, as many as 6 or more, depending upon the size and composition of the meal. Accordingly, if one daily meal is consumed, postabsorptive metabolism would stretch for 18 or more hours of a day. More commonly though, two to three meals are consumed in a day, the successive postprandial phases often fuse one into the next (especially for postprandial lipoprotein metabolism), and the postabsorptive phase may comprise as few as 8 hours of the daily cycle of eating and fasting. A striking characteristic of postabsorptive metabolism is that circulating metabolite concentrations remain quite stable, which is sharply different from the dynamic changes that characterize postprandial metabolism. The stability of metabolite concentration is particularly evident for plasma glucose, and somewhat less so for plasma free fatty acids (FFAs), which have a day-­to-­day variance of approximately 30%, as well as a greater variability for intermediary metabolites such as lactate and ketones. Intuitively, it is appreciated that a stability of circulating levels of substrates during postabsorptive metabolism does not

infer metabolic stasis, even though metabolic rates and energy expenditure are at a relative nadir during resting postabsorptive conditions. Rather, constancy of circulating substrate concentrations reflects the underlying presence of tightly governed rates of substrate flux. Flux can be considered regarding a single enzymatic reaction or along an enzymatic pathway, but in the current context substrate flux refers to its rate of appearance into plasma (from endogenous stores and newly synthesized substrate) and rate of utilization, more commonly termed rate of disappearance, from plasma into tissues.1 A persisting stability of substrate concentrations in plasma derives from a well-­balanced equivalency between rates of appearance and disappearance. Briefly, determinations of substrate flux can be performed using an isotopically labeled metabolite and measurement of its dilution in the unlabeled (endogenous) pool. This is a largely a research method rather than a clinically pragmatic determination, but an understanding of the findings, qualitatively and, in select instances, quantitatively, provides mechanistic insights for understanding postabsorptive intermediary metabolism. Physiological governance of flux will be one focus of this section, with emphasis upon its endocrine regulation, and this understanding of flux and interorgan exchange begins with a review of the energy stores of fat, carbohydrate, and protein within the major tissues, as shown in Table 22.1. A second key characteristic of postabsorptive metabolism is an approximate and sustained balance between the oxidation of carbohydrate versus that of lipid. This balance between glucose and fat oxidation is not a true equipoise, as approximately 60% of energy production derives from fat oxidation. But this sustained pattern too is a sharp difference from postprandial metabolism, where there is a pronounced

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TABLE 22.1  Fuel Reserves in a Prototypical

70-­kg Male

AVAILABLE ENERGY (kcal) Organ

Glucose or Glycogen

Free Fatty Acids or Triglycerides

Protein

Blood Liver Brain Adipose Muscle

18 400 8 80 1200

80 450 0 135,000 350

0 400 0 40 24,000

early suppression of fat oxidation and stimulation of glucose oxidation, with a more gradual resumption of fat oxidation and coordinate decline of glucose oxidation as the transition toward postabsorptive metabolism begins to occur. Delineating the tissue responsible for glucose and fat oxidation will also be a focus of this section, and in relation to overall rates of energy expenditure. As a prelude, the concept to be developed is that endocrine governance of substrate oxidation during postabsorptive metabolism is mostly indirect, in the sense that rates of glucose uptake and oxidation are not directly insulin-­stimulated (as opposed

to postprandial conditions), nor is fat oxidation directly controlled by endocrine signals. Instead, glucose uptake and oxidation are largely “insulin-­independent,” and that of fatty acids is substrate-­driven, with endocrine control exerted through governing rates of release of glucose and fatty acids. A schematic depicting the primary systems involved in postabsorptive energy metabolism is shown in Fig. 22.1.

Resting Energy Expenditure and Postabsorptive Respiratory Quotient Energy expenditure is comprised of three principal components: resting energy expenditure (REE), physical activity, and the thermic effects of food ingestion.2 The rate of REE is approximately 1 kcal/min, or approximately 1400 to 1500 kcal per day. REE accounts for approximately 60% of daily energy expenditure, and the thermic effect contributes approximately 10%, but considering that energy expenditure due to physical activity can vary greatly, these proportionalities are generalizations. Notably, REE is proportional to body size, revealing strong linear correlation with fat-­free mass, which is comprised of the more metabolically active tissues. This is similar for fasting rates of hepatic glucose production (HGP; discussed subsequently). Thus, both REE and HGP reveal a vital communication between body composition, the tissues that mostly define energy requirements, and governance of postabsorptive metabolism.3 However, as will also be addressed, the relationship between fat mass and rates of fatty acid flux is less rigorous than that for the relationships of HGP or REE and fat-­free mass.

Ketones Glucose

Glucose

Glycogenolysis

Gluconeogenesis

Lactate Alanine

FFA Glycerol

FFA

Glucagon Insulin

Fig. 22.1  Postabsorptive (fasting) metabolism. The driver of glucose flux during postabsorptive intermediary metabolism is hepatic glucose production, control over which is mediated by a relatively low ratio of insulin to glucagon. Because of low peripheral circulating insulin and in relation to the molecular physiology of tissue glucose transporter, glucose utilization in peripheral tissues is largely insulin-­independent, and, apart from the central nervous system, is lower than the utilization of fatty acids. Low peripheral circulating insulin enables lipolysis in adipose tissue to proceed supporting a rate of free fatty acid (FFA) flux that is roughly double the circulating energy equivalence of that for glucose and underlying the 60% contribution of fat oxidation to resting rates of energy expenditure.

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise Measurement of REE (and the other components of energy expenditure) is most commonly performed by measurement of oxygen consumption and carbon dioxide production using the method of indirect calorimetry. An alternative is a doubly-­labeled water isotopic method that can be used over short intervals in freely living individuals.4 Each approach has advantages and limitations. One of the advantages of indirect calorimetry is that it can provide much more finite or precise temporal resolution of patterns of energy expenditure and substrate oxidation rates. Gas exchange can be measured in dedicated, specifically engineered calorimetry chambers, or less precisely but more accessibly at the bedside with a hood canopy or on a treadmill with a mouthpiece to collect respired air that feeds into gas analyzers. The equations of calculation of energy expenditure are referenced, together with a critique of some of the nuances and limitations of indirect calorimetry.5 Beyond its use in metabolic research, one of the pragmatic clinical uses of indirect calorimetry is in assessing metabolic responses to enteral or parenteral nutrition in critically ill patients. Using these determinations, the quotient of carbon dioxide (CO2) production to oxygen (O2) consumption can be examined; this is termed the respiratory quotient (RQ). Values for RQ denote the relative reliance upon carbohydrate versus lipid oxidation, and with some additional determinations (e.g., urinary nitrogen), the contribution of protein oxidation, usually a modest clarifying adjustment, can also be estimated. Using rates of O2 consumption, CO2 production, and urinary nitrogen, actual rates of glucose and fat oxidation can be calculated. However, to examine concepts germane to integration of glucose and fat oxidation during postabsorptive and postprandial conditions (as well as during exercise), the RQ itself is a useful and straightforward parameter. The principles underlying the RQ stem from the chemical composition of oxidative substrates. Oxidation of glucose (a polymer of six CH2O) to six CO2 and six H2O requires a consumption of an equimolar quantity of six O2; hence, the corresponding RQ has a value of 1.0. Oxidation of fatty acid (a polymer of CH2 units) requires a proportionately greater amount of oxygen for complete combustion and yields approximately in 7 moles of CO2 production relative to a requirement for 10 moles of O2 consumption; hence, the RQ for fat oxidation is 0.70. Intermediate RQ values reflect an admixture of glucose and fat oxidation. In a lean, healthy human, a typical fasting RQ value is in the range of 0.78 to 0.83. It can be estimated from this range of RQ that, during resting postabsorptive conditions, the oxidation of fat provides energy that accounts for approximately 60% of REE. KEY POINTS:  Resting Energy Expenditure • Resting energy expenditure (REE) is approximately 1 kcal/min, 1400–1500 kcal/day, though proportional in amount to fat-free mass, which contains the metabolically active tissues that contribute to REE. • REE generally accounts for approximately 60% of daily total energy expenditure, though the proportion will vary in relation to levels of energy expended in physical activity. • Measurement of oxygen consumption and carbon dioxide production for estimation of energy expenditure also can be used to estimate respective rates of glucose and fat oxidation.

Postabsorptive Glucose Utilization There is a well understood persistent and obligate requirement of the central nervous system (CNS) for glucose uptake from plasma and its oxidation to meet its bioenergetic needs. The brain does not store glycogen, and without continuous glucose delivery becomes deprived of its requisite substrate. This rate of CNS glucose uptake is approximately 70 to 80 mg/min or approximately 1 mg/min-­kg body weight

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of glucose flux, which is about half of the postabsorptive rate of glucose flux (as further described). The predominant glucose transporter of the CNS is GLUT3, a high-­affinity (low Km) glucose transporter, and as such GLUT3 maximizes glucose transport within the normal (i.e., euglycemic) range of fasting plasma glucose.6 CNS glucose uptake is not further increased at higher plasma concentrations, as during postprandial conditions. As a further consequence of this molecular physiology of GLUT3, CNS glucose uptake can become critically reduced with a 25% to 30% reduction in fasting plasma glucose concentrations, as there is a sharp decline in CNS glucose uptake as plasma glucose concentration declines to levels lower than this. Due to the properties of GLUT3, glucose uptake and oxidation by the brain is categorized as “insulin-­independent,” as is the great majority of the remaining glucose flux during postabsorptive conditions. Other than the brain, there is no single peripheral tissue that predominates in rates of postabsorptive glucose uptake; instead, glucose uptake is dispersed somewhat evenly across tissues, each at a relatively low rate and with an obligate need by red blood cells. Only a minor proportion of the peripheral tissues’ (i.e., non-­CNS) directed glucose flux, also approximately 1 mg/kg-­min, undergoes oxidation; instead, it is mostly metabolized by glycolysis to the level of trioses (e.g., pyruvate and interconversion to lactate), while some undergoes a transamination reaction to form alanine, and these trioses reenter circulation as part of an interorgan exchange. A major fate is as a substrate for gluconeogenesis in the liver, completing an ongoing, recurring cycle within postabsorptive metabolism. KEY POINTS: Molecular Physiology of Glucose Transporters as a Determinant of Glucose Utilization During Postabsorptive Conditions • GLUT3, expressed in the CNS, is a high-affinity isoform that maximizes glucose uptake across the normal range of fasting glucose and does not further increase as blood glucose rises. • GLUT4, expressed in skeletal muscle, heart, and adipose tissue, is sequestered in cytosolic vesicles under resting postabsorptive conditions, leading to low rates of glucose use by these tissues. • GLUT2, expressed in liver and in pancreatic β cells, is a low-affinity isoform that enables blood glucose–dependent rates of transport, an attribute that further serves as a glucose sensor within these tissues that have key roles in responding to blood glucose changes.

Postabsorptive Fatty Acid Utilization Nearly all tissues and organs have a capacity to oxidize fatty acids, with notable exceptions being red blood cells that lack mitochondria and the CNS, due to inability of fatty acids to traverse the blood–brain barrier. Kidney, heart, and liver are key tissues accounting for uptake and oxidation of fatty acids during postabsorptive metabolism, as is skeletal muscle. The persistent reliance of the CNS on glucose oxidation has already been stated, and, in an integrated physiology context, it is fat oxidation that accounts for a major proportion of energy production in peripheral tissues during postabsorptive conditions; as noted earlier, it accounts for approximately 60% of REE. It is worthwhile to delve a bit deeper into skeletal muscle metabolism, as an example of a tissue that has a high postabsorptive utilization of fatty acids and yet also has a high capacity for glucose utilization, a capacity to transition or toggle between fuels that is manifest by other tissues, though perhaps not in as pronounced a manner as skeletal muscle. Thus, skeletal muscle has a robust capacity to “switch” fuels, and this metabolic flexibility is an important manifestation of metabolic health,7,8 in appraising governance and response in transitions between postabsorptive and postprandial metabolism. The governing of this switching of fuels will be more fully addressed later.

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During resting postabsorptive conditions, skeletal muscle has a quite low rate of glucose uptake and relies instead chiefly upon fatty acid oxidation. It has been estimated, based on arteriovenous differences across an arm or a leg (tissue beds that are predominated by, though not exclusively comprised of, skeletal muscle) that fractional extraction of plasma glucose is only 1% to 2%, whereas that for plasma fatty acids is approximately 30% to 35% during resting postabsorptive conditions.9 The low rate of glucose usage by skeletal muscle during fasting conditions is attributable to low circulating insulin levels during postabsorptive conditions, inadequate to stimulate glucose uptake by skeletal muscle. Moreover, during fasting conditions there is a net efflux of pyruvate, lactate, and alanine from skeletal muscle, that as an aggregate molar amount of efflux approximates the uptake of glucose, leaving a net zero balance for glucose-­derived carbons. The uptake and glycolytic metabolism of glucose by skeletal muscle, returning lactate to venous blood and for uptake in the liver as a substrate for gluconeogenesis, represents the well-­described Cori cycle.10 There is as well a low intrinsic rate of glycogenolysis in resting skeletal muscle, which contrasts with more active postabsorptive glycogenolysis in liver. Though muscle glycogen reserves, which in aggregate approximate 400 g (dry weight), are abundant and constitute approximately 80% of systemic glycogen reserves, the pool is held for mobilization in response to physical activity, especially strenuous exercise, as will be delineated later. Furthermore, skeletal muscle lacks glucose-­6-­phosphatase and cannot release free glucose. A series of now classic human physiology studies identified that fatty acid oxidation is the principal energy source of resting skeletal muscle during postabsorptive conditions.11 This finding was based upon the measurement of the RQ across the forearm, based on regional determinations of O2 uptake and CO2 production across the limb (rather than the systemic gas exchange earlier described). An RQ value in the range of 0.70 to 0.76 was observed in healthy, lean individuals, lower than systemic RQ (not surprising given the exclusion of CNS glucose oxidation from the regional measurement) and denoting a nearly exclusive reliance upon fat oxidation. Skeletal muscle, like most nonadipose tissues, does contain lipid droplets (LDs), comprised mostly of triglyceride (TG), and fatty acids can be mobilized from these droplets, as well as exchange with the TG in the droplets on uptake into myocytes, and uptake of plasma FFAs is the principal source for oxidation during resting postabsorptive conditions. The uptake of FFAs into skeletal muscle and other tissues is not directly governed by hormonal actions at the sarcolemma, unlike the direct actions of insulin to stimulate glucose transport into muscle; rather, endocrine governance of fatty acid uptake into muscle and other tissues is indirect, mediated by endocrine control of lipolysis, and hence governance of plasma FFA concentrations. Plasma concentration of FFAs is the key factor governing rates of uptake into muscle and other tissues. During postabsorptive conditions, a typical circulating concentration of plasma FFAs is 0.5 mmol/L, or approximately one tenth the molar concentration of plasma glucose. Moreover, FFAs, being strongly hydrophobic molecules, circulate noncovalently bound to albumin; there are generally one to three fatty acids bound per albumin molecule at concentrations common in postabsorptive conditions. Fatty acids taken up by tissues are from the much smaller “free fraction” of unbound fatty acids that exists in equilibrium with albumin-­bound fatty acids. A comparison of the respective concentrations of glucose and FFAs in plasma (e.g., 5.0 vs. 0.5 mmol/L, respectively) as an index of relative availability during postabsorptive metabolism is somewhat misleading, because it overlooks the respective underlying rates of glucose and FFA flux. As earlier noted, a typical postabsorptive rate of plasma glucose flux is 2 mg/min-­kg body weight (10 μmol/min-­kg body weight; in aggregate approximately 700 μmol/min for a 70-­kg individual). The

rate of appearance of fatty acids into plasma is not too dissimilar, at approximately 1.7 mg/min-­kg (6–8 μmol/min-­kg body weight; in aggregate approximately 400–500 μmol/min in a 70-­kg individual).12,13 This rough equivalency in flux, somewhat lower for fatty acids than for glucose, reflects a more rapid turnover rate (shorter half-­life) for FFAs than for glucose; the half-­lives are 2 minutes and 15 minutes, respectively, for plasma FFAs and glucose. Also, the higher energy density of FFA relative to glucose (9 compared with 4 kcal/g, respectively) means that the potential circulating energy yield of FFA is nearly twice that of glucose. Taking each of these factors into consideration and recognizing the considerably more rapid turnover rate of the plasma FFA pool than that of glucose, it can be more clearly understood how it is that the oxidation of plasma FFAs accounts for approximately 60% of REE during postabsorptive conditions, despite an order of magnitude difference in plasma concentrations and an exclusive use of glucose by the CNS. To delve a bit further into postabsorptive kinetics of glucose and fatty acid flux, it was earlier pointed out that rates of fasting HGP are linearly proportional to body weight, and more tightly so to fat-­free mass. There is a linear relationship between postabsorptive rates of fatty acid rate of appearance and fat mass, the latter reflecting of course the tissue of origin from which fatty acids are released via lipolysis. This correlation accounts for roughly 40% of the interindividual variance in postabsorptive fatty acid flux, at least across a cohort of men and women ranging from lean to obese,14 so it is an association of moderate strength only. Also, of interest, there is a rather strong negative, curvilinear correlation between postabsorptive fatty acid flux (normalized to per kg fat mass) and obesity, suggesting that the rate of release of fatty acid from adipose, per unit weight of fat, is reduced in obese relative to leaner individuals. Another way to express this is that, in lean individuals, the mobilization rate (i.e., lipolytic rate) per kilogram of fat mass is substantially accelerated compared with that in obese individuals. Yet, despite this, plasma fatty acid levels tend to be somewhat increased in obese compared with lean individuals, and thus, even though there appears to be a per unit of fat greater stringency in control of lipolysis, this does not appear to fully compensate for the overall expansion of fat mass. There are also findings indicating that body fat topography is associated with variances in rates of postabsorptive fatty acid flux, with higher rates in obese women who have an upper body fat distribution compared with obese women with a lower body fat distribution, the latter not differing in postabsorptive fatty acid flux from lean women.15 At the cellular level, unbound FFAs enter a cell down a concentration gradient (plasma to cytosol) by “flip-­flop” diffusion across the cell membrane, and there is facilitation by tissue-­specific families of fatty acid transporters. Once within the cytosol, fatty acids noncovalently bind to fatty acid–binding proteins, which protects the cell from detergent-­like, damaging properties of unbound FFAs. Intracellular fatty acids are quickly “activated” by esterification to CoA, to form fatty acyl-­CoA, an adenine triphosphate (ATP) requiring reaction catalyzed by the enzyme acyl-­CoA-­synthase, a reaction that is essential in maintaining a concentration gradient relative to plasma. Fatty acyl-­CoA can be partitioned toward reesterification to form TG in LDs or other pathways of lipid synthesis (e.g., phospholipids, sphingolipids), but during postabsorptive conditions a considerable fraction are partitioned toward β-­oxidation. β-­oxidation is a repeating four-­step catabolic set of enzymatic reactions that, with each cycle, progressively cleaves the full-­length fatty acid into two-­carbon fragments, acetyl-­ CoA moieties, which can then enter the TCA cycle to complete final oxidation. Both processes occur within the mitochondria matrix, yet fatty acyl-­CoAs are unable to diffuse across the outer and inner mitochondrial membranes, which possess a phospholipid composition unique from that of the cell membrane. Instead of diffusion, a specific a three-­component transport complex, the carnitine-­palmitoyl transferase complex (CPT), mediates this entry of fatty acyl-­CoA. Greater

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise details of the intracellular catabolism of fatty acids are beyond the scope of this chapter, but the biochemistry is well described.16 There is one aspect of this molecular physiology that warrants highlighting, because it is a fulcrum for the governance of fat oxidation, and hence pivotal as well in the governance of intermediary metabolism. This aspect is the role of malonyl CoA in allosteric modulation of the activity of CPT-­1, the enzyme that mediates transfer of an acyl moiety from acyl-­CoA to carnitine, and it is the carnitine-­acyl that is shuttled into the mitochondrial matrix, where acyl-­CoA is reformed before entry into the β-­oxidation cycle. The activity of CPT-­1 is strongly inhibited by malonyl CoA, though there are some tissue differences in this sensitivity, with the isoform of CPT-­1 in skeletal muscle being more sensitive to malonyl CoA inhibition than the isoform in liver. Malonyl CoA is a crucial intermediate in the process of de novo lipogenesis, derived in turn from a surfeit of acetyl-­CoA, generated by glycolysis and hence conversion from pyruvate. Even in tissues like skeletal muscle that have a low overall capacity for de novo lipogenesis, the generation of malonyl CoA serves an important substrate-­based signaling function in a reciprocal on-­off toggling of glucose and fatty acid oxidation.17 During postabsorptive conditions, malonyl CoA concentrations in skeletal muscle (and other tissues) are low, and CPT-­1 is unimpeded, and this, together with relatively avid uptake of plasma fatty acids, drives fat oxidation. Glucose oxidation is low, due primarily to the low rates of glucose uptake and glycolysis, and activity of the pyruvate dehydrogenase complex, which catalyzes formation of acetyl-­CoA (for entry into the TCA cycle) from pyruvate upon the latter’s entry into mitochondria, is correspondingly low as compared with insulin-­ stimulated conditions. In the liver, the good availability of fatty acids during postabsorptive conditions, low concentrations of malonyl CoA, and, thus, relatively unimpeded CPT complex activity, create conditions wherein a surfeit of acetyl-­CoA derived from β-­oxidation of acyl-­CoA can give rise to ketogenesis. Ketone bodies are the most water-­soluble form of lipid-­derived metabolites and are formed in the liver from the condensation of two acetyl-­CoAs. Ketones (acetoacetate and β-­hydroxybutyrate) can be utilized by numerous tissues and production rates, and blood concentrations rise with prolongation of fasting. With prolonged fasting, the CNS adapts for utilization of ketones, which provides an additional mechanism for sparing the use of glucose. Clinically, in severe insulin deficiency, accelerated rates of ketone production cause diabetic ketoacidosis. KEY POINTS: Postabsorptive Fatty Acid Rate of Appearance and Utilization • Usual concentrations of plasma fatty acids are one tenth of those of plasma glucose, but the rate of appearance (in mg/min-kg body weight) is roughly equivalent to glucose, and the energy yield is nearly 2-fold higher than for glucose. • The half-life of plasma fatty acids is just 2 min, nearly an 8-fold more rapid turnover of plasma fatty acids than glucose. • As a fraction of stored triglyceride in adipose tissue, the rate of lipolysis is a miniscule fraction (∼10-5), a thousandth of the rate of release of hepatic glycogen, reflecting tight, minute-to-minute governance of lipolysis. • The endocrine control of lipolysis mostly represent the opposing actions of insulin (restraining) and catecholamines (stimulating) lipolysis.

Lipolysis and Control of Fatty Acid Availability During Postabsorptive Conditions In the introduction to this section on postabsorptive intermediary metabolism, emphasis was placed upon the stability of fasting substrate concentrations, especially that of plasma glucose. In comparison, greater variability on a day-­to-­day basis is observed for plasma

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concentration of fatty acids, and this greater variability relative to glucose can also be observed within any given interval of postabsorptive metabolism, wherein fluctuations of plasma fatty acid concentrations are more evident than those for glucose. However, a conclusion that governance of fasting rates of fatty acid flux is less stringent than that of fasting rates of glucose flux is arguably incorrect, and the context to refute this notion is outlined here. A first context is to consider the size of the potential fatty acid pool (i.e., the reservoir of stored TG) and the extent to which a fasting rate of fatty acid flux represents a fractional turnover of this pool. Second, the rapid turnover rate of plasma fatty acids (half-­life of approximately 2 minutes) is such that any modulation of rates of appearance of fatty acids quickly resettles into a near steady-­state equilibrium and can accordingly manifest as a change in fatty acid concentration. Thus, a change in plasma fatty acids reveals acute changes in the level of control of lipolysis, and perhaps as well variance in rates of clearance by tissues of utilization, not per se a loss or absence of control. Third, and as will be delineated next, the specific steps controlling lipolysis are multifaceted and complex and, while still very much an area of new discoveries, denote intricate governance.18 Even in a lean individual there is a considerable reserve of stored energy in the form of TG in white adipose tissue. In a 70-kg lean adult, fat mass comprises approximately 10% of body weight in a man and somewhat more, approximately 30% in a healthy lean woman; certainly, these percentages are much greater in overweight and obese men and women. Each gram of fat, fully combusted, yields 9 kcal; thus, 7 kg of fat mass in a lean, 70-kg male or 20 kg of fat mass in a lean, 70-kg woman equates to a potential energy reserve (estimated at 7,700 kcal/kg adipose) of 77,000 kcal and 231,000 kcal, respectively, in a man or a woman. This far exceeds the caloric equivalent of 400 kcal corresponding to a replete hepatic glycogen content of 100 g and the 1600 kcal corresponding to a replete skeletal muscle glycogen content of 400 g. Thus, a fatty acid flux of approximately 120 mg/min in a 70-­kg adult represents a small minute-­to-­minute fractional release (turnover) of the 10 to 15 kg of adipose tissue in a man or woman. In white adipose tissue, adipocytes contain one large LD with a narrow perimeter of cytosol and organelles. The hydrophobic core of the LD is largely TG, its surface covered by a phospholipid monolayer coated with protein chaperones, notably perilipins, that can, in a regulated manner, constrain access by lipases to TG.18 There are three key lipases that can carry out lipolysis of TG in a LD. These start with adipose triglyceride lipase (ATGL): TG is its preferred substrate, its activity is modulated by a protein cofactor, release of which is governed by and related to conformational changes in perilipins induced by endocrine stimulators of lipolysis, and its reaction yields a fatty acid and diacylglycerol. Upon the latter, hormone-­sensitive lipase (HSL) next acts to yield a fatty acid and monoacylglycerol, and HSL activity, which has been well-­studied, is increased by endocrine stimulators of lipolysis and inhibited by insulin, the endocrine suppressor of lipolysis. The third lipase is monoacylglycerol lipase, which cleaves the remaining fatty acid and thereby also yields glycerol. The overall yield of lipolysis is three fatty acids and free glycerol. Free glycerol exits the adipocyte and serves as a gluconeogenic precursor in the liver. Increased cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate second messengers generated by catecholamines, growth hormone, atrial natriuretic peptide, and potentially glucagon (though the physiologic relevance of glucagon in vivo is unclear) signal transduction, and are the endocrine stimulators of lipolysis. These regulate reversible phosphorylation that induces conformational changes in perilipins and HSL activation. Insulin signaling induces the opposite set of responses. It is worth noting that, while catecholamines can stimulate lipolysis in adipose tissues and glucose production by the liver, the adipose response is more sensitive. This also

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applies to the suppressive effects of insulin: adipose is more responsive compared with liver. In plasma, fat circulates in two forms, as FFAs and as TG contained in lipoproteins; most of this circulating TG during postprandial conditions is contained in very low density lipoproteins (VLDL) and with the addition of chylomicrons during postprandial conditions. Lipoprotein metabolism is addressed comprehensively in Chapter 25. TG catabolism certainly occurs during postabsorptive conditions and can accordingly supply fatty acids for oxidative metabolism, but a large majority of fat oxidation, perhaps as great as 90% during postabsorptive conditions, derives from circulating fatty acids. Brown adipose tissue too contains LDs, and LDs are found in numerous nonadipose tissues, but in aggregate these are minor reserves relative to white adipose tissue, and in general the fatty acids liberated from these LDs are consumed within the tissues in which these are present and not released into circulation, analogous therefore to the situation with muscle glycogen. KEY POINTS:  Lipolysis of Triglyceride Contained in the Adipocyte Lipid Droplet • In addition to the phospholipid monolayer of lipid droplets (LDs), perilipin proteins are on the surface of LDs and can limit access of lipases to the TG contained in the core of LDs. • In response to endocrine stimulators of lipolysis (catecholamines, growth hormone, atrial natriuretic peptide), the increase in second messenger cyclic adenosine monophosphate (or cyclic guanosine monophosphate) induces conformation changes in the perilipin coating of LD and makes accessible the protein cofactor for activation of adipose triglyceride lipase, the lipase that cleaves free one fatty acid to generate diacylglycerol. • Stimulators of lipolysis also activate hormone-sensitive lipase (HSL), which cleaves a fatty acid from diacylglycerol; the activation of HSL and its inactivation by insulin (the sole endocrine suppressor of lipolysis) is a fulcrum of control over rates of lipolysis. • The remaining fatty acid is cleaved by the action of monoacylglycerol lipase, and this too generates free glycerol. Adipose tissue lacks glycerol kinase, so glycerol liberated by lipolysis cannot be activated to form glycerol-3-phosphate, and thus cannot form a new backbone for TG synthesis. Instead, glycerol freed by lipolysis exits adipose to serve as a gluconeogenic precursor. Glycerol-3-phosphate for TG synthesis is derived from glycolysis in the adipocyte.

Fatty Acid Reesterification as a Component of Flux There is another aspect of fatty acid flux that is germane to understanding postabsorptive intermediary metabolism, and this is fatty acid reesterification (to TG). Fatty acid esterification, an anabolic process, is the predominant pathway in adipocytes during postprandial conditions (when lipolysis is strongly suppressed), but some partitioning of newly liberated fatty acids to reesterification occurs in adipocytes during postabsorptive conditions, though mostly this takes place in other organs, notably the liver, to which fatty acids are taken up, and fatty acyl-­CoAs are partitioned between pathways of esterification and oxidation. Theoretically, following lipolysis of TG, a molar 3:1 ratio of fatty acids to glycerol release from adipose tissue would be anticipated, but the observed ratio is closer to 2 to 2.5:1, denoting an incomplete release of fatty acids. Glycerol newly liberated from TG is released into circulation (and serves as a gluconeogenic precursor in liver), because adipocytes lack glycerol kinase to catalyze the formation of glycerol-­ 3-­phosphate (G-­3P) from glycerol. Instead, G-­3P needs to be generated from the glycolytic intermediate dihydroxyacetone, which in turn depends upon ongoing glucose uptake into adipocytes (which contain scant glycogen and do not carry out gluconeogenesis). When G-­3P is

available, a portion of newly released fatty acids undergo reesterification into TGs, constituting a futile cycle in this pathway. After release into circulation, a substantial portion of circulating fatty acids is oxidized, and this fraction is estimated to represent approximately one half (somewhat less) of fatty acid flux under resting postabsorptive conditions. The balance undergoes reesterification; in the liver this contributes to TG packaged into VLDL as well as LD TG.13 A portion contributes substrate to other pathways of lipid anabolic metabolism, such as phospholipid and sphingolipid synthesis. Considering the “excess” portion of plasma fatty acids, that portion otherwise destined to be partitioned toward reesterification, this portion in the plasma pool can be perceived as an immediately available “reserve” that can quickly “diverted” to meet an arising need for greater fat oxidation, if energy needs dictates, as for instance during the onset of physical activity. This ephemeral plasma pool is more immediately available than allowing for a time lag for mobilization of more fatty acids via an uptick in rates of adipose lipolysis and essentially buys time for this to manifest in increased fatty acid rate of appearance.

Postabsorptive Glucose Production In a healthy 70-­kg human, during postabsorptive conditions, there is at any moment approximately 4 g of glucose in circulation,19 a reserve representing 16 kcal of available energy (∼4 kcal/g glucose). Depletion of just a third of this amount leads to compromised CNS function and morbidity. Thus, the circulating 4 g of glucose is a deceptively thin reserve; consider that this is the glucose content of a “lifesaver” candy. Yet hypoglycemia rarely, if ever, occurs in an otherwise healthy individual, and circulating glucose is replenished continuously by HGP. HGP is governed judiciously, such that the fasting level of plasma glucose is maintained within a tight and stable range, and there are several pathways of metabolism that converge to constitute HGP.20 In addition to HGP, there is a contribution from the kidneys, estimated to account for 10% to 15% of postabsorptive glucose production,21 that is further addressed later. Together, the renal and hepatic contribution comprise endogenous glucose production, a term that somewhat erroneously but commonly is used interchangeably with HGP. During postabsorptive conditions, the rate of HGP is approximately 2 mg/min-­kg body weight (or 140 mg/min for the 70-­kg human). At the transition into postabsorptive metabolism, if the preceding meals fulfilled daily caloric requirements, hepatic glycogen is in a replete state, at a concentration of approximately 100 g (dry weight glycogen, several fold higher due to the hydrated state of glycogen within tissues). Approximately 50% to 60% of HGP can be attributed to hepatic glycogenolysis as postabsorptive metabolism commences. Glycogenolysis is a tightly governed process, acutely regulated by endocrine signaling that coordinates an opposing reciprocal regulation of glycogen synthesis, so that there is little futile cycling of the glycogen pool. Glucagon and catecholamines, through cAMP, activate PKA, which phosphorylates (and activates) glycogen phosphorylase, while also phosphorylating (and inhibiting) glycogen synthase. Hyperglycemia, through allosteric actions of intracellular glucose in the hepatocyte, can inhibit glycogen phosphorylase. Insulin signaling can directly inhibit glycogenolysis, through an oppositely directed impact on reversible enzyme phosphorylation; thus, the fulcrum of control of glycogenolysis is the opposing actions (and relative concentrations) of glucagon and insulin, and with allosteric modulation by glucose. The action of glucose-­6 phosphorylase yields free glucose, which is released into the venous circulation, and the glucose transporter isoform of the liver is GLUT2, a high-­Km isoform that enables rapid transmembrane equilibration of glucose concentrations.6 Together, GLUT2 with the hexokinase isoform of the liver (glucokinase) serve as a “glucose sensor” for the liver. This is also a pairing that acts as a glucose sensor in β cells of the pancreatic islet.

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise Approximately half of HGP can be ascribed to gluconeogenesis, the synthesis of new glucose molecules, using as substrate principally pyruvate, lactate, and alanine, as well as glycerol derived from lipolysis. The biochemistry of gluconeogenesis is largely a reversal of glycolysis, and the detailed biochemistry of this pathway, futile cycles within glycolysis and gluconeogenesis, and other aspects have been carefully delineated.22 Control of gluconeogenesis is somewhat complex and entails components attributable to “substrate push” (i.e., availability of the above triose precursors), direct control by insulin, indirect control by insulin (through governing precursor availability), interaction with opposing effects of glucagon on hepatocytes, and longer-­term transcriptional control over the expression of enzymes that catalyze gluconeogenesis. There is also support for the concept that insulin signaling in the CNS can exert importance governance over rates of HGP. Because of the importance of dysregulated control of gluconeogenesis to the pathogenesis of hyperglycemia in diabetes mellitus, and due to ongoing debate on the key site(s) of control of gluconeogenesis, the interested reader is provided several excellent references for deeper examination of the issues.20,23-­25 Hepatic oxidation of fatty acids, the principal oxidative substrate for the liver during postabsorptive conditions, provides the energy needed for gluconeogenesis, though fatty acid metabolites (i.e., acetyl-­CoA) cannot directly provide substrate for gluconeogenesis. There is evidence that the peripheral action of insulin in inhibiting lipolysis is important in concomitantly controlling rates of gluconeogenesis, both in relation to limiting provision of glycerol as a gluconeogenic substrate and in relation to limiting provision of fatty acid, as an energy source, to the liver. This concept has been termed the “single gateway hypothesis,”23 linking insulin control of lipolysis as a nexus by which control over gluconeogenesis is exerted. While glucagon has a strong acute effect to stimulate glycogenolysis, it also, through induction of cAMP and PKA activation, has a stimulatory effect on gluconeogenesis, mediated by reversible phosphorylation of enzymes in the pathway of gluconeogenesis. Both insulin and glucagon also mediate effects by influencing transcription control of rate-­ limiting enzymes in the pathway of gluconeogenesis. Under usual daily conditions of feeding and fasting, postabsorptive metabolism prevails for 10 to 12 hours, and even this relatively short interval of fasting can result in a meaningful mobilization and depletion of hepatic glycogen (approximately 5 g per hour), especially if there is an interposed session of physical activity. Using the data provided earlier for HGP and fatty acid flux, it can be estimated that the fractional turnover (catalysis) of hepatic glycogen is approximately 0.07% per minute, a relatively small rate of breakdown, but still far greater than the estimated 0.001% per minute fractional turnover of the adipose TG pool. Nonetheless, if glycogenolysis was to proceed at a monotonic rate as postabsorptive metabolism extended, even a liver replete with glycogen would be markedly reduced within 24 hours of fasting. However, hepatic glycogenolysis does not proceed at a fixed, monotonic rate as fasting continues; instead, the proportionality between glycogenolysis and gluconeogenesis (approximately fifty-­fifty proportionality at commencement of postabsorptive metabolism) modulates toward a lesser contribution of glycogenolysis and a larger contribution of gluconeogenesis as the duration of postabsorptive metabolism is extended. Regardless, hepatic glycogen is completely depleted within 48 hours of fasting. This adaptive shift, which serves to conserve remaining hepatic glycogen, occurs while largely maintaining the overall rate of endogenous glucose production. Another aspect of adaptation to a lengthening of the usual period of fasting (e.g., 8–12 hours in a daily cycle) is an increased proportion of endogenous glucose production contributed by renal gluconeogenesis. The contribution of the kidneys to glucose homeostasis has three

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components: (1) renal gluconeogenesis; (2) renal utilization of glucose, especially in the renal medulla; and (3) renal resorption of filtered glucose, which is approximately 180 g per day in a healthy (nondiabetic) individual.21 Therefore, it is the last of these, resorption of filtered glucose, that is the largest of the renal effects on glucose homeostasis. However, the focus here will be on renal gluconeogenesis. Like the liver, the kidney expresses glucose-­6-­phosphatase; these are the only two organs to do so and the only two organs that are capable of gluconeogenesis. Renal gluconeogenesis occurs within proximal tubular cells, which have high energy needs related to ion transport and glucose resorption and a high reliance upon fatty acid oxidation.26 At the start of postabsorptive metabolism, the renal contribution to endogenous glucose production is estimated to comprise approximately 10% to 15%, all derived from gluconeogenesis. As the duration of fasting extends, especially past 48 hours, which causes complete depletion of hepatic glycogen, the relative contribution of renal gluconeogenesis rises to nearly 40% of endogenous glucoses production,27 in essence replacing the now exhausted contribution earlier made from hepatic glycogen. To this is added the glucose-­sparing effects of ketogenesis and increased rates of lipolysis with fasting. Parenthetically, the last citation is regarded as one of the classic physiologic papers addressing the adaptation of intermediary metabolism to prolonged fasting. Glutamine is an important substrate for renal gluconeogenesis, unlike the liver, which has a greater reliance upon alanine, though both liver and kidney use lactate as a precursor. Another difference is that glucagon is considered to have little effect on renal gluconeogenesis, and instead catecholamines, partly or mostly through influencing availability of substrate precursors, affects rates of renal gluconeogenesis. KEY POINTS:  Postabsorptive Glucose Rates of Appearance • The rate of appearance for glucose is approximately 2 mg/min-kg body weight. • 1 mg/min-kg is used by the central nervous system (CNS) and oxidized. • The remaining half is distributed broadly across peripheral tissues and mostly metabolized only by glycolysis to trioses (pyruvate and lactate), which are recycled to liver as gluconeogenic precursors. • The size of the blood glucose pool is approximately 4 g, a thin reserve equivalent to the glucose content of a “lifesaver” candy, and the turnover is relatively brisk, with a half-life for glucose of approximately 15 min.

Hormonal Governance of Glucose Production The opposing actions of insulin and glucagon signaling on hepatocytes are the crucial endocrine regulators of HGP during postabsorptive conditions and have earlier been described with respect to specific pathways involved in HGP. During postabsorptive conditions, the action of insulin serves as a brake upon rates of glucose production, while glucagon serves as an accelerator of glucose production. As earlier noted, glucose utilization during postabsorptive conditions can properly be construed as “insulin-­independent,” and instead it is the action of insulin in governing HGP that is the way insulin governs rates of postabsorptive glucose flux. Thus, during postabsorptive conditions (and in sharp contrast to postprandial conditions or feeding), the physiological role of insulin is more accurately perceived as restraining or controlling rates of catabolism rather than as a stimulating anabolic influence, checking against excessively high release of stored glycogen and of excessive rates of lipolysis of TG in adipose tissue. Pancreatic β cells are astute and sensitive glucose sensors, and even slight increases in circulating glucose evoke increased insulin secretion

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into the portal venous circulation. Conversely, subtle declines of plasma glucose induce corresponding declines of insulin secretion. GLUT2, a high-­Km isoform that enables rapid transmembrane equilibration of glucose concentrations, together with glucokinase, serves as a nexus for “glucose sensing” in β cells, though downstream glucose metabolism is essential for triggering insulin secretion. It should also be noted that increased fatty acids can acutely stimulate insulin secretion (though long-­term exposure to elevated fatty acids has untoward effects), as can amino acids, so β cells are tightly tuned to intermediary metabolism.28 Throughout this chapter emphasis is given to how rates of substrate utilization in tissues are governed by underlying energy expenditure, but in this respect the β cell differs in that its rate of substrate uptake and metabolism is largely a function of substrate availability. This “push” aspect of substrate utilization underlies the “gluco-­stat” sensing function of the β cell in its governance of intermediary metabolism. Another important consideration is that approximately 50% of secreted insulin is extracted by the liver.29 This fraction varies between people and has been estimated to range as high as 80% (though a value nearer 50% is more commonly observed) and to be manifest in part as a heritable factor. Clearly, hepatic first-­pass insulin extraction is a key determinant of the amount of exposure of peripheral tissues to insulin at any given level of insulin secretion; the portal to peripheral gradient for insulin concentration is approximately 3:1. A second important consideration is that insulin secretion is pulsatile30; the pulses of insulin secretion have a periodicity of approximately 5 to 7 minutes and comprise most of the postabsorptive insulin secretion. It is thought that calcium oscillations, perhaps synchronized with glycolytic oscillations, are a mechanism underlying pulsatile insulin secretion. Although some debate continues, the pulsatile delivery of insulin is regarded as contributing to the effectiveness of insulin action in the liver; in the periphery, these pulses are greatly extinguished. Characteristically in a metabolically healthy individual, there is a gradient in the insulin responsiveness of adipose, liver, and skeletal muscle, three key insulin-­responsive tissues.31 This cross-­tissue gradient of insulin action shapes the pattern of postabsorptive metabolism (as well as governing postprandial metabolism, as will be addressed later). Adipose tissue manifests a high level of responsiveness to insulin, a greater “insulin sensitivity” than liver, as reflected in a left-­shifted dose response for suppression of lipolysis relative to suppression of hepatic glucose production. Furthermore, liver is more insulin-­sensitive than skeletal muscle, as reflected in a left-­shifted dose-­response for suppression of hepatic glucose production relative to stimulation of glucose uptake into muscle. Taking this gradient of insulin sensitivity into consideration (adipose > hepatic >> skeletal muscle), together with consideration of the portal delivery and large hepatic extraction of insulin, creating a sharp 3:1 portal to peripheral gradient of insulin concentration, a mechanistic basis can be appreciated, in that adipose and liver are the chief sites of insulin action during postabsorptive conditions, and that skeletal muscle metabolism during postabsorptive conditions is not strongly shaped by insulin action. Pancreatic α cells are also astute and sensitive glucose sensors, but with opposite patterns of response to β cells, and even slight decreases in circulating glucose evoke proportionate, stepwise increases in glucagon secretion. There is a nearly linear response of increased hepatic glucose production to increases of portal venous glucagon.32 And because changes in circulating glucose produce reciprocal responses of insulin and glucagon, these opposing changes in hormone concentration, typically quite subtle (at least as measurable in peripheral

circulation), nonetheless achieve an amplifying physiological effect on hepatic glucose production. KEY POINTS:  Hepatic glucose production during postabsorptive metabolism: • The rate of hepatic glucose production (HGP) after an overnight fast is approximately 2 mg/min-­kg body weight, about 65% of which is contributed by glycogenolysis and the remaining 35% is contributed by gluconeogenesis. • Insulin and glucagon are the key endocrine regulators of HGP. In fact, during postabsorptive conditions, the main action of insulin is as a brake upon HGP and lipolysis as glucose utilization is nearly entirely “insulin-­independent”. • Glucagon, secreted into the portal circulation like insulin, is a potent stimulator of HGP and a crucial component of the endocrine response that comprises the hypoglycemic counter-­regulatory response. • With an extended period of postabsorptive conditions, the liver adapts to rely more upon gluconeogenesis and less upon glycogenolysis to provision HGP.

Glucose Counterregulation Across a lifespan of 80 years or more, assuming mostly good general health, an individual will consume 80,000 meals (with corresponding ascents and descents of plasma glucose) and pass through 30,000 postabsorptive periods, yet never experience an episode of symptomatic hypoglycemia. In fact, an occurrence of hypoglycemia, especially during postabsorptive conditions, is never accepted as normal and instead heralds underlying disease, a dangerous adverse drug effect, or other discoverable extenuating circumstances. While the reciprocal actions of insulin and glucagon provide a foundational understanding of this extraordinary protection against hypoglycemia, there is a multifaceted hormonal counterregulatory response that is a vital additional buttress.33,34 The normative counterregulatory response has been investigated by infusing exogenous insulin, permitting plasma glucose to decline until a symptomatic level of approximately 50 mg/dL is reached, and, after a relatively short interval at this level, halting the insulin infusion is and permitting the plasma glucose to recover. Monitoring of endogenous insulin (via measuring C-­peptide levels in blood), glucagon, epinephrine, norepinephrine, cortisol, and growth hormone during the fall and recovery of plasma glucose delineate the counterregulatory hormone response. Based on this paradigm of insulin-­induced hypoglycemia, the threshold for near-­complete suppression of endogenous insulin secretion is approximately 70 mg/dL, the threshold for robust glucagon secretion is approximately 60 to 65 mg/dL, and that for eliciting epinephrine counterregulation is approximately 60 mg/dL. In response to hypoglycemia, the robust spike of glucagon secretion, much accentuated relative to the more subtle modulations to slight declines of fasting glucose, is only transient in duration, yet achieves prompt robust stimulation of glycogenolysis and a more persistent stimulation of gluconeogenesis. The catecholamine response also acts to increase HGP and accelerate lipolysis. The other key constituents of the counterregulatory hormone response are growth hormone and cortisol, and, relative to glucagon and catecholamines, occur later within the overall counterregulatory hormone response, have a slower onset of effect and a sustained effect. In diabetes mellitus, most especially in type 1 diabetes, there are notable impairments in the counterregulatory hormone response, with impaired glucagon response and, not infrequently, with impaired catecholamine response, and these contribute in a major way to the risk of insulin-­induced hypoglycemia, which is one of the major iatrogenic complications of insulin therapy.35,36

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise

351

Glucose Glucose Glucose

Gluconeogenesis

Glycogen CO2 Lactate

Glucose Uptake Glucose

Incretins

Glucagon Insulin

Lipolysis

Fig. 22.2  Prandial and postprandial (fed state) metabolism. Glucose and proteins digested and absorbed in the gastrointestinal tract enter the portal venous circulation, enabling first-­pass hepatic extraction, while absorbed fat instead enters the circulation via lymphatic entry and in essence undergo first-­pass extraction in adipose tissue. One prominent effect of chemosensing of nutrients by enteroendocrine cells is secretion of incretins, which potentiates nutrient-­stimulated insulin secretion (and suppression of glucagon secretion). Downstream actions of this pancreatic islet response include suppression of hepatic glucose production, stimulation of hepatic glucose uptake, suppression of lipolysis, and stimulation of glucose uptake into skeletal muscle. Glucose oxidation largely meets bioenergy needs during at least the first half of postprandial metabolism, and glucose availability is such that its storage as glycogen is a prominent pathway in the liver and muscle. CO2, Carbon dioxide.

PRANDIAL AND POSTPRANDIAL INTERMEDIARY METABOLISM Introduction Ingestion of food is necessary for survival and brings pleasure (mostly), and there are complex neural circuits, gut hormone circuits, and diverse peripherally generated cues that signal hunger and elicit behaviors to seek food and respond to food ingestion.37 Knowledge of prandial and postprandial intermediary metabolism is but one aspect of this complex physiology, yet it is foundational for understanding governance of appetite, energy balance, and weight regulation. This knowledge can also inform the dialog as to what comprises a healthy versus an unhealthy diet, though it is of course the longer-­term outcomes rather than near-­term changes in intermediary metabolism that are truly informative. In terms of definition, the term “prandial” refers to the period of eating, while the term “postprandial” refers to the subsequent period, when digestion and absorption are ongoing and exogenous nutrients are undergoing metabolic disposition in the liver and peripheral tissues. The duration of prandial conditions is approximately 0.5 hour, while postprandial metabolism, depending upon the size and macronutrient composition of the meal, can extend from several to 6 to 8 hours, before a transition is completed back into postabsorptive conditions (or until the next meal is ingested, starting the postprandial process anew). The main areas of focus of this section will continue to be glucose and fatty acid metabolism within the context of endocrine regulators (Fig. 22.2). Admittedly, this focus on glucose and fatty acid metabolism can be faulted as a “rounding up of the usual suspects”; nontargeted metabolomic surveys indicate at least several thousand components, likely closer

to 10,000 discrete small molecule metabolites, and thus comprise a broad spectrum of intermediary metabolism relevant to both postabsorptive and postprandial phases.38 Considerable progress has been made in identifying biomarkers of metabolic disease and disease risk within the metabolome, one notable example being that of branched-­chain amino acids, which cluster with insulin resistance and risk for type 2 diabetes mellitus. Another provocative finding from the metabolome surveys is that fatty acid length and number of double bonds can be a strong predicative risk factor for type 2 diabetes mellitus.39 However, maturing an understanding of metabolome changes to yield mechanistic insights into the regulation of intermediary metabolism is still a work in progress. The focus here will be upon pathways of glucose and fatty acid metabolism and the context of responses to a prototypical meal, namely one that is a 7-­to 10-­kcal/kg caloric load (around 40% of daily calorie consumption), comprised of approximately 50% carbohydrate, 30% protein, and 20% fat. Clearly, there is considerable contemporary interest in how variations in macronutrient content and proportions may influence health, modify disease and disease risk, and influence weight regulation. However, an adequate consideration of how dietary variations further influence postprandial intermediary metabolism is beyond the scope of this section. Hopefully, a good grounding in the response to this putative standard meal will enable a balanced and critical appraisal of changes induced by diet modification.

Digestion, Absorption, and Enteroendocrine Cells The aggregate surface area for absorption in the small intestine is estimated to be approximately 200 m2, roughly the size of a tennis court, though more recent findings suggest it is only half this surface area.40 Regardless, it is of a remarkable dimension, especially considering that

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it is folded in a fully functional capacity within the relatively tight confines of the abdominal cavity. This origami of folding of the surface area reflects the recurring pattern of millions of small intestine villi that amplify the surface area of the lumen, which is further expanded 10-­fold by the brush border of the apical surface of enterocytes. A great majority of the cells of the small intestine epithelium are enterocytes, the workhorses of nutrient absorption, and these are supported by the protective action of a smaller population of mucous-­secreting goblet cells. However, as a text focused upon endocrinology, it is important to call out the 1% of the gut epithelial population that are enteroendocrine cells (EECs). As a proportion of the cell population of villi, EECs are indeed a miniscule fraction, but in aggregate EECs constitute the largest endocrine organ of the body. EECs are chemical sensors of nutrients and, in response to carbohydrate, lipid, protein, and other components of intestinal chyme, release peptide hormones and neurotransmitters.41 What is conceptually unique about EECs relative to other endocrine glands is that, whereas the latter sense and respond to the internal milieu to maintain homeostasis, EECs respond to the external challenge presented by ingested food to prepare the gastrointestinal system for digestion and absorption, prepare peripheral tissues for metabolism of food, prepare pancreatic islets for insulin and glucagon responses, and carry out gut–brain signaling governing satiety.42 Thus, the EECs are a complex endocrine system that is central in governing postprandial intermediary metabolism. EECs are a highly diverse cell population. Formerly it was thought that there was one EEC cell type for each gut hormone, but recent advances in single-­cell transcriptomics and other approaches now clearly demonstrate that an individual EEC can secrete several gut hormones in a coordinated and consistent manner, and that EEC subsets are distributed in an orchestrated anatomic pattern; the functional ramification of all this remains to be more fully comprehended. Several of the gut hormones secreted by EECs govern the satiety response to eating, including glucagon-­like peptide-­1 (GLP-­1), peptide tyrosine tyrosine, and cholecystokinin, while ghrelin signals hunger. Some of these and other various gut hormones have a key role in regulating gut motility, bile acid release, pancreatic exocrine secretion, and intestinal water and electrolyte secretion. Here, we will focus upon the component of EEC response characterized as the incretin axis, and this is presented subsequently in connection with insulin and glucagon responses to meal ingestion. Returning to the issues of digestion and absorption of food, the essential chemical reaction that renders food into its constituent components so that these are in a state that can be absorbed by small intestine enterocytes is hydrolysis. Hydrolysis cleaves starches and smaller carbohydrate polymers, cleaves acyl groups from the glycerol backbone of TGs (and from cholesterol esters), and breaks up ingested protein by cleaving amide linkages between amino acids. An array of enzymes, many secreted and others residing on the brush border surface of enterocytes, catalyze hydrolysis of ingested macromolecule nutrients. For the interested reader, more detailed information on the digestive and absorptive process is referenced.43 The process is facilitated by and dependent upon bulk secretion of water and electrolytes that modulate pH and disperse nutrients within the gut lumen and by bile that emulsifies fat. Amylase is secreted by the exocrine pancreas in sufficient quantity to rapidly hydrolyze starch into disaccharides that are then efficiently cleaved by brush border enzymes into sugar monomers; mostly it is the monomer glucose that is absorbed and quickly released into the portal venous circulation. This abundant enzymatic capacity enables rapid entry of carbohydrate into circulation, a rate determinant being more gastric emptying than hydrolytic enzyme capacity for carbohydrate. A considerable proportion of glucose absorption is mediated by coupling with sodium absorption through the sodium-­glucose transporter-­1, and there is also contribution from

GLUT2. Pepsin, secreted within the stomach and with optimal activity in the acidic environment of the stomach, dissembles collagen within proteins, providing pancreatic peptidases in the small intestine with more ready access to cleave protein into smaller fragments that are ultimately rendered to individual amino acids by enterocyte brush border peptidases. Amino acids, mostly similar to glucose but with some exceptions, appear relatively quickly in the portal circulation. The situation with digestion and absorption of fat differs in several respects from that of carbohydrate and protein, nutrients that are more polar than fat and more water-­soluble, and this physiology of intestinal lipid metabolism has recently been reviewed.44 Briefly, the strong hydrophobicity of TG, the major component of ingested fat, is an impediment to its digestion. This hydrophobicity limits the surface area of fat globules and thus constrains access of pancreatic lipase and cholesterol esterase. Emulsification of fat by bile helps to form small lipid micelles, vastly expanding the surface area on which lipase can act. Parenthetically, bile acids are now recognized to have an important signaling role, clearly akin to motifs of endocrine signaling, and are the ligands for receptor-­ mediated actions in liver and peripheral tissues.45 Accordingly, it is appropriate to include bile acid signaling as part of the multifaceted panoply of gut-­derived messages that govern intermediary metabolism, intestinal physiology, systemic energy balance, and appetitive behavior. Fatty acids and monoacyl-­ glycerol are absorbed into enterocytes, where long-­chain acyl group are reesterified, and the newly formed TG is packaged with apoB48 into large chylomicron particles. Chylomicrons enter lacteals contained within small intestine villi and enter systemic circulation via the lymphatic thoracic duct, thereby foregoing first-­pass hepatic extraction, unlike glucose and amino acids. Interestingly, large chylomicrons released by enterocytes have too large a diameter to permit passage through the fenestrations within hepatic capillaries, and, as will be addressed later, it is as chylomicron remnants, reduced in size by catabolism in peripheral tissues and notably adipose, that such passage becomes possible. Another difference from carbohydrate and protein absorption is that TG entry into systemic circulation is significantly delayed, enough that ingested glucose and fat entries may, at least figuratively, be considered somewhat separate phases. It can be speculated that this temporal separation, together with the different ports of entry (portal vs. systemic) may act to diminish the fatty acid competition in the early prandial period that is predominated by glucose metabolism, and that later, with relatively prompt clearance of ingested glucose, the potential for glucose competition during metabolism of ingested fat. The notion of glucose–fatty acid substrate competition as a governing principle of intermediary metabolism is further addressed subsequently.

KEY POINTS:  Digestion and Absorption of Nutrients • The surface area of the small intestine is 100-fold greater than that of the body’s skin (250 vs. 2 m2), but food must be cleaved by hydrolysis into monomeric constituents to be absorbed by enterocytes. • The more polar, water-soluble nutrients of glucose and amino acids are absorbed into enterocytes and released at their basal surface into the portal venous circulation for first-pass uptake and metabolism by the liver. • The less polar, hydrophobic lipid nutrients are absorbed into enterocytes after emulsification with bile, after which lipase cleaves triglyceride (TG) to fatty acids and monoacylglycerol (MAG). On entering enterocytes, fatty acids and MAG undergo reesterification to form TG, and this is packaged into large chylomicrons. • Chylomicrons are released into lacteals of the intestinal villi and enter systemic circulation at the level of the thoracic lymph duct, thus initially bypassing the liver, unlike carbohydrate and protein.

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise

Insulin and Glucagon Responses It is difficult to overstate the importance of insulin as a determinant of the metabolic response to feeding and in evoking the metabolic shift from fasting patterns of metabolism. Insulin secretion increases promptly upon eating, and by approximately 5-­to 10-­fold during prandial versus fasting conditions, dependent upon meal size and carbohydrate content. The sharp rise in insulin secretion has a mirror image in the suppression of glucagon secretion, the latter reflecting a regulatory role of the sharp rise in insulin to reduce glucagon secretion. Certainly, the vigorous insulin secretion is in response to a rise in plasma glucose, and with potentiation by other nutrients (e.g., amino acids). Yet, a century ago, perceptive investigators postulated an additional impetus, at the time speculatively termed the incretin effect, the components of which were later identified.46 One way to capsulize the effect of the incretin effect is to recount a crucial experimental finding: when a postprandial (peripheral) glucose concentration was simulated by a carefully modulated infusion of glucose, the insulin secretory response was clearly lower, by more than half, than when equivalent hyperglycemia occurred naturally in response to ingestion of glucose. This difference in the amount of insulin secreted suggested the action of a signal(s) additional to hyperglycemia itself; the components of the incretin axis were later identified as the incretin hormones (incretins) glucose-­dependent insulinotropic polypeptide (GIP) and GLP-­1. As noted earlier, GIP and GLP-­1 are secreted by EECs. The EECs secreting GIP are predominately located in the small intestine, while EECs capable of secreting GLP-­1 have a more extensive distribution in both the small and large intestines. In addition, there can be cosecretion of other gut hormones in conjunction with that of GIP and GLP-­1. These EECs function as nutrient chemoreceptors responsive to ingested glucose, fatty acids and fatty acid metabolites, and amino acids. The secretion of GIP and GLP-­1 is further modulated by G protein–coupled receptors (GPCRs) expressed on GIP-­and GLP-­ 1–secreting EECs. Prominent among these GPCRs are a family of lipid-­sensing receptors (i.e., GPR40, GPR120, GPR119, and others) that respond to fatty acids of differing chain length and to fatty acid metabolites, the generation of which can also derive from metabolic activity in adjacent enterocytes, signifying operation of paracrine signaling.47 In turn, many of these lipid-­sensing receptors are also expressed on pancreatic β cells (and, for some, on pancreatic α cells). The receptors for GIP and GLP-­1 are expressed on pancreatic β cells and α cells. Increases in GIP and GLP-­1 secretion occur as early as 5 to 10 minutes after the start of eating and continue for most of the prandial period, and secretion of GIP is more sustained into the postprandial period. As alluded to earlier, GIP and GLP-­1 strongly potentiate glucose-­induced insulin secretion, which is also to state there is only a nominal effect to stimulate insulin secretion at euglycemia or hypoglycemia. There are emerging findings that GIP may amplify the glucagon response to hypoglycemia,48 and that the GPCR GPR119 has a similar effect.49 These latter findings raise the provocative notion that there is an aspect of incretin biology that helps to defend against hypoglycemia, perhaps acting in a physiological context in the descending arm of plasma glucose during postprandial metabolism by heightening the sensitivity of an α cell response. It has been estimated that, in healthy individuals, the actions of these incretin hormone account for more than half of the prandial increase in insulin secretion. GLP-­1 also has receptors in the CNS and a role in governing satiety and appetite; whether GIP has a similar role is less clear. GLP-­1 also has effects on gut motility. It suffices to state that the incretin axis, and more broadly the EEC-­derived hormones, have a major role in the overall endocrine governance of postprandial metabolism.

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KEY POINTS:  Entero-endocrine Cells and the Incretin Response • Enteroendocrine cells (EECs) are distributed throughout the stomach, small intestine, and large intestine in a regulated distribution of pluripotential hormone secreting cells that act as chemosensors of nutrients and nutrient metabolites. • EECs comprise just 1% of the epithelial cell population of the small intestine, but in aggregate constitute the largest endocrine organ in the body. • One aspect of the EECs constitutes the incretin axis, composed of two gut hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like polypeptide-1 (GLP-1), that are secreted in response to meal ingestion. • In a glucose-dependent manner, GIP and GLP-1 robustly amplify glucose-dependent insulin secretion, likely accounting for 50% of the meal-related increase in insulin secretion. The incretin effects are minor at fasting glucose levels due to low secretion of GIP and GLP-1 and because of their glucose-dependent action on insulin secretion. • A number of lipid-sensing G protein–coupled receptors modulate GIP and GLP-1 secretion and can also modulate insulin secretion directly via receptor expression on β cells.

Effects on Energy Expenditure and Respiratory Quotient During prandial conditions, extending into the postprandial period, there is a modest increase in systemic energy expenditure. This is termed the thermic effect of food and in aggregate comprises approximately 10% of daily energy expenditure. The processes of ingestion, digestion, and absorption, as well as downstream metabolism of nutrients, are considered to account for the increase in energy expenditure that comprises the thermic effect. The magnitude of this effect is partially dependent on the type of nutrient consumed, being less for carbohydrate than for protein or fat. In addition to the thermic effect of food ingestion, there is characteristically a robust effect of meal consumption on the RQ, the quotient of CO2 production to O2 consumption, described earlier, and a parameter reflecting relative contributions of carbohydrate versus fat combustion. And, as earlier noted, a typical fasting RQ in a lean, healthy individual is approximately 0.80 ± 0.03, and this can characteristically increase to a value approaching or achieving 1.0 (signifying complete reliance upon glucose oxidation) within an hour of eating, especially if carbohydrate comprise 50% or more of the calories consumed.7 This indicates that, in the early postprandial period, glucose oxidation nearly completely accounts for energy expenditure. As the postprandial period continues, the RQ value gradually recedes toward a resumption of postabsorptive values. Peak rates of glucose oxidation after a meal approach 3 mg/kg-­min, or nearly 50% of the peak rate of systemic glucose appearance (∼6–8 mg/kg-­min) following meal ingestion, which in turn is a 3-­to 4-­fold elevation compared with fasting HGP rates (2 mg/kg-­min).50,51 In aggregate, across the prandial and postprandial periods, it has been estimated that oxidation of glucose accounts for 40% to 50% of the metabolic disposition of ingested glucose, with the remainder partitioned to “non-­oxidative” metabolism, largely glycogen storage in liver and muscle, together with a smaller fraction that is metabolized glycolytically but not oxidized.

Hepatic Glucose Metabolism In response to the ingestion of a mixed meal or a predominately glucose load, the liver promptly switches from its role during fasting as the primary producer of glucose to one of robust glucose uptake, accruing glycogen while concomitantly suppressing release of glucose. The activation of robust glucose uptake by the liver requires a combination of both hyperinsulinemia and hyperglycemia.20,52 Experimentally

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raising insulin while maintaining euglycemic levels of plasma glucose (i.e., 80–100 mg/dL) does not substantially increase glucose uptake by the liver, though these conditions do suppress hepatic glucose production. Similarly, hyperglycemia by itself (e.g., experimentally controlling insulin concentration through concomitant somatostatin and exogenous insulin infusion) does not elicit strong glucose uptake by the liver, though this suppresses hepatic glycogenolysis. Yet, the combination of hyperinsulinemia and hyperglycemia is a strong stimulus for hepatic glucose uptake. It is important in this context to note that, during the absorptive phase of a meal, portal concentrations of glucose exceed those in the periphery, and this acts to potentiate the effect of the portal to peripheral gradient for insulin in stimulating hepatic glucose uptake. It has been further postulated that a portal to arterial glucose gradient generates a portal signal amplifying hepatic uptake of glucose; the exact nature of the putative portal signal has not been firmly identified.53 Following a 12-­hour or longer postabsorptive phase, a single meal does not fully replenish hepatic glycogen, but, incrementally, over the course of two to three meals in a day, hepatic glycogen becomes fully replete, with a content of approximately 70 to 100 grams, dry weight (Table 22.1). Both increased intracellular glucose availability and insulin signaling activate the enzyme glycogen synthase in hepatocytes while reciprocally inhibiting glycogen phosphorylase; both enzymes are governed by phosphorylation and dephosphorylation cascades previously described. Because the hepatocyte glucose transporter, GLUT2, enables rapid exchange of glucose across the full range of glucose concentrations, glucose transport is not a rate-­ limiting step per se for rates of glycogen synthesis in the liver, but rather metabolic control is attributed to downstream steps, notably glucokinase and glycogen synthesis. This differs from skeletal muscle, as will be addressed later. Overall, it is estimated that glucose uptake by the liver accounts for approximately one third of the disposition of ingested glucose. This alone is a sizeable contribution, but by itself is still an underestimation of how the prandial transitions of hepatic glucose metabolism shape glucose tolerance. Suppression of hepatic glucose production during prandial and postprandial conditions is the additional crucial determinant of glucose tolerance and indirectly governs disposition of the portion of ingested glucose that is not taken up by the liver and instead enters the peripheral circulation. Suppression of HGP occurs promptly during the prandial and postprandial phases; HGP reaches a nadir within an hour of eating, and there is a gradual return to fasting rates of HGP over 4 to 6 hours following meal ingestion.50,51 Regarding governance of HGP suppression, hyperinsulinemia has a strong effect to suppress glycogenolysis, as noted earlier. Interestingly, gluconeogenesis is reduced but not nearly as completely suppressed as glycogenolysis. However, the output of newly synthesized glucose is redirected toward glycogen synthesis rather than as release from the hepatocyte into circulation. This contribution to glycogen synthesis, derived from glucose created by gluconeogenesis, is termed the “indirect pathway” of glycogen formation to distinguish it from the direct pathway based on hepatic glucose uptake and direct incorporation of glucose-­6-­phosphate into the glycogen synthesis pathway. The indirect pathway of glycogen formation is a meaningful component of glycogen repletion during postprandial conditions.

Postprandial Peripheral and Central Nervous System Glucose Utilization The patterns of peripheral tissue utilization of glucose divide into those that are insulin-­ independent and those that are insulin-­ dependent. CNS consumption of glucose, as described earlier, is insulin-­independent and continues unchecked during fed conditions,

and, with a low-­Km glucose transporter (i.e., GLUT3), this rate does not increase due to hyperglycemia. Nonetheless, the CNS does make an important contribution to the disposition of ingested glucose, accounting for an estimated 25% or more of the ingested amount. This partitioning of ingested glucose to the CNS is an “indirect effect” consequent to, and dependent upon, suppression of HGP and the replacement of circulating glucose with that absorbed from the meal. This principle of ingested glucose substituting for reduced HGP pertains to other tissues in accounting for a relatively rapid distribution and uptake of ingested glucose. There is also a potentiating effect on glucose utilization caused by a reduced availability of plasma fatty acids. With the sharp increase in insulin during feeding, there is robust suppression of lipolysis in adipose tissue and a consequent sharp reduction in plasma fatty acids. Though ingested fat packaged into chylomicron delivers fatty acids to peripheral tissue (as will be discussed later), the temporal delay in entry of chylomicrons relative to ingested glucose creates a situation such that, in the early phase of postprandial metabolism (for approximately the first 2 hours), glucose is the more predominately available substrate. Thus, the many tissue and organs that have capacity to take up and utilize fatty acids have diminished access to these, and alternatively rely more fully upon circulating glucose. The increased glucose flux during postprandial conditions is associated with a postprandial rise in circulating lactate levels, an approximate doubling, which provides gluconeogenic substrate for the indirect pathway of hepatic glycogen synthesis described above. In the preceding section on postabsorptive metabolism, skeletal muscle was highlighted as an exemplar of fatty acid uptake and oxidation and equally, but oppositely, during postprandial metabolism, skeletal muscle is an exemplar of insulin-­dependent glucose uptake. Glucose transport across the sarcolemma is regarded as the rate-­ limiting step for insulin-­stimulated glucose utilization by muscle54,55; physiological levels of hyperinsulinemia increase the kinetics of glucose transport in muscle by an order of magnitude compared with postabsorptive conditions. The postprandial increase in plasma insulin is sufficient to initiate the signal transduction cascade that greatly increases glucose transport capacity, and this stems from the molecular physiology of GLUT4, the glucose transporter predominant in skeletal muscle. Under postabsorptive conditions, GLUT4 is mostly contained within cytosolic vesicles, sequestered away from the sarcolemma. The insulin signaling cascade triggers translocation of GLUT4 to insertion within the sarcolemma, where it can manifest functionality to enable bidirectional facilitated transport of glucose.6 Across a limb, the postprandial fractional extraction of glucose increases nearly 20-­fold or more compared with postabsorptive conditions. Also, under resting conditions, not all capillaries in skeletal muscle are patent and receive equal perfusion: a sizeable fraction is closed at any instance during resting conditions, and in response to increased insulin a process of capillary recruitment and increased perfusion occurs.56 Taken together, the enhanced delivery and distribution of insulin and glucose into the interstitial space of skeletal muscle potentiate glucose transport, the rate-­limiting step for glucose metabolism in muscle. It is estimated that skeletal muscle accounts for the disposition of approximately one third of ingested glucose during postprandial conditions. Thus, the trilogy of brain, liver, and muscle accounts for much of the disposition, approximately 90%, of ingested glucose. It is estimated that glycogen formation is the metabolic fate that accounts for approximately half of postprandial glucose uptake into muscle.50,57 The role of insulin signaling in myocytes largely parallels that outlined for hepatocytes in activating glycogen synthesis, albeit without contribution from the indirect pathway utilizing gluconeogenesis that does not occur in muscle. Glucose oxidation accounts

l

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise for approximately 40% of insulin-­stimulated glucose uptake, and the remainder, less than 10%, is released from muscle as pyruvate, lactate, or alanine, thus continuing or increasing the Cori cycle. Like the rise in systemic RQ during feeding, in skeletal muscle regional indirect calorimetry reveals a sharp rise in RQ, and it is the rise in muscle RQ that in good measure underlies the rise of systemic RQ.50 Cardiac muscle during postprandial conditions manifests a metabolic response similar as that of skeletal muscle, increasing glucose uptake and oxidation and increasing glycogen formation, while reducing reliance upon fatty acid oxidation. The decrease in fat oxidation is also mediated by a suppression of lipolysis and reduced availability of plasma fatty acid, and intracellularly by generation of malonyl CoA from increased glycolytic flux, thus inhibiting CPT-­1, crucial for translocation of acyl-­CoA into mitochondria. The importance of substrate competition in governing glucose versus fatty acid metabolism, an important concept of intermediary metabolism, was initially articulated by Randle.58 This substrate competition can be empirically demonstrated in humans as well as in in vitro laboratory studies. Experimentally maintaining fasting levels of plasma fatty acids (by lipid infusion) even while raising insulin into the upper physiological range impairs insulin-­stimulated glucose uptake into muscle and lessens suppression of HGP.9,59 In fact, the acutely induced pattern of insulin resistance caused by fatty acids under these experimental conditions bears a strong resemblance to that observed in obesity and type 2 diabetes mellitus and has been a strong impetus for deeper inquiry into the “lipotoxic” contributions to insulin resistance and disease pathogenesis.60 Indeed, in a provocative and classic essay it was argued that, if not for the ease of the water-­soluble chemistry of glucose relative to the complexity of lipid analyses, emphasis may not have been placed so strongly on perturbed glucose metabolism in studying diabetes, and instead more focus may have been allocated to perturbation of fatty acid metabolism in the pathogenesis of diabetes.61 The shift from a reliance upon fat oxidation during postabsorptive conditions to strong reliance upon glucose oxidation during postprandial conditions has been termed “metabolic flexibility,”8,62 and is a prominent aspect or manifestation of healthy metabolic transition, especially for postabsorptive to postprandial transitions. Earlier, the substrate signaling role of malonyl-­CoA was addressed in mediating the transition from fatty acid to glucose oxidation.63 Further, hyperglycemia itself, independent of a concomitant rise in insulin, can evoke a rise in carbohydrate oxidation and a corresponding reduction in fat oxidation, systemically and within skeletal muscle.64,65 Thus, the concept of substrate competition is bidirectional, with fatty acids impeding insulin-­stimulated glucose metabolism and hyperglycemia perturbing postabsorptive patterns of fatty metabolism. This is an important concept within regulation of intermediary metabolism and is relevant to metabolic diseases like type 2 diabetes mellitus.

KEY POINTS:  Postabsorptive Metabolism • The relative stability and constancy of plasma glucose and fatty acid concentrations derives from tightly governed rates of appearance, mobilized from endogenous sources and rates of disappearance from plasma into tissues. • Endocrine–substrate feedback loops are a primary mechanism for control of glucose and fatty acid rates of appearance, whereas rates of glucose and fatty acid utilization are concentration-driven and governed by energy requirements. • There is a relative parity in oxidation of fatty acids and glucose during postabsorptive metabolism, the latter occurring mainly in the CNS, but the former broadly distributed across peripheral tissues.

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Postprandial Adipose Tissue Metabolism Glucose uptake into adipose tissues increases during postprandial conditions of hyperglycemia and elevated insulin. Like skeletal muscle, adipose tissue expresses the GLUT4 transporter isoform that undergoes translocation to the cell surface under insulin stimulation. However, it has been challenging to quantify rates of glucose uptake into adipose tissue, at least with the same clarity that has been achieved for skeletal muscle; arteriovenous balance studies are technically difficult due to the small vessel diameters in adipose, microdialysis has provided good insight, and, more recently, metabolic imaging approaches have yielded quantitative estimates.66,67 The emerging consensus appears to be that adipose tissue may account for approximately 5% to 10% of glucose utilization during postprandial conditions. However, adipose tissue has a relatively low rate of oxygen consumption and contains minor amounts of glycogen, and the chief metabolic fate of glucose taken up by adipose is glycolysis, with the generation of G-­3P to serve as a backbone for TG synthesis. Insulin-­stimulated glucose uptake into adipose can properly be regarded as providing a requisite foundation for the crucial role of adipose in postprandial disposition of ingested TG. There is also efflux of lactate from adipose into venous circulation, which continues during postprandial metabolism, indeed at rates that exceed postabsorptive conditions. Special notation should be made of brown adipose tissue (BAT) and its capacity for substrate utilization, which considerably exceeds that of white adipose tissue when considered on a per cell or per unit weight basis. Indeed, while skepticism had once seemed to prevail about the existence of meaningful amounts of BAT depots in humans, metabolic imaging with glucose analogs convincingly demonstrated both the existence and heightened metabolic rates of BAT.68 The postprandial response of BAT is a contributor to the aggregate thermic effect of food in keeping with the uncoupling of BAT mitochondria and this tissue’s thermogenic function. During the postprandial phase there is strong insulin-­induced suppression of lipolysis and a shift toward TG storage. The drop in plasma fatty acids and glycerol is evident promptly in response to feeding, reaches a nadir at approximately 1 hour after the start of eating, and persists, only gradually returning to usual postabsorptive levels over the duration of the postprandial period. This is a pattern that parallels that for suppression of HGP and is evocative of the single-­gateway hypothesis of insulin governance of lipolysis and HGP that was cited earlier. However, quantitatively, the most important aspect of adipose metabolism following meal ingestion concerns lipoprotein metabolism. Following fat ingestion, chylomicrons enter systemic circulation (via lymphatic drainage from splanchnic tissues), beginning to appear an hour or so after the meal, reaching a peak several hours after meal ingestion and remaining elevated for up to 6 to 8 hours if the amount of fat ingestion was relatively large. Chylomicrons are too large to exit capillaries, yet adipose tissue has a major role in the metabolism of chylomicrons, through its expression, synthesis, and release of lipoprotein lipase (LpL).12 LpL released from adipocytes binds to the luminal surface of adjacent capillaries. The release of LpL from adipocytes is stimulated by insulin, and accordingly recedes during postabsorptive conditions. Thus, the molecular physiology of insulin action in adipose tissue may be construed as establishing a “first-­pass” capacity for the partitioning of ingested fat toward this tissue by its catabolic effect on chylomicron TG. The armatures of LpL enable engagement with chylomicrons and catalyze hydrolysis of TGs into fatty acids that can be taken up by adipocytes, where, in conjunction with G-­3P generated by glycolysis metabolism, provide substrate for esterification into TG and storage in the adipocyte LD. Though there is a potential for spillover into systemic circulation of fatty acids liberated by LpL, the amount is relatively minor, denoting an efficient capacity for fatty acid uptake in insulin-­stimulated adipocytes. Nonetheless, catabolism of chylomicrons is only partially completed

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during a passage through adipose capillaries, and the resulting remnant chylomicrons, now of diminished diameter and with a change in composition (decreased ratio of TG to phospholipid), are further acted upon in the liver (as discussed later) and in subsequent passages through adipose and other tissues. Skeletal muscle, heart, and other tissues express LpL. However, in skeletal muscle, LpL activity generally decreases in response to increased insulin and is higher during fasting conditions, thus directed toward VLDL rather than chylomicron catabolism. KEY POINTS:  Tissue Distribution of Ingested Glucose During Postprandial Metabolism • The combination of hyperglycemia and hyperinsulinemia, accentuated within portal circulation relative to the peripheral circulation, triggers a shift in the liver from glucose export to robust glucose uptake, with a predominant metabolic fate being repletion of hepatic glycogen. Liver accounts for approximately one third of the disposition of ingested glucose. • The strong, rapid, and sustained suppression of HGP after meal ingestion effectively substitutes ingested glucose for that of endogenous glucose as the source for CNS glucose oxidation, accounting for approximately one quarter of the disposition of ingested glucose. • The prandial rise in insulin signals recruitment of GLUT4 to the cell surface in muscle and adipose. Glucose uptake by muscle accounts for a third or more of ingested glucose disposition, and the metabolic fate of this is directed toward glycogen formation in muscle and a marked shift from fat to glucose oxidation. • Adipose glucose uptake may account for a tenth of glucose uptake during postprandial conditions and has a key role in providing generation of glycerol-3-phosphate to provide the backbone for triglyceride formation. • Overall, roughly half of glucose metabolism during postprandial conditions is consumed in glucose oxidation, and the remainder is stored as glycogen, including a large portion of the gluconeogenic flux occurring in liver, the indirect pathway for glycogen synthesis.

Hepatic Metabolism of Fatty Acids and Lipoproteins In considering the contributions of various organs and tissues to postprandial metabolism, we began with outlining hepatic glucose uptake and glycogen repletion, and, in beginning to conclude this section, a turn to another aspect of the postprandial hepatic response is warranted. The liver is the main metabolic “clearing house,” and hepatic metabolism of remnant chylomicrons is an important aspect of postprandial intermediary metabolism and lipoprotein metabolism, one that is crucial for removing chylomicron remnants and restoring the predominance of VLDL particles as TG-­rich lipoproteins during postabsorptive conditions. As noted earlier, newly formed chylomicrons are too large to traverse the 100-­nm fenestrations of hepatic capillaries, but, in a reduced size after partial hydrolysis by adipose LpL, remnant chylomicrons (now approximately 75 nm in diameter) can be metabolized in the liver and undergo receptor-­mediated uptake into hepatocytes for degradation. Some of the lipid components are recycled into newly formed VLDL, and a proportion enter other lipid metabolism pathways, including sphingolipid and phospholipid pathways and TG storage in hepatic LDs, and a portion undergo oxidation. Thus, there is a complex map of potential lipid metabolism pathways that operate in hepatocytes and that are important in completing the transition from postprandial to postabsorptive metabolism.12 The role of the liver in ketogenesis was addressed in the section on postabsorptive metabolism, as well as how ketogenesis increases as fasting is extended in duration. Conversely, during postprandial hepatic metabolism the pathway of de novo lipogenesis (DNL) can increase if

the carbohydrate load is large and is consumed in conjunction with high caloric intake.69 Attention has increasingly turned to the role of DNL in the pathogenesis of hepatic steatosis associated with obesity and insulin resistance, termed nonalcoholic fatty liver disease. When there is a surfeit of pyruvate-­derived acetyl-­CoA, sufficient to drive formation of mitochondrial citrate and its export from the mitochondria to the cytosol, then the enzyme acetyl-­CoA carboxylase, under the influence of hyperinsulinemia, catalyzes the formation of malonyl-­CoA. An important role for malonyl-­CoA as an allosteric inhibitor of CPT-­1 and fatty acid translocation into the mitochondria has been described earlier. In addition, the enzyme fatty acid synthase carries out DNL by repetitively using malonyl-­CoA to construct fatty acids, most commonly C-­16 palmitate. Yet another important contribution of malonyl-­CoA is to provide substrate for fatty acid elongation to form very long chain fatty acids (i.e., chain length >22 carbons). Very long chain fatty acids serve specialized functions in membrane formation, and long chain fatty acids, particularly polyunsaturated fatty acids, can have a signaling function in transcriptional control of hepatic lipid metabolism.

Methodologic Approaches to Investigating Postprandial Conditions Methods for assessing postabsorptive metabolism, notably isotope dilution flux studies and indirect calorimetry, were discussed in a previous section, and here attention is turned to investigations of postprandial intermediary metabolism. Descriptive studies are highly informative and of practical clinical utility, and are commonly done with measurements of metabolites and hormones following glucose challenges and mixed-­composition meal ingestion. Flux studies of glucose metabolism can be integrated into these by including labeled glucose in the ingested load (to trace its entry into peripheral circulation and estimate its uptake in the splanchnic tissues, notably liver), while concomitantly infusing a separately labeled glucose isotope to enable determination of HGP and overall glucose rate of disappearance. Several citations that utilize this approach have been provided.50,51 Also, indirect calorimetry can reveal patterns and rates of fatty acid and glucose oxidation. Measurements of arteriovenous differences provide further insights into the contributions of various tissue beds, notably for skeletal muscle, and with some application to the splanchnic bed and adipose tissues. However, there remains a main limitation to these approaches regarding measuring insulin sensitivity. The pronounced dynamic changes in glucose, fatty acids, and insulin concentrations, together with interindividual differences in respective levels, makes it quite challenging to “isolate” how efficiently (or inefficiently) insulin stimulates metabolism. Mathematical modeling approaches that have used the data from an oral glucose challenge or mixed meal challenge and consider insulin secretory rates have derived estimations of insulin action.70 From these parameters a disposition index can be determined that capsulizes the integrated efficiency of glucose disposition. A paradigm shift in quantifying response to insulin was the development of an experimental platform widely termed the “glucose clamp.” Initially designed by Andres and colleagues,71 and later refined, modified, and evolved by countless investigators, the core concept is to attempt to hold fixed (i.e., steady state) concentrations of insulin and plasma glucose, achieved by constant infusion of insulin and an adjustable infusion of glucose, adjusting the rate of glucose infusion based on frequent measurements of plasma glucose to hold steady or “clamped” blood glucose concentrations.72 The key outcome is the steady-­state rate of glucose infusion, which, in the context of stable insulin and glucose concentrations, provides a quantitative index of insulin sensitivity in stimulating glucose utilization. This allows comparison across individuals and groups of a parameter of insulin action. This platform can also employ isotope labelled glucose infusion to estimate the effect of insulin on HGP, can employ indirect calorimetry to study oxidative metabolism, and study

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise effect on lipolysis and fatty acid metabolism, as well as other parameters. So, in many respects, a glucose clamp recapitulates a metabolic context analogous to postprandial conditions, but of course is not a full recapitulation, because it experimentally fixes glucose and insulin concentrations rather than allowing the dynamic changes that characterize postprandial intermediary metabolism. Moreover, the peripheral infusion of insulin (and of glucose) does not recreate the portal to peripheral gradients that are crucial factors in shaping postprandial hepatic metabolism. Perhaps the greatest utility of the glucose clamp approach has been to enable studies of insulin-­stimulated skeletal muscle metabolism. Under the conditions of hyperinsulinemia, and while maintaining euglycemia with the clamp method, skeletal muscle accounts for nearly 70% to 80% of overall glucose utilization. Also, the metabolic fate of glucose utilization during euglycemia clamp conditions fairly closely mirrors that observed under postprandial conditions: half of more of glucose taken up is stored as glycogen, a large fraction is immediately oxidized, and a smaller fraction undergoes glycolysis and release as lactate and related trioses. Arteriovenous limb balance studies can provide additional clarity or specificity regarding skeletal muscle metabolism.9,57 Another methodological advance has been to use the glucose clamp as a physiologic platform upon which concomitant imaging can be done. One prominent example has been to use magnetic resonance spectroscopy to image the accretion of glycogen in skeletal muscle or liver.73 Another approach, cited earlier, has been to use positron emission tomography in conjunction with glucose clamps and specific positron-­labeled isotopes to measure the kinetic steps of glucose delivery, transport, and phosphorylation in skeletal muscle and to image glucose metabolism in adipose tissues.66,67

PROTEIN AND AMINO ACID METABOLISM The 20 amino acids that are the building blocks underlying protein metabolism are an important aspect of intermediary metabolism. However, the details of the metabolic pathway of each amino acid (or class of amino acids) is a diverse topic, beyond the scope of this chapter, and these pathways are well described.74 Instead, in this section, the interface or interdigitation of amino acids with carbohydrate and fatty acid metabolism will be addressed, notably when amino acid metabolism yields precursors for gluconeogenesis and under the metabolic conditions when amino acids are consumed as substrates for energy production. However, unlike carbohydrate and lipid intermediary metabolism, for which arguably the primary purpose is combustion for energy production, the main purpose of amino acid metabolism is to conserve the overall mass of proteins and support continued turnover or replacement of proteins to ensure intact functional capacity is maintained, and concepts on protein turnover (degradation and synthesis) will be addressed. Collectively, amino acids circulate in plasma at an approximate concentration ranging between 35 to 65 mg/dL, divisible roughly evenly across the 20 constituents,43 with some like glutamate and alanine trending toward higher concentrations, as these are integral to nitrogen exchange between tissues. There are active transport mechanisms involved both in the absorption of individual amino acids from the intestinal tract and from plasma into cells. To support protein synthesis the intracellular pool of amino acids must also reflect the entirety of the amino acid alphabet, including both essential and nonessential amino acids. Thus, part of the intermediary metabolism of amino acids, including crucial transamination pathways, is to ensure appropriate proportionality and availability across the amino acid pool, compensating or adjusting for dietary variances, and thereby enabling protein synthesis. The essential amino acids are the ten amino acids for which endogenous synthetic pathways are mostly insufficient to maintain adequate availability, and thus, there is an essential reliance on dietary availability. It is in this context of meeting the dietary requirements for essential amino acids, recommendations are for roughly 60 g

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of protein consumption daily. Though a daily consumption exceeding 100 g is quite common, and it is with these higher levels of consumption that partitioning amino acids toward oxidation and for gluconeogenesis (or even lipogenesis) tends to be a more prominent facet of postabsorptive and postprandial intermediary metabolism. Upon absorption from the small intestine, amino acids enter the portal venous circulation, a process that tends to lag temporally behind that of glucose. There is a high splanchnic and hepatic fractional extraction of amino acids. These are both sites of relatively high protein turnover, in contrast to the large amount of protein in skeletal muscle, that also turns over but at a significantly slower rate, as does collagen in bone and other tissues. Enterocytes that cover the extensive intestinal epithelial surface themselves have a cell cycle of only several days, and this rapid rate of cellular turnover, together with their rich complement of transporters and enzymes, constitutes a high need for protein replacement; it has been estimated that splanchnic protein turnover is nearly 50% per day. The liver produces circulating plasma proteins (e.g., albumin, clotting factors, transporter proteins), as well as its own abundant enzymatic capacities, and these have a relatively rapid turnover. Albumin, as an example of a hepatic-­synthesized protein, has a turnover of approximately 20% per day, while that of immunoglobins is not too dissimilar. In contrast, the turnover of skeletal muscle protein is estimated to be 1% to 2% per day. These are sharp contrasts, essentially dividing the patterns of protein turnover into categories of a fast-­turnover (e.g., hepatic, and splanchnic) and a slow-­turnover pool.1 There is not a dedicated storage form for amino acids, akin to TG for fatty acids or glycogen for glucose, that sequesters amino acids and can then later be mobilized. Instead, proteins are synthesized to fulfill specific essential functions and in accordance with turnover rates for that specific protein. In this sense, the overall mass of protein in the body, approximately 20% of body weight (Table 22.1), is a reserve of amino acids and indeed supplies nearly three quarters of the amino acids utilized in daily patterns of protein breakdown and synthesis, estimated at 300 to 400 g daily, with the remaining one quarter of amino acids utilized daily coming from food ingestion.10 Excess amino acids beyond immediate needs for protein synthesis must be otherwise metabolized. To enter these pathways, the amino group is deaminated, and ammonia nitrogen is transferred to an α-­keto acid, most typically α-­ketogluturate. The resulting deaminated product is metabolized to acetyl-­CoA, which can then enter oxidative metabolism to yield energy or serve as a substrate for conversion to newly synthesized glucose or to support de novo lipogenesis. This exchange of nitrogen carried out by transamination is quite vigorous.74 In a seminal series of experiments, Schoenheimer and colleagues conducted labeling studies with the stable isotope 15N and observed that, if the label were administered contained in only a single protein or compound, the isotope 15N would nonetheless rapidly and extensively appear in numerous other proteins and amino acids, denoting rapid exchanges within the nitrogen pool of amino acids.75 There is also an overriding tightly governed balance of nitrogen and an excess of amino acids, created either by higher rates of protein degradation or, more commonly, by ingestion of protein in surplus of that needed for protein balance, lead to higher nitrogen excretion and diversion of the resultant carbon backbone to other pathways. Urea is the pathway for disposal of the ammonia liberated by transamination that accounts for 80% of nitrogen excretion, the high solubility of urea in water making this efficient for nitrogen excretion. Urea synthesis occurs in the liver, and its excretion occurs of course in the kidney. In skeletal muscle, nitrogen liberated by transamination of amino acids can be carried to the liver via the previously cited Cahill cycle, as pyruvate can accept the nitrogen to form alanine, thereby carrying both a triose carbon backbone for gluconeogenesis and shuttling the nitrogen for urea synthesis. Another fate for the α-­ketogluturate that is generated from an amino acid undergoing deamination is to be oxidized. An estimate of the contribution of protein to substrate oxidation can be based on the amount of urinary nitrogen. Approximately 1 g of urinary nitrogen arises from the oxidation

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of approximately 6.25 g of protein oxidation.5 On a protein intake of approximately 1 g per kg body weight, typical of a modern diet, nitrogen excretion is approximately 10 g per day, representing approximately 60 g of protein oxidation daily. This compares with an average daily combustion by the brain alone of 120 g of glucose but is about on par with a combustion of about 60 to 70 g of fat daily, though the energy yield of fat is more than twice that of protein. Altogether it is estimated that there is approximately 300 to 400 g of protein turnover daily, thus greatly exceeding the 60 to 100 g of daily protein ingestion.1,10 Yet, despite this relatively large daily turnover of protein, the protein mass of the body remains highly conserved and is large, comprising 20% of body weight; viewed from another perspective, it comprises approximately three quarters of the solid mass of the body (this of course excludes the 60% of weight attributable to water and that due to anhydrous fat). For instance, skeletal muscle comprises approximately 40% of weight in a lean adult, and approximately 20% of muscle weight is represented by protein, and thus 5 to 6 kg of protein in muscle in a 70-­kg adult. At the 1% turnover rate, it can be estimated that approximately 50 to 60 mg of skeletal muscle protein is degraded and synthesized in a balanced homeostasis each day. The role of exercise in influencing contractile protein turnover in muscle will be addressed later. Endocrine regulators of protein synthesis are insulin, growth hormone, and testosterone, which have an anabolic effect, whereas cortisol has a catabolic effect. Also of obvious importance for protein synthesis is a high availability of amino acids, and so in this sense, protein synthesis can be generally regarded within the panoply of postprandial anabolism. Exercise can stimulate protein synthesis in skeletal muscle (and in the heart), especially for several hours in the postexercise period when a previous bout of exercise can augment the effect of food and protein ingestion to stimulate muscle protein synthesis. As well, exercise can shape the remodeling adaptation of a specific muscle to adjust to repeated exposure to an exercise, and these effects are more pronounced in response to resistance types of exercise. KEY POINTS:  Protein and Amino Acid Metabolism • Key function of amino acid metabolism is to ensure appropriate availability across the amino acid pool to facilitate protein synthesis, compensating or adjusting for dietary variances. • Essential amino acids are designated as the ten amino acids for which there is an essential reliance on dietary availability. • Protein stores in the body supply most of the 300-­400 g of amino acids required for daily protein breakdown and synthesis, the remaining amino acids are supplied from ingested food. • The liver produces circulating plasma proteins. • Amino acids not utilized for protein synthesis are deaminated to yield products for oxidative metabolism, gluconeogenesis or de novo lipogenesis.

EXERCISE METABOLISM Exercise Dramatically Increases Demand For Energy Exercise and other forms of physical activity can dramatically increase energy expenditure, and these robust demands for energy mobilization and utilization involve unique metabolic regulation that require hormonal control as well as hormone-­independent mechanisms (Fig. 22.3). The resting metabolic rate or energy expenditure is approximately 5 mL O2 consumption per kg body weight per minute. During moderate exercise O2 consumption can increase 10-­fold, and in elite athletes O2 consumption during maximal exercise can be as high as 90 mL per kg body weight per minute, an astounding 18-­fold increase from rest. This increase is almost exclusively due to the increase in energy demand by contracting skeletal muscle, and to a lesser extent the rapidly beating heart.

KEY POINTS:  Exercise Metabolism • Increases in epinephrine during moderate to high intensity exercise stimulates glycogen breakdown to supply glucose to contracting skeletal muscle. • Low circulating insulin and elevated glucagon during exercise help to increase hepatic glucose output to maintain blood glucose levels during exercise. • Lactate and amino acid production by exercising muscle can be converted back to glucose in the liver where it is subsequently delivered back to exercising muscle via the circulation. • The need for exogenous insulin and impairments in counterregulatory responses to hypoglycemia in Type 1 diabetes makes the regulation of blood glucose during exercise problematic for these patients. • Low circulating insulin and elevated glucagon during exercise act to stimulate lipolysis of triglycerides within adipose tissue to increases fatty acids in the circulation for delivery to exercising muscle. • Elevated epinephrine during moderate to high intensity exercise stimulates hydrolysis of triglycerides stored within skeletal muscle to supply fatty acids to contracting skeletal muscle. • Exercise training increases the availability of carbohydrate and lipid fuels and the metabolic machinery to store, mobilize and utilize these fuels to support the energy demands of exercise. Exercise training also shifts fuel preference towards fatty acid oxidation in the postabsorptive (fasting) state and increases insulin sensitivity in the postprandial (fed) state.

Energy Metabolism During Exercise The control of metabolism to supply energy during exercise involves most of the same systems of hormonal regulation described for postabsorptive metabolism, but often on a different level of magnitude and also involving unique mechanisms, some of which are only beginning to be understood. The increase in energy expenditure during exercise is supported by multiple systems in skeletal muscle. During high intensity exercise lasting up to several seconds, such as sprinting or weightlifting, stored phosphocreatine supplies energy in the form of ATP catalyzed by the creatine kinase (CK) enzymes. An additional function of CK is thought to be a buffer against increasing adenine diphosphate (ADP) concentrations, because ADP is a key regulator of many enzymatic reactions in the cell. CK is highly abundant in skeletal and cardiac muscle, and clinically, elevated CK levels in blood are used to diagnose damaged cardiac muscle (myocardial infarction) and skeletal muscle disease (muscular dystrophy).

Glucose Metabolism During Exercise During exercise of lower to moderate intensity, energy can be provided by both glucose and fatty acids. Exercise of moderate to relatively high intensity relies predominantly on glycolytic or anaerobic metabolism, predominantly through the breakdown of glycogen stores in muscle.76 This rapid increase in glycogen breakdown is regulated by mechanisms involving catecholamine stimulation of the adenylate cyclase reaction to ultimately activate glycogen phosphorylase, along with endogenous calcium activation released through muscle contraction.77 Glycogen stores in skeletal muscle (Fig. 22.4) are limited to approxiamtely 400 g (1600 Kcal) (Table 22.1) and can be a limiting factor in marathon running or other long-­duration endurance sports. Fortunately, other fuel sources can supply energy for exercise. The liver is remarkable in its ability to increase HGP to maintain blood glucose levels even during strenuous exercise. Even during moderate intensity exercise there are increases in glucagon and catecholamines, and a concomitant decrease in insulin secretion, that in concert enable increased HGP; HGP can increase many fold.78 This effect on HGP and the accompanying endocrine response is more subtle during mild intensity exercise, such as walking, even when sustained. During a bout of moderate exercise

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise

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Glucose Gluconeogenesis

Glycogenolysis

Glucose Lactate Alanine

Glucose Uptake

FFA

Glucagon Insulin

Lipolysis

Fig. 22.3  Exercise metabolism. Glycogenolysis and insulin-­independent glucose uptake are rapidly accelerated during exercise and compensate for the low circulating insulin levels, which also serve to release the suppression of lipolysis within adipose tissue and enable increased HGP. This synergizes with higher epinephrine and norepinephrine, which act to increase triglyceride hydrolysis in adipose tissue and skeletal muscle and increase glycogenolysis in skeletal muscle. The high rates of glucose metabolism result in the release of lactate and alanine, particularly during higher intensity exercise, which are then transported to the liver and converted back to glucose via the Cori and Cahill cycles, all of which markedly increase energy supply to skeletal muscle. A caveat to this description of intermediary metabolism during exercise is that the fuel admixture combusted during physical activity is strong influenced by the intensity of the exercise, the duration of exercise, and the underlying fitness of the individual. In general, lower to midlevel intensity exercise, a longer duration of such exercise, and a higher level of fitness are factors that act to increase reliance upon fatty acid oxidation rather than upon glucose metabolism. FFA, Free fatty acid.

Mitochondria Glycogen Triglyceride droplet

Fig. 22.4  Electron micrograph of human skeletal muscle (vastus lateralis) illustrating fuel stored as glycogen and as triglyceride in lipid droplets, and held in close proximity to mitochondria for efficient oxidative metabolism.

muscle cells take up and utilize glucose from the circulation supplied by the liver. This process is dependent on glucose transport via the GLUT4 glucose transporter, described in the sections on postabsorptive and postprandial metabolism, whereby these GLUT4-­containing vesicles are translocated from the cytosol to the sarcoplasmic membrane and T-­ tubules. The mechanisms governing this GLUT4

translocation and glucose transport differ from that described above for insulin-­stimulated glucose transport after feeding. Circulating insulin levels during exercise are quite low, necessitating a separate mechanism involving the signaling molecules AMPK, calcium, and nitric oxide.79 The fate of this glucose once inside the cell can be complete mitochondrial oxidation or, during higher intensity exercise where oxygen is limiting, the glucose supplies energy through glycolysis but with pyruvate converting to lactate rather than continuing to oxidative pathways. If this high intensity exercise continues, the lactate in muscle is exported into the blood, where it can be produced at a faster rate than it can be cleared by the liver and converted back to glucose via the Cori cycle, thus resulting in an accumulation of lactate in the blood. In this scenario, lactate levels can increase from a resting level of approximately 1 mmol/L to upwards of 15 to 20 mmol/L. It is this acute increase in lactate during exercise with the increase in associated H+ (acid) that reduces the blood pH significantly and results in muscle fatigue and the need to reduce exercise intensity or to completely stop exercise. Patients with type 1 diabetes mellitus require insulin for survival, and this need makes the regulation of blood glucose during exercise problematic for these individuals. Because insulin and exercise itself have independent effects on glucose uptake, having an inappropriately higher amount of circulating insulin due to giving too much insulin prior to exercise will effectively add to the independent effects of muscle contraction on glucose uptake and cause excessive decreases in blood glucose, and hypoglycemia may ensue. Also, a relative excess

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of circulating insulin impedes the increase of HGP. Furthermore, the counterregulatory responses to hypoglycemia are impaired in type 1 diabetes mellitus, i.e., increases in catecholamines and glucagon.80 Thus, this hormonal dysregulation and absolute lack of insulin in type 1 diabetes mellitus highlights the independent effects of exercise and insulin on glucose uptake and why having the right balance of exogenous insulin before, during, and after exercise is critical in type 1 diabetes mellitus.

Fatty Acid Metabolism During Exercise The increase in the demand for energy during exercise can also be supported by fatty acids. The source of these fatty acids can vary according to availability, as well as the intensity and duration of exercise. A considerable portion of the fatty acids comes from the circulation, resulting from their release by adipose tissue TG hydrolysis. Low circulating insulin levels during exercise act to release the brake on lipolysis, thereby allowing more fatty acids to be released into the circulation and taken up by exercising muscle. In addition, epinephrine stimulates lipolysis and hydrolysis of adipose tissue TG by activating HSL as well as TG within circulating VLDL. The fatty acids that are free in circulation are taken up and oxidized by skeletal and cardiac muscle through beta oxidation and mitochondrial oxidative phosphorylation. During low-­intensity exercise such as walking, plasma fatty acids comprise the major fuel source for exercising muscle and accordingly, given the enormous fuel reserve of TG in adipose, fuel availability is not a constraining factor, as contrasted with the much more finite reserve of muscle glycogen needed to support high intensity exercise. Fatty acid oxidation during exercise can also be supported by the hydrolysis of TG stored inside muscle cells themselves (Fig. 22.4). This source of energy can supply approximately 50% of the fatty acid oxidation in exercising muscle.81 Fig. 22.4 illustrates the location of these TG droplets adjacent to mitochondria. These LDs replete with TG are accompanied by LD proteins, perilipins, as well as lipases that are critical for TG hydrolysis and release of fatty acids close to mitochondria.82 Given the need for fatty acid transport proteins in the aqueous cellular environment, this subcellular geography is important for the process of fatty acid storage, mobilization, and oxidation during exercise.

Effects of Exercise Training on Intermediary Metabolism Chronic exercise training results in a plethora of changes and adaptations, which largely act to enhance both the availability of carbohydrate and lipid fuels and the metabolic machinery to store, mobilize, and utilize these fuels to support the energy demands of acute exercise bouts. Exercise training increases insulin sensitivity (see earlier section on postprandial metabolism), which serves to enhance rates of glycogen synthesis, and, along with increased enzymes regulating glycogen storage, culminates in an increase in the amount of glycogen storage. Because low muscle glycogen limits long-­duration exercise performance, increasing glycogen storage in muscle can enhance exercise performance.83 This limitation in muscle glycogen storage and in endogenous glucose production by the liver led to decades of research and billions of dollars spent on sports nutrition products to supplement glucose availability during bouts of exercise. Exercise training also enhances storage of TGs in skeletal muscle, an important fuel source for exercising muscle. Interestingly, excess storage of muscle TGs in conditions of low physical activity and overnutrition, i.e., obesity, is associated with insulin resistance and increased risk for type 2 diabetes.84 One of the most important adaptations that can occur with exercise training is the increase in the cellular machinery to mobilize, deliver, transport, and utilize both carbohydrate and fat fuels. Prominent among these adaptations are robust increases in mitochondria content and associated enzymes to enhance capacity

for oxidative metabolism. These changes induced by training consequently enhance the capacity to perform exercise longer and at a higher intensity. Moreover, exercise training enhances regulation of both postabsorptive and postprandial metabolism, as discussed earlier, e.g., enhanced insulin sensitivity, fatty acid oxidation, and overall metabolic flexibility, and has a multitude of effects that reduce body fat and reduced risk for type 2 diabetes and cardiovascular diseases.

SUMMARY AND CONCLUDING REMARKS Intermediary metabolism is a complex constellation of processes, principally enzymatic reactions, by which nutrition is converted into components that can be utilized to produce energy, stored in forms that can later be mobilized, and that can serve to support anabolic actions in the building of cellular and organ structure. In this chapter, emphasis was placed upon the roles of various organs, notably, brain, liver, adipose, and skeletal muscle, and how the integration of, and exchanges between, these individual organs comprise systemic intermediary metabolism. In particular, the concept of interorgan substrate flux was delineated as an integrated physiological aspect of metabolism, and it was emphasized how endocrine governance of intermediary metabolism can be largely understood by examining the effects on modulating rates of glucose and fatty acid flux. Another fulcrum of control in intermediary metabolism that was emphasized was the control of glucose and fat oxidation, how this partitioning is most fundamentally controlled by relative substrate availability and can manifest competition between carbohydrate and lipid metabolism, and how this substrate competition is kept salutary by endocrine actions. Hopefully, it has been useful to frame the delineation of intermediary metabolism in a context of three common daily experiences––fasting, feeding, and physical activity––and explore the deceptively, or seemingly silent, sharp changes that mark transitions from feeding to fasting and then back to fasting, and in response to exercise.

REFERENCES 1. Wolfe RR, Chinkes DL, eds Calculation of substrate kinetics: single-­pool model. In Isotope Tracers in Metabolic Research. Hoboken: John Wiley & Sons, Inc.; 2005:21–50. 2. Westerterp KR. Control of energy expenditure in humans. In: Feingold KR, et al., ed. South Dartmouth. Endotext; 2000. 3. Heymsfield SB, et al. The anatomy of resting energy expenditure: body composition mechanisms. Eur J Clin Nutr. 2019;73:166–171. 4. Schutz Y. Respiration chamber calorimetry and doubly labeled water: two complementary aspects of energy expenditure? Eur J Clin Nutr. 2018;72:1310–1313. 5. Frayn K. Calculation of substrate oxidation rates in vivo from gaseous exchange. J Appl Physiol Respirat Environment Exer Physiol. 1983;55:628– 634. 6. Mueckler M, Thorens B. The SLC2 (GLUT) family of membrane transporters. Mol Aspects Med. 2013;34:121–138. 7. Goodpaster B, Sparks LM. Metabolic flexibility in health and disease. Cell Metab. 2017;25:1027–1036. 8. Kelley D, Mandarino LJ. Fuel selection in human skeletal muscle in insulin resistance: a reexamination. Diabetes. 2000;49:677–683. 9. Kelley D, Mokan M, Simoneau J-­A, et al. Interaction between glucose and free fatty acid metabolism in human skeletal muscle. J Clin Invest. 1993;92:91–98. 10. Frayn K, Evans R. Integration of carbohydrate, fat and protein metabolism in normal daily life. In: Frayn K, Evans R, eds. Human Metabolism: A Regulatory Perspective. Hoboken, NJ: Wiley Blackwell; 2019:204–244. 11. Andres R, Cader G, Zierler KL. The quantitatively minor role of carbohydrate in oxidative metabolism by skeletal muscle in intact man in the basal state. Measurements of oxygen and glucose uptake and carbon dioxide

CHAPTER 22  Regulation of Intermediary Metabolism During Fasting, Feeding, and Exercise and lactate production in the forearm. J Clin Invest. 1956;35: 671–682. 12. Frayn K, Evans R. Lipoprotein metabolism and atherosclerosis. In: Frayn K, Evans R, eds. Human Metabolism: A Regulatory Perspective. Wiley Blackwell; 2019:302–324. 13. Jensen M, Ekberg K, Landau BR. Lipid metabolism during fasting. Am J Physiol Endocrinol Metab. 2001;281:E789–E793. 14. Mittendorfer B, Magkos F, Fabbrini E, et al. Relationship between body fat mass and free fatty acid kinetics in men and women. Obesity. 2009;17:1872–1877. 15. Jensen M, Haymond MW, Rizza RA, et al. Influence of body fat distribution on free fatty acid metabolism in obesity. J Clin Invest. 1989;83:1168– 1173. 16. Snider M, McGarry JD, Hanson RW. Lipid metabolism I: synthesis, storage, and utilization of fatty acids and triacylglycerols. In: Devlin T, ed. Textbook of Biochemistry with Clinical Correlations. New York, NY: A. John Wiley & Sons, Inc.; 2010. 17. McGarry JD, Foster DW. In support of the roles of malonyl-­CoA and carnitine acyltransferase I in the regulation of hepatic fatty acid oxidation and ketogenesis. J Biol Chem. 1979;254:8163–8168. 18. Yang A, Mottillo EP. Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics. Biochem J. 2020;477:985–1008. 19. Wasserman DH. Four grams of glucose. Am J Physiol Endocrinol Metab. 2009;296:E11–E21. 20. Petersen M, Vatner DF, Shulman GI. Regulation of hepatic glucose metabolism in health and disease. Nat Rev Endocrinol. 2017;13:572–587. 21. Gerich J. The role of the kidney in normal glucose homeostasis and in the hyperglycemia of diabetes mellitus: therapeutic implications. Diabetic Med. 2010;27:136–142. 22. Harris R. Carbohydrate metabolism I: major metabolic pathways and their control. In: Devlin T, ed. Textbook of Biochemistry with Clinical Correlations. New York, NY: A. John Wiley & Sons, Inc.; 2010. 23. Bergman RN, Iyer MS. Indirect regulation of endogenous glucose production by insulin: the single gateway hypothesis revisited. Diabetes. 2017;66:1742–1747. 24. Edgerton D, Lautz M, Scott M, et al. Insulin’s direct effects on the liver dominate the control of hepatic glucose production. J Clin Invest. 2006;116:521–527. 25. Hatting M, Tavares CDJ, Sharabi K, et al. Insulin regulation of gluconeogenesis. Ann NY Acad Sci. 2018;1411:21–35. 26. Sasaki M, Sasako T, Kubota N, et al. Dual regulation of gluconeogenesis by insulin and glucose in the proximal tubules of the kidney. Diabetes. 2017;66:2339–2350. 27. Owen O, Felig P, Morgan AP, et al. Liver and kidney metabolism during prolonged starvation. J Clin Invest. 1969;48:574–583. 28. Prentki M, Matschinsky FM, Madiraju SRM. Metabolic signaling in fuel-­ induced insulin secretion. Cell Metab. 2013;18:162–185. 29. Asare-­Bediako I, et al. Variability of directly measured first-­pass hepatic insulin extraction and its association with insulin sensitivity and plasma insulin. Diabetes. 2018;67:1495–1503. 30. Bertram R, Satin LS, Sherman AS. Closing in on the mechanisms of pulsatile insulin secretion. Diabetes. 2018;67:351–359. 31. Rizza R, Mandarino LJ, Gerich J. Dose-­response characteristics for effects of insulin on production and utilization of glucose in man. Am J Physiol Endocrinol Metab. 1981;240:E630–E639. 32. Cherrington AD, Lacy WW, Chiasson J-­L. Effect of glucagon on glucose production during insulin deficiency in the dog. J Clin Invest. 1978;62:664–677. 33. Gerich JE. Glucose counterregulation and its impact on diabetes mellitus. Diabetes. 1988;37:1608–1617. 34. Rizza RA, Cryer PE, Gerich JE. Role of glucagon, catecholamines, and growth hormone in human glucose counter-­regulation. J Clin Invest. 1979;64:62–71. 35. Cryer P. Mechanisms of hypoglycemia-­associated autonomic failure and its component syndromes in diabetes. Diabetes. 2005;54:3592–3601. 36. Gerich JLM, Noacco C, Karam J, et al. Lack of glucagon response to hypoglycemia in diabetes: evidence for an intrinsic pancreatic alpha-­cell defect. Science. 1973;182:171–173.

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37. MacLean P, Blundell JE, Mennella JA, et al. Biologic control of appetite: a daunting complexity. Obesity. 2017;25:S8–S16. 38. Newgard C. Metabolomics and metabolic diseases: where do we stand? Cell Metab. 2017;10:43–56. 39. Rhee E, Cheng S, Larson MG, et al. Lipid profiling identifies a triacylglycerol signature of insulin resistance and improves diabetes prediction in humans. J Clin Invest. 2011;121:1402–1411. 40. Helander HF, Fandriks L. Surface area of the digestive tract -­revisited. Scand J Gastroenterol. 2014;49:681–689. 41. Psichas A, Reimann F, Gribble FM. Gut chemosensing mechanisms. J Clin Invest. 2015;125:908–917. 42. Latorre R, Sternini C, De Giorgio R, et al. Enteroendocrine cells: a review of their role in brain-­gut communication. Neurogastroenterol Motil. 2016;28:620–630. 43. Guyton A, Hall JE. Digestion and absorption in the gastrointestinal tract. In: Guyton A, Hall JE, eds. Textbook of Medical Physiology. Philadelphia: Elsevier Saunders, Inc.; 2006:808–818. 44. Ko CW, Qu J, Black DD, et al. Regulation of intestinal lipid metabolism: current concepts and relevance to disease. Nat Rev Gastroenterol Hepatol. 2020;17:169–183. 45. Molinaro A, Wahistrom A, Marschall H-­U. Role of bile acids in metabolic control. Trend Endocrinol Metab. 2018;29:31–41. 46. Pais R, Gribble FM, Reimann F. Stimulation of incretin secreting cells. Therap Adv Endocrinol Metab. 2016;7:24–42. 47. Miyauchi S, et al. New frontiers in gut nutrient sensor research: free fatty acid sensing in the gastrointestinal tract. J Pharmacol Sci. 2010;112:19–24. 48. Christensen MCS, Sparre-­Ulrich AH, Kristensen PL, et al. Glucose-­ dependent insulinotropic polypeptide augments glucagon responses to hypoglycemia in type 1 diabetes. Diabetes Care. 2015;64:72–78. 49. Li NXBS, Kowalski T, Yang L, et al. GPR119 agonism increases glucagon secretion during insulin-­induced hypoglycemia. Diabetes. 2018;67:1401–1413. 50. Kelley D, Mitrakou A, Marsh H, et al. Skeletal muscle glycolysis, oxidation, and storage of an oral glucose load. J Clin Invest. 1988;81:1563–1571. 51. Basu R, DiCamillo B, Toffolo G, et al. Use of a novel triple-­tracer approach to assess postprandial glucose metabolism. Am J Physiol Endocrinol Metab. 2003;284:E55–E69. 52. Moore MC, et al. Regulation of hepatic glucose uptake and storage in vivo. Adv Nutr. 2012;3:286–294. 53. Dicostanzo CA, et al. Role of the hepatic sympathetic nerves in the regulation of net hepatic glucose uptake and the mediation of the portal glucose signal. Am J Physiol Endocrinol Metab. 2006;290:E9–E16. 54. Bertoldo A, Pencek RR, Azuma K, et al. Interactions between delivery, transport, and phosphorylation of glucose in governing uptake into human skeletal muscle. Diabetes. 2006;55:3028–3037. 55. Pencek R, Bertoldo A, Price J, et al. Dose-­responsive insulin regulation of glucose transport in human skeletal muscle. Am J Physiol Endocrinol Metab. 2006;290:E1124–E1130. 56. Zhang L, et al. Insulin sensitivity of muscle capillary recruitment in vivo. Diabetes. 2004;53:447–453. 57. Kelley D, Reilly JP, Veneman T, et al. Effects of insulin on skeletal muscle glucose storage, oxidation, and glycolysis in humans. Am J Physiol Endocrinol Metab. 1990;258:E923–E929. 58. Randle PJ. Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years. Diabetes Metab Rev. 1998;14:263–283. 59. Boden G. Obesity, insulin resistance and free fatty acids. Curr Opin Endocrinol Diabetes Obes. 2011;18:139–143. 60. McGarry J. Banting Lecture 2001: Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes. 2002;51:7–18. 61. McGarry J. What if Minkowski had been ageusic? An alternative angle on diabetes. Science. 1992;258:766–770. 62. Goodpaster BH, Sparks LM. Metabolic flexibility in health and disease. Cell Metab. 2017;25:1027–1036. 63. Saha A, Vavvas D, Kurowski TG, et al. Malonyl-­CoA regulation in skeletal muscle: its link to cell citrate and the glucose-­fatty acid cycle. Am J Physiol Endocrinol Metab. 1997;272:E641–E648. 64. Mandarino L, Consoli A, Kelley DE. Differential regulation of intracellular glucose metabolism by glucose and insulin in human muscle. Am J Physiol (Endocrinol Metab). 1993;265:E898–E905.

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65. Yki-­Jarvinen H, Bogardus C, Howard B. Hyperglycemia stimulates carbohydrate oxidation in humans. Am J Physiol Endocrinol Metab. 1987;253:E376–E382. 66. Ng JM, et al. PET imaging reveals distinctive roles for different regional adipose tissue depots in systemic glucose metabolism in nonobese humans. Am J Physiol Endocrinol Metab. 2012;303:E1134–E1141. 67. Virtanen KA, et al. Glucose uptake and perfusion in subcutaneous and visceral adipose tissue during insulin stimulation in nonobese and obese humans. J Clin Endocrinol Metab. 2002;87:3902–3910. 68. Cypess AM, et al. Identification and importance of brown adipose tissue in adult humans. N Engl J Med. 2009;360:1509–1517. 69. Sanders FW, Griffin JL. De novo lipogenesis in the liver in health and disease: more than just a shunting yard for glucose. Biol Rev Camb Philos Soc. 2016;91:452–468. 70. Cobelli C, et al. The oral minimal model method. Diabetes. 2014;63:1203– 1213. 71. Sherwin RS, et al. A model of the kinetics of insulin in man. J Clin Invest. 1974;53:1481–1492. 72. DeFronzo R, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol Endocrinol Metab. 1979;237:E214–E223. 73. Petersen KF, Shulman GI. Etiology of insulin resistance. Am J Med. 2006;119:S10–S16. 74. Mehler AH. Amino acid metabolism II: metabolism of the individual amino acids. In: Devlin TM, ed. Textbook of Biochemistry: With Clinical Correlations. New York: John Wiley & Sons Inc.; 1986:462–464.

75. Schoenheimer R, Ratner S, Rittenberg D. The process of continuous deamination and reamination of amino acids in the proteins of normal animals. Science. 1939;89:272–273. 76. Romijn JA, et al. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am J Physiol. 1993;265:E380–E391. 77. Gollnick PD, Hermansen L. Biochemical adaptations to exercise: anaerobic metabolism. Exerc Sport Sci Rev. 1973;1:1–43. 78. Trefts E, Williams AS, Wasserman DH. Exercise and the regulation of hepatic metabolism. Prog Mol Biol Transl Sci. 2015;135:203–225. 79. Richter EA, Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 2013;93:993–1017. 80. Camacho RC, et al. Glucoregulation during and after exercise in health and insulin-­dependent diabetes. Exerc Sport Sci Rev. 2005;33:17–23. 81. Goodpaster BH, Wolfe RR, Kelley DE. Effects of obesity on substrate utilization during exercise. Obes Res. 2002;10:575–584. 82. Gemmink A, et al. Decoration of myocellular lipid droplets with perilipins as a marker for in vivo lipid droplet dynamics: a super-­resolution microscopy study in trained athletes and insulin resistant individuals. Biochim Biophys Acta Mol Cell Biol Lipids. 2021;1866:158852. 83. Hargreaves M, et al. Effect of carbohydrate feedings on muscle glycogen utilization and exercise performance. Med Sci Sports Exerc. 1984;16:219– 222. 84. Kelley DE, Goodpaster BH. Skeletal muscle triglyceride. An aspect of regional adiposity and insulin resistance. Diabetes Care. 2001;24:933–941.

23 Adipose Tissue Function: Metabolic and Endocrine Ulf Smith, Peter Arner, and Mikael Ryden

OUTLINE Obesity and its Health Consequences, 363 Different Types of Obesity and Associated Health Risks, 363 Summary, 365 Adipogenesis and its Regulation, 365 Adipose Cell Differentiation and Role of Genetic Factors, 365 Role of Aging and Cell Senescence, 366 Summary, 366 Immunometabolism: Its Regulation and Role in Insulin Resistance, Inflammation, and Disease, 366 Linking Inflammation to Insulin Resistance, 367 Causal Drivers of Insulin Resistance and Inflammation in Human Adipose Tissue?, 368 Summary, 368 Lipid Metabolism in White Adipose Tissue, 368 Lipid Turnover, 368 Methodology, 369

Lipid Uptake and Lipogenesis in Fat Cells, 369 Regulation of Lipolysis, 370 Glucose Metabolism, 371 Summary, 371 Human Brown Adipose Tissue––Brown or Beige?, 372 Brown versus Beige/Brown Adipose Cells, 372 Beige/Brown Adipose Cells in the Subcutaneous White Adipose Tissue––A Possible Target in Obesity?, 372 Aging Reduces Brown and Beige Adipose Cells, 373 Summary, 373 Adipose Tissue Endocrine Functions and Changes in Obesity, 373 Summary, 374 Adipose Tissue as a Therapeutic Target, 375 Pharmacotherapy in Obesity, 376 Summary, 376

 

OBESITY AND ITS HEALTH CONSEQUENCES Our changed lifestyles with less physical activity and easy access to calorie-­dense fast foods have caused a global epidemic of obesity. This has also led to an increase in obesity-­associated chronic diseases, including cardiovascular disease, diabetes, fatty liver disease (nonalcoholic fatty liver disease [NAFLD]/nonalcoholic steatohepatitis [NASH] and nonalcoholic liver cirrhosis), osteoarthritis, and dementia, as well as liver, breast, and colon cancer. Fig. 23.1 shows a typical Caucasian individual with obesity and summarizes some common chronic disorders and characteristics. Epidemiologically, the degree of obesity is usually expressed as body mass index (BMI), which is measured as body weight in kg/ height in meters2. Table 23.1 shows current BMI definitions of normal weight, overweight, and different degrees of obesity. Based upon current US trends, it is predicted that by 2030 around 50% of adults will be obese, and, of these, 25% will have severe obesity, with a BMI over 35 kg/m2.1 In addition to the rising prevalence of obesity, an additional concern is that obesity also manifests earlier in life, resulting in an earlier initiation and greater duration of the associated chronic diseases. The prevalence of obesity is increasing rapidly in several Asian countries, while it seems to have reached its peak in many European countries. Nevertheless, the current global obesity epidemic is a major challenge to population health and entails rising health costs. A large international age-­adjusted study of global mortality in relation to relative degree of obesity measured as BMI showed that mortality in adults increased almost linearly with increasing BMI. Compared with a normal BMI of 18.5 to 25 kg/m2, set to a hazard ratio (HR) of 1.0,

overweight increased HR to 1.2, grade 1 obesity to 1.45, grade 2 obesity to 1.94, and grade 3 obesity to 2.76.2 This documents the global health problem of obesity. Although death from myocardial infarction has declined in several countries due to reduced smoking, better hypertension/lipid control, and technological advances in cardiac interventions, the prevalence of heart failure increases due to obesity. Other comorbidities associated with obesity have also become global epidemics, in particular diabetes and its different complications. The International Diabetes Federation3 estimates that in 2019 the global prevalence of diagnosed diabetes was 463 million people, while an additional 232 million had undiagnosed diabetes. The global health expenditures due to diabetes are estimated to constitute around 10% of the total healthcare costs.3 Thus, obesity is a major global health problem, and the healthcare system has to a large degree failed to establish treatments resulting in sustained body weight loss. While bariatric surgery is efficient in some of the most severe cases, it will never constitute a global treatment option. The pharmaceutical industry is therefore keen to developing new agents targeting appetite regulation and energy expenditure. In the United States, unlike in most other Western countries, life expectancy has been decreasing in recent years. Although many factors may contribute, the high incidence of obesity in this country is likely to be a causal factor.

Different Types of Obesity and Associated Health Risks Although BMI is used as an indicator of relative obesity, it has long been recognized that the condition is not homogeneous, and that different types of obesity, related to body fat distribution, are also associated with a lower or higher risk of developing cardiovascular disease,

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Features of abdominal obesity Low-grade inflammation

Genetics + lifestyle

Prothrombotic state

Epi-/genetic factors Environment

Weight gain

Adipocyte hypertrophy • Adipogenesis • Adipose tissue dysfunction and inflammation • Impaired lipid storage capacity

CVD

Type 2 diabetes

Hypertension

Dyslipidemia

Adipose tissue dysfunction Insulin resistance Adipocyte hypertrophy • Lipid overflow • Lipotoxicity • Ectopic lipid accumulation

Fig. 23.1  Features of Abdominal Obesity. CVD, Cardiovascular disease.

TABLE 23.1  Definitions of Obesity and Waist Circumference Thresholds Relative Degree of Obesity, Bod Mass Index, kg/m2

Waist Circumference Thresholds, cm

Waist Circumference Thresholds, cm

Normal weight 18.5–24.9 Overweight 25–29.9 Obesity, Grade 1 30–34.9 Obesity, Grade 2 35–39.9 Obesity, Grade 3 40–60

Males >90 >100 >105 >125 >125

Females >80 >90 >110 >115 >115

diabetes, and other complications. In fact, adipose tissue is a good example of something of which both too much and too little can be harmful, as individuals with lipodystrophy, i.e., lacking areas of adipose tissue, show the same health risks and similar metabolic aberrations as individuals with obesity and expanded adipose tissue. The subcutaneous adipose tissue (SAT) is the largest adipose depot of the body and also the most efficient and least harmful site to deposit excess lipids. The SAT expands by recruiting new adipose cells (hyperplasia) and/or by expanding the size of available cells (hypertrophy). Adipose cells are unique in their ability to expand and store lipids, as they can inflate in size by at least 10-­fold! There is a continuous turnover of adipocytes in SAT of around 10%/year. Recruitment of new cells is essentially limited to before puberty,4 although it seems that thigh/gluteal SAT can continue to recruit new cells in adult females.5 Expansion of SAT by hyperplasia is less harmful, while hypertrophic growth is metabolically harmful and leads to a dysfunctional and inflamed adipose tissue, development of dyslipidemia, systemic inflammation, and local and systemic insulin resistance which, in turn, promote the risk of developing obesity-­associated complications.6 There are also other adipose tissue depots, including the visceral (intraabdominal) site, which has long been considered a marker of metabolically unhealthy obesity due to its close association with diabetes, cardiovascular disease, and NAFLD/NASH.7 Over 50 years ago, this prompted the subdivision into “apple” versus “pear”-­shaped obesity, where the former (also termed “abdominal type,” see Fig. 23.1) is more closely associated with health complications. The reason for each individual’s propensity to develop apple/abdominal or pear/peripheral obesity is unclear, but genetic factors play a role, in addition to gender, as women are more likely to have peripheral, and therefore less harmful, obesity, at least

Adipocyte hyperplasia • Normal adipocyte function and lipid storage • Healthy metabolism

Visceral/ intra-abdominal adipose tissue

Fig. 23.2  Adipose Tissue Expansion Through Hypertrophy or Hyperplasia and Consequences for Ectopic Fat Accumulation.

before the menopause. After menopause, women can also develop an abdominal type of obesity.7 The most simple and commonly used measure of abdominal versus peripheral obesity is waist circumference. There is ample evidence establishing the increased health risk of a large waist circumference for any given BMI. This has led to a recent Consensus Statement by the International Atherosclerosis Society that risk assessments of developing cardiovascular disease and other obesity-­associated complications should include BMI and waist circumference.8 Other institutions and governmental bodies, such as the World Health Organization and National Institutes of Health, have also issued recommendations on how waist circumference should be measured and stated that waist circumferences over 88 cm in women and 102 cm in men indicate an increased risk of complications, independent of BMI. However, it is also clear that increased obesity itself leads to expansion of the waist circumference, suggesting that the waist circumference should also be stratified according to BMI, and this is included in the Consensus Statement.8 Importantly, there are also ethnic differences, and the recommended waist circumference thresholds in Japanese and Asian populations are generally lower than those in Caucasians. Increased waist circumference is a marker of expanded visceral/ intraabdominal adipose tissue, which reflects the available SAT cells becoming saturated with stored excess lipids, which, in turn, leads to increased ectopic lipid storage in other organs/tissues that can store lipids.9 This is supported by the close association between the amounts of visceral fat and liver lipids (NAFLD/NASH) and also with the accumulation of ectopic lipids in skeletal muscles, pancreas, and heart (Fig. 23.2), with negative metabolic consequences and health risks. The ability of the subcutaneous adipocytes to expand makes their size an excellent marker of adipose tissue health and function, and also of

CHAPTER 23  Adipose Tissue Function: Metabolic and Endocrine systemic consequences, including degree of insulin resistance. This has been shown in many studies and is exemplified by the finding that the degree of reduction of subcutaneous cell size following bariatric surgery is a considerably better marker of improved insulin resistance than the amount of weight lost.10 Extensive in vivo characterization has shown that subcutaneous adipose cell size is a marker of both the amount of ectopic fat accumulation and the degree of systemic insulin resistance.11 It should also be stressed that BMI is not a sufficiently sensitive measure of dysmetabolic state and health risk, given that around 20% to 30% of individuals with normal BMI exhibit insulin resistance and increased risk of developing diabetes and cardiovascular disease. Conversely, a similar proportion of people with obesity can be metabolically healthy; although epidemiological studies have shown that they still have an increased risk of developing diabetes and cardiovascular disease, this risk is considerably less than in weight-­matched individuals who display a dysmetabolic state.12 These data again show that BMI is not sufficiently sensitive at an individual level and indicates situations where additional markers such as waist circumference, or preferably subcutaneous adipose cell size (although not easy to measure in routine clinical settings), are helpful. It would be valuable to have early, sensitive, and clinically useful markers of an individual’s that could predict the health risk associated with future weight increase and identify SAT dysfunction at an early stage. The only currently identified genetic marker of adipose cell size and health risk is related to the KLF14 gene, primarily in females,13 although another study14 suggested that it was related to the risk of developing cardiovascular disease in both genders. Several other genes have also been shown to be associated with adipose cell differentiation and storage capacity, but with little individual impact.15 KEY POINTS  • Subcutaneous adipose tissue can expand through hyperplasia and, most commonly in adults, through hypertrophy. Expanded subcutaneous adipose cell size and increased waist circumference are indicators of the future risk of developing diabetes and cardiovascular disease.

Another condition of inherently dysfunctional adipose tissue has been identified in individuals with a genetic propensity for type 2 diabetes. Around 30% of first-­degree relatives (FDRs) of individuals with type 2 diabetes exhibit an inappropriate increase in subcutaneous adipocyte size for any given BMI, indicating that they can be metabolically obese even at a normal BMI. It is also well-­established that FDRs have a very high risk of developing both type 2 diabetes and cardiovascular disease.16 This sensitivity to the environment is proposed to depend on an inherent (possibly genetic) reduction in the number of available SAT progenitor cells able to undergo differentiation, thus promoting inappropriate hypertrophic expansion of the adipocytes, resulting in dyslipidemia and insulin resistance. This is best seen in the nonobese state,17,18 because adipose cells also have a finite expansion capability in obesity, and is also seen in nonobese individuals with type 2 diabetes.19 However, much work needs to be done to better understand the interactions between the environment and adipose tissue genetics.

Summary The current global epidemic of obesity is a major threat to both individual health and health expenditures. Obesity promotes metabolic dysfunctions including insulin resistance and dyslipidemia and is a major risk factor for developing cardiovascular disease, diabetes, liver disease, cognitive dysfunction and several forms of cancer. While the degree of obesity is commonly stratified by BMI, an individual risk

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score should also include waist circumference. The SAT has a limited ability to expand and, when exceeded, it leads to accumulation of ectopic fat in many organs/tissues enhancing health risks.

ADIPOGENESIS AND ITS REGULATION Adipose Cell Differentiation and Role of Genetic Factors White adipose tissue is a complex tissue consisting of several different cell types that exhibit close crosstalk with and balance among each other to ensure a healthy and functional tissue. The tissue contains uncommitted mesenchymal stem cells, committed preadipocytes at different stages of differentiation, immune cells of different types, and endothelial cells that regulate lipid transport to/from the blood and fibroblasts. As described above, adipose tissue expansion is dependent on the increase in size of differentiated and mature adipocytes (hypertrophy) and/or the induction of commitment and further differentiation of new adipocytes (hyperplasia) from existing progenitor cells, as well as further differentiation of the preadipocytes. In humans, the uncommitted progenitor cells are both of mesenchymal stem cell origin, as well as of hematopoietic (bone marrow) origin. Surprisingly, studies have shown that, in humans, around 10% of the progenitor cells are derived from the bone marrow.20 The undifferentiated mesenchymal stem cells are multipotent and can be differentiated in vitro to adipocytes, osteocytes, and chondrocytes, depending on environment and specific spatiotemporal differentiation signals. Commitment of both human and murine stem cells into the white adipogenic lineage is dependent on bone morphogenetic protein (BMP) 4,21 while brown adipose cell (see later) commitment seems to be more dependent on BMP 7.22 Following initial commitment into the adipose cell lineage, a complicated sequence of events involving a number of different temporally induced transcription factors promotes full differentiation. Among these, C/EBPα and PPARγ are essential to inducing and maintaining a differentiated adipocyte phenotype. Many studies have shown that adipose tissue is a major regulator of whole-­body insulin sensitivity, inflammation, and metabolism through its secretion of different adipokines and cytokines, as well as lipid storage and regulation of lipolysis. Dysfunctional adipose tissue is characterized by insulin resistance, elevated lipid levels, and increased systemic inflammation. Hypertrophic, rather than hyperplastic, expansion leads to dysfunctional adipose tissue and a dysmetabolic state, with increased risk of developing cardiovascular disease, diabetes, and other complications. It is clear from both in vivo23 and in vitro studies24 in humans that hypertrophic expansion of subcutaneous adipose cells is a consequence of a reduced ability to recruit and differentiate new adipose cells, and that there is also a negative correlation between estimated adipose cell number and cell size in humans.25 Several studies have identified a large number of genes related to an individual’s ability to safely enhance SAT lipid storage and shown that these genes are also associated with a reduced risk of developing diabetes.26 Genes related to adipose tissue distribution are also markers of adipose tissue cell size and metabolism.25 As discussed earlier, individuals with a genetic predisposition for type 2 diabetes (FDRs) are also characterized by markers of reduced subcutaneous adipogenesis and inappropriately expanded cells for a given BMI. This is most clearly seen in nonobese individuals, because there is finite expandability of adipose cells in obesity. Together, these data support the concept that genetic factors regulate SAT expansion and distribution, and that these factors are associated with the metabolic state and insulin sensitivity. There is much additional experimental support for this concept; for instance, it has been shown that functional mutations of PPARγ, the key transcriptional regulator of adipogenesis, are associated with

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an approximately 8-­fold increase in the risk of developing diabetes.27 Although these mutations are rare, they support the importance of a properly functioning SAT in reducing the risk of developing type 2 diabetes. As discussed later (in the Therapy section), this is also supported by the clinical use of the PPARγ ligands, the thiazolidinediones (TZDs), to treat type 2 diabetes. TZDs enhance differentiation of existing preadipocytes, thereby improving lipid storage capacity in the SAT and the release of beneficial adipokines such as adiponectin, while tissue inflammation is reduced. This leads to improved insulin sensitivity, lower plasma glucose levels, and reduced ectopic lipids in the liver and elsewhere while the SAT expands.28 KEY POINTS  • Adipose tissue is a major regulator of whole-­body insulin sensitivity, and expanded adipose cell size is a marker of dysfunctional tissue. PPARγ ligands (thiazolidinediones) can increase recruitment of new adipose cells, and this is associated with enhanced insulin sensitivity.

Role of Aging and Cell Senescence The most important general risk factor for many chronic diseases, including cardiovascular disease and diabetes, is aging. Aging (age >75 years) is associated with a reduced number of functional cells in many tissues, including heart, skeletal muscle, and adipose tissue, and an increased in fibrosis and tissue inflammation. The reason for this is that normally proliferating progenitor cells undergo proliferative senescence with aging that inhibits their ability to proliferate, differentiate, and replace dying cells. Senescent cells have left the cell cycle and become proinflammatory, and also secrete a number of different cytokines and chemokines, proteases, DNA, microRNA, and fibrotic factors: the so-­ called senescence-­ associated secretory phenotype (SASP).29 Secretion of SASP-­associated factors to neighboring cells, and systemically, also means that normal, nonsenescent cells become targets and inhibited from undergoing normal differentiation. Many animal studies have shown that, with aging (defined as mice older than 12 months), the adipose tissue becomes smaller and displays fewer cells.30. In humans, it is more difficult to define a specific age for adipose-­associated senescence, given that lifestyles are very variable; for instance, levels of physical activity, which prevents induction of cell senescence,31 may remain high even in individuals of advanced age. However, pooling large databases showed a gradual increase in the relative amount of visceral fat with age, which probably also reflects changes in the amount and cell size of the SAT, even though this was not measured.7 Insulin sensitivity is also reduced with aging in humans, but, again, defining specific ages is difficult, given that lifestyle has such an important impact on insulin sensitivity. Nevertheless, aging becomes gradually associated with reduced numbers of functional cells in most tissues and combined with enhanced inflammation in both animals and humans. However, aging is only one of several factors that promote cell senescence; other factors include DNA damage, inflammation, increased reactive oxygen species, and others.32 As discussed above, the number of subcutaneous adipose cells measured in the abdominal site apparently becomes fixed around the time of puberty.4 This means that, when these cells have become fully differentiated to adipose cells and accumulated lipids, additional expansion is primarily achieved by hypertrophy, with the metabolic consequences discussed. However, there is also regular turnover of the adipose cells, with a half-­time of around 10 years. With aging and increasing cell senescence there will be fewer functional progenitor cells to replace the dying adipose cells, thus leading to further expansion of the available cells. Hypertrophic expansion of the adipose cells is thus a marker of

reduced availability of new progenitor cells to differentiate, as has been observed clinically.33 Similarly, extensive studies with human adipose progenitor cells in vitro have shown that individuals with hypertrophic expansion of the adipose cells are also characterized by impaired differentiation of new cells.24 Because the number of progenitor cells is apparently not reduced, these data suggest that hypertrophic expansion of the adipose cells in adult humans could be associated with increased cell senescence preventing normal differentiation of progenitor cells. In fact, this is exactly what was found with progenitor cells from individuals with hypertrophic obesity, and this was even further pronounced in cells from type 2 diabetic individuals.34 Importantly, increased cell senescence was also seen in nondiabetic individuals with a genetic predisposition for type 2 diabetes (FDRs), who also exhibited markers of inappropriate expansion of the subcutaneous adipose cells, as discussed. KEY POINTS  • Increased adipose cell senescence is associated with aging and chronic diseases and means that the adipose progenitor cells can not undergo proliferation as needed to replace dying cells. Senescent cells become proinflammatory and secrete inflammatory and other factors (senescence-­ associated secretory phenotype) that target and alter ambient cells.

Although senescent progenitor cells cannot undergo differentiation, the number of such cells in human adipose tissue is usually not more than around 10%, even in type 2 diabetic individuals. However, the consequences of cell senescence are amplified by their secretion of SASP-­associated factors, which target neighboring, as well as peripheral cells, inhibiting their normal differentiation via para-­and endocrine effects. Thus, conditioned tissue culture medium from senescent cells added to nonsenescent cells inhibits normal differentiation of the latter. Interestingly, it is not only the adipose progenitor cells that become senescent, but also the endothelial cells in adipose tissue, which has negative consequences for lipid transport in and out of the tissue35,36 and contributes to systemic dyslipidemia. To what extent other cells in the adipose tissue also become senescent and what consequences this has needs to be studied.

Summary Adipose tissue is a major regulator of whole-­body insulin sensitivity, but when adipose cells expand (hypertrophic obesity), insulin sensitivity is reduced and inflammation and lipid levels increase, together with the risk of developing cardiovascular disease, type 2 diabetes, liver disease, and other conditions. This risk can be reduced by taking measures to reduce body weight and/or increase adipose cell storage capacity, including treatment with TZDs. Recent studies have identified cell senescence as a major inhibitor of normal cell differentiation and have found that aging, hypertrophic obesity, and type 2 diabetes involve increased cell senescence in the adipose tissue. Use of senolytic agents in animal models has shown that deletion of senescent cells markedly improves several chronic conditions, raising the expectations for future human treatments when potential safety issues have been clarified.

IMMUNOMETABOLISM: ITS REGULATION AND ROLE IN INSULIN RESISTANCE, INFLAMMATION, AND DISEASE Inflammation is a physiological consequence that follows exposure to external and internal stressors and involves an acute response characterized by redness (rubor), heat (calor), pain (dolor), swelling (tumor), and dysregulated cellular/organ function (functio laesa).

CHAPTER 23  Adipose Tissue Function: Metabolic and Endocrine That inflammation could be linked to metabolic diseases such as obesity and type 2 diabetes was known already in the nineteenth century, when salicylate was proposed as a treatment against obesity-­associated diabetes. However, whether this effect was linked to adipose tissue was unknown. While the notion of inflammation as a culprit driver was subsequently established in a number of other diseases, its role in white adipose tissue was for a long time disregarded. It was not until the early 1990s that it was demonstrated in murine models that obesity and insulin resistance are linked to increased expression and local release of tumor necrosis factor alpha (TNFα) in white adipose tissue.37 This sparked an intense line of research trying to understand the pathophysiological mechanisms and the role of this response. What soon became clear from these early studies in animals, and later also in human adipose tissue, was that obesity/insulin resistance is associated with a chronic low-­grade inflammation that lacks most of the classical symptoms of acute inflammation. Instead, there is increased expression and release of cyto-­and chemokines expressed and released from several different cell types, and some of these can act as chemoattractants to promote further infiltration of different immune cells. A plethora of studies have confirmed that adipocytes can indeed express and release several proinflammatory factors, including CCL2 (also termed monocyte chemoattractant protein-­1) and interleukin-­6 (IL6), while TNFα and IL1β are secreted locally, predominantly from adipose tissue–resident macrophages. The latter are central cell types in the innate immune system and are among the most abundant leukocytes in obese/insulin resistant adipose tissue. Typically, macrophages in the adipose tissue from subjects with obesity/insulin resistance are centered around what are considered to be “dying” adipocytes in crown-­like structures. Traditionally, these macrophages display a phenotype more similar to a proinflammatory M1 subtype than to the tissue repair–associated M2 cells. Admittedly, the subdivision into M1/ M2 is an oversimplification, and inflamed adipose tissue can display a large panorama of different macrophage subtypes,38 the roles of which remain somewhat unclear. Moreover, while macrophages are the most abundant white blood cells in inflamed adipose tissue, most types of immune cells can be found therein, including T-­cells, B-­cells, and different forms of granulocytes. The inflammatory phenotype described above is linked to a number of morphological changes in adipose tissue, including increased adipocyte size (see also earlier), collagen deposition, fibrosis, and possibly reduced vascularization and hypoxia,39 although the latter is still debated regarding human white adipose tissue.40 The functional changes in adipocytes linked to inflammation include increased basal but attenuated hormone-­stimulated lipolysis KEY POINTS  • White adipose tissue displays a proinflammatory response in the obese/ insulin-­resistant state that is characterized by an increase in the expression and secretion of chemoattractants in the adipocytes themselves, as well as an increased abundance of resident leukocytes.

(see also section on lipid turnover), reduced insulin-­induced glucose uptake and lipogenesis, and lower adipogenesis. These are all processes where insulin plays a major regulatory role in promoting lipid accumulation, and thereby energy storage. Collectively, this reduction in adipocyte insulin sensitivity41 will result in reduced lipid storage capacity, and it is therefore possible that the inflammatory response induced under caloric oversupply is an evolutionarily conserved response that helps limit adipose tissue expansion. This response becomes maladaptive under a constant hypercaloric state. It should also be mentioned that data in

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animal models suggest that a (possibly transient) proinflammatory response may be required for adipose tissue expansion, and that inhibiting this process may result in a pernicious metabolic profile.42 KEY POINTS  • The proinflammatory response in white adipose tissue is linked to morphological and functional changes in tissue phenotype. While this chronic activation is detrimental, a transient inflammatory response may be important to allow white adipose tissue expansion.

Linking Inflammation to Insulin Resistance A number of studies have identified the different mechanisms through which inflammation attenuates insulin signaling. These involve both transcriptional (mediated, e.g., by NF-­kB activation) and posttranscriptional effects on different phosphorylation steps in the insulin signaling cascade.43 Combined approaches, using both cyto-­/chemokine blocking antibodies in vivo as well as genetically modified animal models (primarily tissue-­specific knockouts and transgenic mice), have established the causal role that adipose tissue inflammation plays on insulin resistance. This has spurred substantial interest in targeting adipose inflammation in humans. Several clinical studies have been performed with blocking antibodies targeting cytokines. The first approach to be tested was to inhibit TNFα with a single intravenous injection, which proved unsuccessful in improving metabolic control.44 Subsequent studies using more established TNFα inhibitors (e.g., infliximab, used routinely in the treatment of rheumatic and inflammatory bowel disease) have not demonstrated significant metabolic effects in subjects with type 2 diabetes. The reasons for this could be multiple and include the possibility that the antibody concentrations were insufficient, the fact that adipose tissue does not release TNFα into the circulation, and/or that antibodies penetrate poorly into the adipose tissue. Moreover, TNFα inhibition in other conditions can cause severe side effects such as serious/fatal infections (e.g., reactivation of tuberculosis). Instead, based on its central role in attenuating cell function in organs such as adipose tissue and the pancreas, several clinical studies have focused on inhibiting IL1β. The IL1 receptor antagonist anakinra and the IL1β-­inhibitory antibody canakinumab, originally developed for use in rheumatic diseases, have been studied in randomized, placebo-­controlled trials with glycemia and type 2 diabetes incidence as primary outcomes. Anakinra was shown to reduce systemic inflammation (measured as circulating C-­reactive protein and IL6 levels) and improve glycated hemoglobin and insulin secretion, although the effects were less impressive than currently available antidiabetic drugs.45 Canakinumab reduced the incidence of cardiovascular events46 but had no effect in reducing the incidence of type 2 diabetes in patients with prediabetes at baseline.47 Moreover, the rate of fatal infections was significantly higher in the canakinumab arm of the study. Altogether, while the effects on adipose tissue were not assessed in either of these studies, the results suggest that targeting inflammation is not a very efficient treatment for obesity—associated insulin resistance and/or type 2 diabetes and the rate of serious adverse events are not negligeble.48 This indicates that inflammation in adipose tissue may be a consequence rather than a causal factor, and that efficient treatments targeting adipose tissue should focus on the mechanisms that drive inflammation in the first place. In line with this notion, data in genetically modified animals suggest that insulin resistance in adipocytes promotes the development of inflammation, not the other way around.49

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KEY POINTS  • Therapies targeting white adipose tissue inflammation are not particularly efficient in improving insulin sensitivity/glucose control, suggesting that inflammation is a consequence rather than a causal factor underlying the development of insulin resistance/type 2 diabetes.

Causal Drivers of Insulin Resistance and Inflammation in Human Adipose Tissue?

Lipid Turnover

These aspects have prompted the quest for identification of the drivers of insulin resistance and inflammation in adipose tissue. Much focus has been put on adipocyte size, as cell hypertrophy is closely linked to a proinflammatory adipose phenotype with increased secretion of, e.g., TNFα and CCL2 and reduced insulin sensitivity.19 It is still unclear how adipocyte size could impact inflammation, but it has been proposed that hypertrophy is a result of reduced adipogenesis, fibrosis, and possibly local hypoxia.50 Others have suggested that changes in adipocyte metabolism result in an epigenetically-­driven change in gene transcription promoting a proinflammatory state. For example, obesity and adipocyte hypertrophy are characterized by lower glutamine levels in the tissue, which promotes an increase in O-­GlcNAcylation, a posttranslational modification of proteins and chromatin that activates the transcription and secretion of IL6 and IL1β.51 This, as well as other changes in adipocyte metabolism, could be part of an evolutionarily conserved response to increases in adipocyte size in order to prepare the tissue for tissue expansion, as discussed previously. KEY POINTS  • The drivers of WAT inflammation remain unclear but increased fat cell size and disturbances in adipocyte metabolism are believed to be important.

Summary While adipose tissue inflammation is closely linked to insulin resistance and a pernicious metabolic profile, the causal mechanisms remain unclear and its role as a therapeutic target are probably negligible. Instead, future efforts should aim at identifying the culprit pathways that enable this response which could possibly pave the wave for other treatment approaches.

LIPID METABOLISM IN WHITE ADIPOSE TISSUE Adipose tissue was long thought to be an inert organ harboring lipids for energy storage and isolation purposes. In the middle of the Alimentation

twentieth century it became apparent that the fat cells within white adipose were very active, displaying marked metabolism of lipids, i.e., storage and release of fatty acids, to control energy homeostasis. These processes are relatively complex with regard to both regulation and physiological/pathophysiological impact, and have been reviewed in detail elsewhere.52,53 Herein, the most important aspects are discussed, i.e., lipid turnover, lipid synthesis, lipid mobilization, and the impact of glucose metabolism.

Fatty acids are the most energy-­rich molecules in the body and are mainly stored as triglycerides in fat cells and liberated through hydrolysis of the triglycerides. Although the fatty acid turnover is mainly driven by storage/release in adipose tissue, other sources are also of importance. These include ingestion during meals, usage for very low density lipoprotein–triglyceride production by the liver, trapping (see later), and oxidation during physical or mental performance. Lipids are stored in mature white fat cells in a single lipid droplet mainly composed of triglycerides. This is different from all other cell types, which display multiple lipid droplets.54 There is constant turnover of these lipids (Fig. 23.3), which constitutes a single metabolic pool within the lipid droplet in human fat cells.55 During the 10-­year lifespan of a fat cell, triglycerides are turned over approximately six times, irrespective of the size of the lipid droplet. Two adipocyte factors determine the turnover rate, namely the abilities to synthesize (Kin) and hydrolyze (Kout) triglycerides. The turnover is constant when Kin = Kout. In addition to these kinetic parameters, the turnover is governed by external factors, namely alimentation (mainly influencing Kin) and peripheral utilization (mainly influencing Kout). Changes in fat cell lipid turnover have clinical implications. Overweight and obesity are characterized by decreased turnover due to increased Kin and decreased Kout. Dyslipidemia, in particular a genetic form termed familial combined hyperlipidemia, is associated with decreased turnover that is linked to a combination of low Kin and Kout. It is speculated that this dual kinetic effect “uncouples” fat cells from fatty acid uptake/release and thereby increases the exposure of the liver to fatty acids, which are major substrates for the triglyceride component in lipoprotein synthesis. Differences in lipid turnover can also be observed between adipose depots. As discussed earlier, adipose tissue is distributed into different regions, which display very different sizes. The subcutaneous area is by far the largest (approximately 80% of all adipose tissue). It is believed that this region is a metabolic buffer with high capacity to retain lipids that otherwise would spill over to other tissues such as liver, skeletal muscle, and pancreatic β cells and thereby cause metabolic aberrations.53,56 The “buffering” Peripheral utilization

Adipose tissue events

LIVER

CIRCULATION Glycerol TG Glucose FA TG

FA LPL (glycerol)

Turnover ↓ excess body fat ↑ body weight loss

Lipoproteins FA

Oxidation SKELETAL MUSCLE

Fig. 23.3  Adipose Tissue Events. FA, Fatty acids; TG, triglycerides; LDL, low density lipoprotein.

CHAPTER 23  Adipose Tissue Function: Metabolic and Endocrine capacity is most pronounced in the “lower body” (below the waist line) SAT,57 which may explain, at least in part, why “upper body” (above the waist line) obesity is more pernicious from a cardiometabolic point of view.58,59 The size and distribution of SAT is subject to sex dimorphism and differences in lipid kinetics between men and women.60 In general the depot is larger in women than in men. In addition, women tend to expand lower SAT more than men, while men are prone to accumulate fat in the upper body region. There are no clear-­cut differences between the sexes in subcutaneous adipose lipid turnover, although men appear to have less capacity to store and release fatty acids than women in vivo. From a pathophysiological point of view the smaller visceral adipose depot has a unique role.58,61 It is drained by the portal vein to the liver, so fatty acids can directly be delivered to this organ from visceral fat cells and thereby influence lipoprotein and glucose metabolism. The lipid mobilization activity is greater in visceral adipose tissue than in the subcutaneous fat region. This is evident in upper body obesity, and in particular among men, who often have marked visceral adipose accumulation. The consequence is that large amounts of fatty acids are delivered to the liver, causing glucose intolerance and dyslipidemia according to mechanisms detailed elsewhere.52,53,61 Enhanced peripheral delivery from upper SAT of fatty acids to the liver is also involved in the described pathophysiology. The regional differences in lipid turnover occur in vivo.62 During the gradual transition from leanness to obesity, lipid turnover is lowered in SAT already in the overweight state. Visceral adipose tissue does not change in this way until marked obesity is present. This regional variation may be a factor responsible for higher lipid mobilization from visceral than SAT in less extreme conditions of excess body fat. Finally, lipid turnover is directly involved in changes of subcutaneous fat mass over time.63 Aging is associated with decreased turnover, mainly due to a decrease in Kout, at least in part caused by an age-­ dependent decrease in catecholamine-­ stimulated lipolysis. If this is not compensated by a decrease in Kin, the subcutaneous fat mass will be expand. Conversely, an increase in Kout, such as occurs following bariatric surgery, is associated with long-­term maintenance of reduced body weight. Finally, a recently recognized and potentially important aspect of adipose lipid turnover is so-­called fatty acid trapping.64 These lipid species are not only stored in adipose tissue as triglycerides, they can also be retained within the extracellular space of adipose tissue bound to carriers such as albumin or specific transporter proteins. During esterification and lipolysis (see later) some fatty acids are trapped and not immediately taken up by fat cells or released into the circulation. This is most evident in obesity and dyslipidemia, where fatty acids are trapped in increased amounts and may cause postprandial dysmetabolism according to mechanisms discussed in detail elsewhere,64,65

KEY POINTS  • There is a rapid turnover of the triglycerides in the lipid droplet of human fat cells. During the 10-­year lifespan of a fat cell, its lipid droplet triglycerides are renewed around six times. Changes in the rate of turnover are important for the regulation of adipose tissue. Decreased turnover is associated with enlargement of the fat mass and with dysmetabolic features.

Methodology Human fat cell lipid (and glucose) metabolism has been intensely investigated thanks to advanced in vivo and in vitro methods summarized in Table 23.2. By determining the 14C content in lipids it is possible to calculate their age and estimate the turnover. Fatty acid, glycerol, or glucose tracers can be infused intravenously and used in combination with catheterization or position emission tomography to determine uptake and release of relevant metabolites. Small water-­soluble molecules (mainly glycerol, glucose, and lactate) can be measured in situ in the water space of SAT by means of microdialysis. Adipose tissue can be excised for ex in vivo analyses. Often freshly isolated fat cells are prepared for short-­term (hours or a few days) metabolic studies. Finally, precursor cells such as uncommitted mesenchymal stem cells or committed so-­called preadipocytes can be isolated from the stroma vascular compartment and cultured for a long time. These precursor cells have been instrumental for understanding the molecular regulation of fat cells, because they can be subjected to genetic transfer and editing.

Lipid Uptake and Lipogenesis in Fat Cells Fat cells can synthesize triglycerides in several ways; the stepwise esterification starting with glucose and fatty acids is quantitatively the most important one (Fig. 23.3). Fatty acids and triglycerides in chylomicrons or very low density lipoprotein enter the extracellular adipose compartment from the bloodstream. The triglycerides are hydrolyzed by lipoprotein lipase.66 This enzyme is synthesized by fat cells and excreted to the extracellular space of adipose tissue, where it catalyzes the breakdown of blood-­derived triglycerides into fatty acids and glycerol. Glucose is transported into fat cells and metabolized to alpha glycerol phosphate, which is the initial glycerol backbone of triglycerides. The extracellular fatty acids derived from the lipoprotein lipase action, as well as the free fatty acids bound to albumin in the circulation, are transported into fat cells. They are esterified onto the glycerol backbone to form triglycerides. Fatty acids produced by lipolysis (see later) can also be used for triglyceride synthesis in the pathway mentioned earlier instead of leaving the fat cells. This is called reesterification and is quantitatively important; up to 30% of fatty acids produced by lipolysis may

TABLE 23.2  Methods for Studying White Adipose Tissue Metabolism in Humans Method

Body Level

Main Use

Intravenous infusion of tracers, mainly labeled fatty acids and dexoyglucose in combination with positron emission tomography or catheterization Catheterization of subcutaneous adipose tissue veins

In vivo Whole body or regions

Lipolysis or uptake of glucose and fatty acids by adipose tissue

In vivo Abdominal or gluteal regions In situ Abdominal or gluteal regions Ex vivo (in removed tissue samples) In vitro in living fat cells In vitro using cell cultures of precursor cells

Uptake, release, or retention of fatty acids

Microdialysis of subcutaneous adipose tissue Determination of lipid age by 14C dating Isolated mature fat cells Differentiated fat cells

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Lipolysis (glycerol levels) Lipid turnover studies Molecular and metabolic studies Molecular, genetic, and metabolic studies

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TABLE 23.3  Species Differences in White Fat Cell Lipid Metabolism Event

Humans

Rodents

Hormonal stimulation of lipolysis in adults Catecholamine signaling

Only catecholamines and natriuretic peptides Mainly lipolytic beta 1 and 2 adrenoceptors and antilipolytic alpha-­2 adrenoceptors Through insulin receptor substrate 1 and 2 Involves cell death–inducing DNA fragmentation factor alpha-­like effector A Little in mature fat cells Important for lipid metabolism

Numerous hormones, but not natriuretic peptides Lipolytic beta 1, 2, and 3 adrenoceptors; none of the antilipolytic alpha-­2 type Through insulin receptor substrate 1, 2, and 3 Involves Gi proteins

Insulin signaling TNFα signaling to lipolysis De novo fatty acid synthesis Cell death–inducing DNA fragmentation factor alpha-­like effector A

be reesterified. There are also alternative pathways for triglyceride synthesis in fat cells, but they are quantitatively less important in humans. Glucose can be metabolized to fatty acids (de novo lipogenesis), and other metabolites such as pyruvate can be converted to glycerol (glyceroneogenesis). The glycerol molecules can be directly phosphorylated to alpha glycerol phosphate and used for fatty acid esterification. The major regulator of the adipocyte lipid synthesis is insulin. The hormone activates lipoprotein lipase and stimulates fat cell uptake and metabolism of glucose. Insulin may also directly stimulate the enzymes involved in fatty acid esterification. Following food intake, insulin levels are elevated, and the different regulatory factors described above are activated. During food deprivation, insulin levels are low and, consequently, the triglyceride accumulation in fat cells diminished. Obesity per se is associated with increased activity of lipoprotein lipase, which may facilitate net storage of triglycerides in fat cells.

Regulation of Lipolysis KEY POINTS  • The quantitatively most important factor for lipid storage within fat cells is uptake and esterification of fatty acids derived from triglyceride-­containing particles in the circulation. In addition, uptake of free fatty acids originating from the circulation or derived from fat cell lipolysis is also important. However, de novo lipid synthesis from glucose (lipogenesis) is only a minor contributor to total lipid storage in fat cells.

The signal and metabolic pathways regulating lipid mobilization are well recognized but subject to important species differences (Table 23.3). The regulation in human fat cells is depicted in Fig. 23.4. Triglycerides are stepwise broken down during a process termed lipolysis.67 A specific lipase termed adipose triglyceride lipase (ATGL) and its cofactor truncated abhydrolase domain–containing protein-­5 (CGI-­58) catalyzes the breakdown of triglycerides into diglycerides and fatty acids. The diglycerides are hydrolyzed to monoglycerides and fatty acids by another enzyme, hormone-­sensitive lipase (HSL). Finally, monoglyceride lipase catalyzes the final lipolysis step, namely formation of glycerol and fatty acids from the breakdown of monoglycerides. Despite being a relatively simple process, lipolysis is regulated in different manners by several hormonal and metabolic factors.68,69 Five different signal transduction systems have been described, all using specific cell surface receptors. A lipolytic and an antilipolytic system are coupled to cell membrane–bound adenylyl cyclase via inhibitory Gi-­ and stimulatory Gs-­coupled membrane proteins. Catecholamines are the most important hormones causing lipolysis stimulation, which occurs through beta-­ adrenergic receptors (in humans mainly type 1 and 2) and Gs-­coupled proteins. However, they can also bind to alpha2A

Marked in mature fat cells Not expressed

adrenergic antilipolytic receptors, which are coupled to Gi. Therefore, the net effect of catecholamines on lipolysis is determined by the balance between beta (usually dominant) and alpha effects. Other antilipolytic Gi-­coupled hormones and metabolites are adenosine, prostaglandins, and lactate, which are produced locally and therefore act in a paracrine/ autocrine fashion. The physiological role of these regulators is not well understood. Gs activation stimulates adenylyl cyclase, which increases the production of cyclic adenosine monophosphate (AMP) from adenosine triphosphate (ATP) and, in turn, activates an enzyme complex termed protein kinase A. Inhibition of adenylyl cyclase has the opposite effect. This mode of action also operates for two other endogenous substances, niacin and beta-­hydroxybutyrate. They share the same Gi-­ coupled receptor, HCA2. Binding of the agonists to the fat cell surface receptors inhibits lipolysis through the described inhibitory pathway. Another class of prolipolytic hormones is the family of natriuretic peptides, which bind to cell surface receptors linked to cell membrane-­ associated guanylyl cyclase. This generates cyclic GMP which, in turn, activates the enzyme complex protein kinase G. Insulin is the most important antilipolytic hormone. After receptor binding it activates receptor-­associated proteins termed insulin receptor substrates. They activate the enzyme phosphoinositide 3-­kinase and further downstream signaling, which ultimately stimulates the enzyme phosphodiesterase 3B. This hydrolyzes cyclic AMP into inactive 5’ AMP so that protein kinase A becomes less active. Protein kinase A and G have the same effects on the lipases discussed earlier. Thus, both kinases phosphorylate and activate HSL, as well as the ATGL– CGI-­58 complex. In addition, a lipid droplet coating phosphoprotein, perilipin-­1, is phosphorylated, which allows the lipases to access the lipid droplet surface for triglyceride hydrolysis. In addition to the hormone-­regulated pathways described earlier, a signal pathway through TNFα is important for controlling the chronic (basal) lipolytic activity. The cytokine is produced by fat cells and inflammatory cells within adipose tissue and is thus an autocrine/ paracrine factor. The TNFα receptor signal pathway has two actions. Firstly, it inhibits phosphodiesterase 3B, which elevates the cyclic AMP level. Secondly, it activates another lipid droplet coating protein called cell death–inducing DNA fragmentation factor alpha-­like effector A. Both cytokine effects increase basal lipolytic activity. Although not definitively proven, changes in lipolysis are likely to have a clinical impact. Perhaps the most obvious factor is the relation to fat cell size.70 The rates of spontaneous and catecholamine-­stimulated lipolysis are increased, while the antilipolytic effect of insulin is decreased, in large fat cells. This may be because large fat cells contain more cyclic AMP than small ones. Obesity is often characterized by large fat cells, which would intuitively cause increased lipolysis rates. However, the impact of obesity on lipolysis is complex. The basal rate is elevated, probably as a consequence of inflammation and thereby enhanced local production of tumor necrosis factor alpha. The antilipolytic effect of

CHAPTER 23  Adipose Tissue Function: Metabolic and Endocrine Catecholamines Catecholamines Prostaglandins Adenosine Lactate,...... Insulin

R

IR

Gi

GsR se Gs

Gi

IRS

R

Gu

an yl

yl

PKA

cAMP

PDE3B

CG

ATG L

PL

P

DG

e

P

HSL

P

TG

as

PKG

P

1

cl

cGMP

PLIN

I58

cy

P

TNFalpha

NP

cAMP

P

TNER

Natriuretic peptides

ycla

lyl c

ny Ade

371

Glycerol FA

MG

IN

Transporter

Glycerol FA

1

CIDEA

Fig. 23.4  Regulation in Human Fat Cells.

insulin is decreased, but the same is true for the prolipolytic effect of catecholamines and natriuretic peptides. The effects of excess body fat on lipolysis-­regulating hormones may explain why fasting circulating fatty acid levels are only modestly elevated in the obese state. On the other hand, animal studies and short-­term clinical studies show that inhibition of lipolysis improves insulin sensitivity.71 It is therefore plausible that obesity leads to increased availability of fatty acids, which, in turn, contributes to insulin resistance, impaired insulin secretion, and ultimately type 2 diabetes according to mechanisms described in detail elsewhere.71 On the other hand, prospective studies, which are needed to support a causal role of lipolysis in metabolic diseases, are rare. In one such study it was found that increased basal lipolysis and decreased catecholamine-­ induced lipolysis predict future glucose intolerance and body weight gain.72 The failure of insulin to adequately suppress fatty acid levels in obesity may also lead to so-­called metabolic inflexibility in skeletal muscle.73 The utilization of glucose and fatty acids as fuels for skeletal muscle is switched towards fatty acid use, which may cause lipid toxicity, and thereby insulin resistance, as discussed in detail elsewhere.73 Finally, an association between fat cell lipolysis in vitro and circulating lipid levels has been demonstrated in cross-­sectional studies. KEY POINTS  • In human fat cells, only a few hormone systems have a pronounced, acute effect on lipolysis: the stimulatory catecholamines and natriuretic peptides, plus the inhibitory insulin. In addition, the spontaneous (basal) lipolysis is regulated by local inflammatory factors, in particular TNFα, which increases the lipolytic rate. In obesity and other metabolic conditions, lipolysis regulation is altered. Humans with excess body fat have increased basal lipolysis, decreased ability to activate lipolysis by catecholamines, and resistance to the antilipolytic effect of insulin. Together, these alterations are involved in the metabolic complications of obesity, such as dyslipidemia and insulin resistance. Prospective studies suggest that altered fat cell lipolysis is a factor behind future body weight gain and glucose intolerance.

Glucose Metabolism Fat cells use the same signal system and regulation as skeletal muscle for glucose metabolism. In both cell types, insulin is the major regulatory hormone (stimulation of uptake and further metabolism). However, in quantitative terms, white adipose tissue has a much smaller capacity than skeletal muscle to handle a glucose load. Approximately 10% to 15% of glucose dispersal after food intake is into adipose tissue. There is, on the other hand, one important exception as regards the quantitative role of fat cells in carbohydrate metabolism, namely lactate.74 As a result of glucose metabolism, fat cells produce large amounts of lactate, and this is augmented in large fat cells in obesity. The latter might be due to decreased oxygenation in obese adipose tissue and/ or changes in cellular metabolism leading to activated glycolysis (see earlier). Lactate serves as an autocrine inhibitor of lipolysis (Fig. 23.4). In addition, adipose tissue provides a source for circulating lactate to be used as three-­carbon fuel. KEY POINTS  • Adipose tissue plays a quantitatively minor role in the regulation of whole-­ body glucose uptake. However, glucose metabolism in fat cells has many indirect effects on carbohydrate metabolism, such as on lactate turnover and insulin sensitivity.

Summary Adipocyte storage and release of energy-­rich fatty acids is essential for survival during long periods of food deprivation and in connection with long-­term physical exercise. In addition to being an energy storage organ, white adipose tissue is a reservoir for lipids that may be toxic in other places such as liver, pancreatic β cells, and skeletal muscle. Having too much or too little adipose tissue alters lipid handling and leads to disturbed metabolism, where insulin resistance is the most prominent and important dysfunction.75 Enlarged fat cells and

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unfavorable adipose distribution into the upper body/visceral areas are major culprits in this scenario, and altered lipolysis probably the primary pathophysiological factor.

HUMAN BROWN ADIPOSE TISSUE––BROWN OR BEIGE? Brown adipose tissue (BAT) is primarily found in small mammals and hibernating animals, and brown adipose cells in mice have the same origin as muscle cells.76 BAT regulates body temperature through nonshivering thermogenesis and becomes activated when the animals are exposed to cold or temperatures below their thermoneutrality zone. In mice this is around 30°C, when metabolism is the lowest, and thus considerably higher than the usual room temperature at which mice are housed. Exposure to cold leads to activation of the sympathetic nervous system, which increases lipolysis and the release of fatty acids that are used by the mitochondria-­dense brown adipose cells. Increased sympathetic activation and lipolysis in BAT increase nonshivering heat production through the UCP1 uncoupling protein in the mitochondria. UCP1 uncouples the mitochondria to produce heat rather than the normally generated ATP.77 Many experiments in animals have also shown that sympathetic activation of BAT in the cold consumes enough calories to reduce the amount of adipose tissue, and thus could be a target to treat obesity. BAT is not only present in small mammals, but also in the interscapular region in human infants, where it is critically important for thermogeneration during the first year of life. It then gradually disappears over the first decade. It was long considered to be completely absent in adult humans, with a few exceptions, including patients with pheochromocytomas secreting high amounts of catecholamines and outdoor workers chronically exposed to cold environments. However, in 2007 it was suggested that adult humans may also have some smaller BAT depots.78 This provoked new scientific interest, and, in 2009, several clinical studies using advanced positron emission tomography/computed tomography scans documented the presence of distinct adipose depots in adult humans that were functional and increased glucose and fatty acid uptake, as well as thermogenesis, following stimulation with β3-­adrenergic stimuli or exposure to cold. However, these putative BAT depots were small (around 50–100 g) and primarily found in the cervical, supraclavicular, axillary, mediastinal, paraspinal, and abdominal regions around the kidneys. These findings were followed by many studies suggesting that individuals with larger amounts of BAT also had lower body weights; i.e., it was hypothesized that increasing BAT could be a way of treating obesity. However, several studies performed since then have concluded that BAT in adult humans is not the same BAT observed in rodents, with small lipid droplets and high amounts of mitochondria and UCP1 protein. Instead, it was suggested that human BAT is more of a so-­called “beige/ brite” phenotype with large lipid droplets and low UCP1 expression, and is of unclear importance as a temperature regulator. This has been an issue of long debate, but recent analysis suggests that human BAT is, in fact, similar to the animal tissue, but with a different morphology, because humans live in a thermoneutral temperature and are well insulated and, therefore, do not normally require sympathetic activation and BAT-­induced regulation of thermogenesis.79 Although this may be the case, it is still not likely that human BAT is large enough, contains sufficient UCP1, and/or can be activated to the extent required to actually be a target for body weight reduction. This is clearly in contrast to BAT in mice and other animals.

Brown versus Beige/Brown Adipose Cells As discussed, human brown adipose cells have been characterized as intermediate between white and brown cells, and therefore named

beige/brown cells. However, administration of lipolytically active β3-­agonists was shown to increase metabolism in BAT in humans, although only at very high amounts, when β3-­selectivity was unlikely and associated with increased heart rate and blood pressure.80 This would not be acceptable for an agent to treat obesity. It is well-­ established that β3 receptors are important for activating BAT in mice, but further studies have shown that β2-­adrenergic receptors responding to noradrenaline are mainly expressed in humans. In fact, β3 receptors do not seem to be expressed in human BAT cells at all. However, they are highly expressed in human urinary bladder and gall bladder, as well as in some areas in the heart, which can explain the increased heart rate and blood pressure seen with the administration of β3-­selective agonist.80 The small amount and different morphology of human brown adipose cells (the beige/brown phenotype) have been taken as an indication that the tissue also is thermogenically unimportant. However, it has also been argued that the reason for this is that humans normally live in well-­insulated, thermoneutral environments. In support of this, murine cells also become less active when the animals live in a thermoneutral environment for mice, i.e., around 30°C. Nevertheless, the amount of BAT in man is so small that it is unlikely that it could be activated to a sufficient extent to treat obesity, even if we had access to drugs that could specifically activate human BAT cells. Providing indirect support for this is that fact that the antidiabetic PPARγ agonists, the TZDs, induce beige/brown cells and UCP1 in human adipose cells.81 However, treatment with TZDs is known to increase body weight, rather than decrease it, supporting the idea that human brown cells are a difficult obesity target.

Beige/Brown Adipose Cells in the Subcutaneous White Adipose Tissue––A Possible Target in Obesity? Another scientific approach to find ways to increase oxidative capacity and thermogenesis in human adipose tissue is to increase mitochondria and UCP1 in the conventional white adipose cells through partial transdifferentiation of white cells to a beige/brown oxidative phenotype or to target available specific progenitor cells to become beige/brown and increase their oxidative capacity. It seems that both approaches may be possible, but a key question is if enough UCP1 can be induced to actually increase thermogenesis of sufficient magnitude to also reduce obesity in humans; this remains to be demonstrated. However, both in rodents and in humans, white adipose cells in the large SAT are susceptible to partial transdifferentiation to a beige/ brown phenotype. TZDs can induce a beige/brown phenotype in white SAT cells. Mechanistically, this is due to sirtuin-­dependent deacetylation of PPARγ promoting its interaction with PRDM 16 and EBF 2, leading to its binding to brown-­specific enhancers. In addition to TZDs, thyroid hormones, FGF 21, and BMP4, 7, 8b, and 9 have been shown to increase beige/BAT, and TZDs and BMP4 also target human white adipose cells.79,82 Thus, the white subcutaneous adipose cells are quite susceptible to undergoing partial transdifferentiation from a white to a beige/brown phenotype, but the number of cells capable of being transdifferentiated and the overall consequences for thermogenesis and body weight reduction need to be demonstrated in humans. It has been shown that increased BMP4 levels in mice induce a beige/brown and oxidative phenotype of the white adipose tissue of sufficient magnitude to increase energy expenditure and prevent induction of obesity.83 However, whether these findings can be translated to a possible therapeutic target in humans is currently unclear, considering the complex BMP signaling pathways and potential side effects. It is clear that the quantity of induced beige/brown adipocytes required to increase energy expenditure is considerably higher than

CHAPTER 23  Adipose Tissue Function: Metabolic and Endocrine that required by normal and functional BAT. Considering that the UCP1 mRNA levels are around 1000-­fold higher in brown than in white adipose cells, it has been calculated that a relevant browning agent needs to raise the amount of UCP1 in white adipose tissue by 10-­ fold in order to have around 1% of the amount in BAT.84 Although the pharmaceutical industry is trying to develop browning agents suitable for humans, no such agent is currently available, and it is not clear if this will become a possibility. KEY POINTS  • Brown adipose cells are present in small amounts in humans and are characterized by large lipid droplets and being more beige/brite than brown. Unlike murine cells, they only have small amounts of UCP1 and mitochondria. Although they respond to sympathetic activation, it is unlikely that they can increase metabolism to an extent needed to reduce body weight.

Aging Reduces Brown and Beige Adipose Cells Aging is associated with profound changes in most organs and tissues, including BAT in both rodents and humans.79 This reduction in BAT is more likely to have consequences for thermogenic regulation and propensity to increase body weight in animals than in humans. It has also been shown that the number of beige/brown cells in the white adipose tissue in rodents and humans decrease with age, probably as a consequence of both cell senescence and reduced sympathetic activity. Thus, aging contributes to the loss of both BAT mass and number of beige/brown cells.

Summary BAT is essential for energy homeostasis in rodents. The tissue is present in humans as well, but appears less important from a physiological and pathophysiological point of view. Thus, it is doubtful if human BAT can be used as a target for treating obesity and its comorbidities.

ADIPOSE TISSUE ENDOCRINE FUNCTIONS AND CHANGES IN OBESITY It was already well-­established in the 1960s that white adipose tissue releases glycerol, fatty acids, lactate, and perhaps some less abundant lipid species into the circulation. The pathophysiological role of these factors is described in detail under the lipid turnover section. In contrast, the endocrine function of adipose tissue and release of peptide factors and hormones, collectively termed “adipokines,” long remained unknown. Admittedly, parabiosis experiments in the 1950s using wild-­type and severely obese ob/ob mice suggested that there was an adipose-­derived satiety signal impacting on food intake. However, it was not until 1994, when leptin was cloned, that adipose tissue began to be considered a true endocrine organ.76 Leptin is a peptide hormone belonging to the cytokine family and is primarily released from adipocytes in direct proportion to fat mass. One of its main actions is to attenuate hunger signals in the hypothalamus, and it therefore has a true endocrine function. Leptin is the gene that is disrupted in ob/ob animals, and leptin administration effectively normalizes food intake and body weight in animal models. Big clinical hopes were therefore put on leptin as a treatment modality in common forms of obesity. Unfortunately, leptin levels are very high in people with obesity, and the response to exogenous leptin is nonsignificant, possibly due to “leptin resistance.”85 Leptin missense mutations are uncommon in humans but have been reported in a few families around the world. This results in massive obesity in early childhood, and in these cases recombinant leptin administration (metreleptin) is very effective.

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Leptin is also used in rare lipodystrophic conditions (with low circulating levels) and is believed to exert its action by reducing food and lipid intake, thereby normalizing the hypertriglyceridemia associated with these conditions.86 More recently, metreleptin therapy has also been suggested for other leptin-­deficient states, including anorexia nervosa.87 KEY POINTS  • Leptin remains, to date, the best characterized adipokine with a classical endocrine mechanism of action. While it does not display any relevant effects in the treatment of common forms of obesity, it has an important therapeutic role, particularly in rare forms of leptin deficiency.

The identification of leptin sparked an intense search for additional factors released by adipose tissue. Adipose tissue expresses up to one third of the proteins encoded by the genome, and this includes several hundred potentially secreted proteins. Proteomics screens of different human and murine adipocytes/adipose tissue systems have collectively identified greater than 600 released peptides/proteins.88 This does not imply that all are actively secreted, as some factors may be released through cell necrosis/apoptosis or just under the culture conditions used ex vivo. Peptide factors released from adipose tissue include cyto-­/chemokines (discussed in the immunometabolism section), growth factors (e.g., TGFβ), angiogenic factors (e.g., vascular endothelial growth factor), blood pressure regulators (e.g., angiotensinogen), and structural proteins (e.g., collagens). Importantly, with the exception of leptin and adiponectin (discussed later), very few of these “adipokines” are specific for adipose tissue, and most are expressed in several other organs. Furthermore, in addition to peptides/proteins, adipose tissue releases a large set of nonpolar and polar metabolites, apart from fatty acids, sphingolipids, and prostaglandins; these include, e.g., amino acids, lactate, and nitric oxide.89 The role of some of these factors is briefly discussed later, but it is not possible to cover everything that is released/secreted from adipose tissue. This section will therefore highlight some of the most well-­studied adipokines, which are also summarized in Table 23.4. Among these is adiponectin, a protein belonging to the complement factor family that is secreted almost exclusively from adipocytes. Adiponectin was cloned by several different groups shortly after the discovery of leptin and is secreted as a tri-­, hexa-­, or multimeric complex. It is now generally thought that the multimeric complex is the biologically active form and that it binds and activates two distinct receptors, AdipoR1 and AdipoR2, which display the highest expression in liver and skeletal muscle.90 Adiponectin is released into the circulation, and is therefore considered an endocrine hormone, but in contrast to many other factors secreted from adipose tissue, the levels decrease with increased fat mass and insulin resistance. Adiponectin levels are therefore considered a good marker of metabolic risk. However, despite almost 25 years of research, the pathophysiological role of adiponectin in humans remains unclear, and most studies have been performed in murine models. In rodents, adiponectin improves insulin sensitivity, normalizes adipose tissue lipolysis, reduces hepatic gluconeogenesis, and increases skeletal muscle glucose uptake and fatty acid oxidation.91 Despite these interesting effects, it has proven very difficult to translate this into efficient therapies in humans. Whether this is due to the complex structure of biologically active adiponectin, that it is more difficult to efficiently activate adiponectin receptors in humans, or that it simply plays another role in humans versus rodents is not clear. Leptin and adiponectin remain to date the only true endocrine adipokines, i.e., factors released from the adipose tissue and exerting

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TABLE 23.4 Adipokines Factor, Main Function

Most Important Effects

Free fatty acids, endocrine factors

Increase very low density lipoprotein; gluconeogenesis; reduce glucose uptake in skeletal muscle; effects on insulin secretion? Reduced appetite; increased fatty acid oxidation; plays a role in reproduction

Leptin, endocrine factor

TNFα, autocrine or paracrine factor CCL2, paracrine factor Adiponectin, endocrine factor Interleukins, IL1-­beta, IL6, and IL8, paracrine factors Acylstimulating protein, paracrine factor Plasminogenactivatorinhibitor-­1, endo-­, paracrine factor Visfatin, paracrine factor Osteopontin

Stimulates lipolysis and inhibits insulin action in fat cells Chemoattractant factor that stimulates macrophage infiltration Protective against many pernicious metabolic effects Unclear role; promote an inflammatory role and may attenuate insulin signaling Stimulates lipid deposition; attenuates fatty acid release Promotes thrombosis Regulates inflammation Regulates inflammation

KEY POINTS  • Adiponectin is well-­studied, particularly in rodent models, where it plays important roles in regulating insulin sensitivity. However, its therapeutic role in humans remains unclear, and its druggability is hampered by the large size of the biologically active multimeric complexes.

effects through distinct receptors in another organ. Retinol-­binding protein-­4 (RBP4) is released from adipose tissue and is present at higher levels in the circulation of people with obesity/insulin resistance. RBP4 is a biomarker for insulin resistance and cardiometabolic risk, but to date an established mechanistic link has not been identified.18 Virtually all other adipokines released from adipose tissue are believed to act locally in an auto-­or paracrine fashion. Among these are, e.g., acylstimulating protein (ASP, also termed C3adesArg), plasminogen activator inhibitor-­1 (PAI1), visfatin (also termed pre B-­cell colony-­ enhancing factor [PBEF] or nicotinamide phosphoribosyltransferase [NAMPT]), and osteopontin, to name a few. ASP is formed by enzymatic cleavage of the precursor protein C3 into C3a, initiated by the enzymes adipsin and factor B. ASP seems to play a role in triglyceride synthesis and hydrolysis, and increases fatty acid esterification and inhibits fatty acid release. The function of ASP is considerably attenuated in familial combined hyperlipidemia, although the mechanisms are not yet understood. A caveat in studying ASP is that rodents do not express relevant parts of the ASP system in adipose tissue, which has hampered further investigations. PAI1 is a prothrombogenic factor released from several tissues, including adipose tissue, and the levels are increased in the obese state. As discussed elsewhere, it is also a marker of cellular senescence, and it has been speculated that it could play a role in linking obesity to cardiovascular complications.

Pathophysiology (Changes in Obesity)

Main Cellular Source in Adipose Tissue

Increased release primarily through non–hormone-­stimulated lipolysis, reduced antilipolytic sensitivity to insulin contributes Increased release, primarily from subcutaneous depots; leptin resistance in obesity Increased

Adipocytes

Increased Reduced

Adipocytes

Macrophages and possibly other cells in the stroma vascular fraction Fat cells, immune cells, and other cells in the stroma vascular fraction Adipocytes

Increased

Fat cells, immune cells, and other cells in the stroma vascular fraction

Increased

Fat cells, and other cells in the stroma vascular fraction Cells in the stroma vascular fraction

Increased Increased Increased

Macrophages Fat cells, immune cells, and other cells in the stroma vascular fraction

Visfatin, which is identical to the previously described proteins PBEF and NAMPT, is an enzyme originally described as deriving from visceral fat macrophages. However, visfatin is also expressed in other organs, including muscle and liver. The expression of visfatin is associated with tissue inflammation and a pernicious metabolic profile, as well as atherosclerosis, but the casual links and the role of adipose tissue remain unclear.92 Osteopontin is a glycophosphoprotein expressed in several different cell types, including osteoblasts/osteocytes, where it is involved in bone metabolism and remodelling.93 However, it is also expressed in adipose tissue, where it is believed to regulate the secretion of proinflammatory cytokines from different immune cells. Plasma concentrations of osteopontin and osteopontin mRNA in adipose tissue are upregulated in obesity and insulin resistance. Similar to many other adipokines, the causal role remains to be established.

Summary White adipose tissue expresses at least two adipokines (leptin and adiponectin) with a clear endocrine function, in that they target other organs/tissue via specific receptors. Several other factors are released and present in the circulation, but these are not unique to adipose tissue per se, and the contribution to the circulating concentrations versus other peripheral organs is not yet clear. The latter can only be determined by comparing arteriovenous differences in vessels going to and from different tissues using clinical techniques that only very few groups in the world can perform. In some cases the receptors or signaling mechanisms of these adipokines remain to be elucidated. Most of the adipokines released from adipose tissue are believed to act locally and seem to play important roles in determining tissue function and lipid storage capacity. Obesity perturbs the expression of adipokines and in most cases leads to an increase in the expression of, e.g., leptin, proinflammatory proteins, and extracellular matrix genes.

CHAPTER 23  Adipose Tissue Function: Metabolic and Endocrine

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TABLE 23.5  Adipose Tissue–Targeted Therapies for Human Obesity and its Comorbidities

in Current Use Treatment

Action

Restricted energy intake

1. Reduce adipose mass 2. Bariatric surgery long-­term effective 3. Metabolic effect related to decrease fat cell size 1. Omentectomy has metabolic effects in connection with gastric banding, but not together with gastric bypass 2. Abdominal subcutaneous liposuction has no metabolic effects 1. No important effect on body weight 2. Small metabolic effects 1. Primarily used for diabetes treatment 2. Reduce body weight, sometimes effectively, and are currently used for this purpose. Probably have an indirect effect on adipose tissue 3. Increased subcutaneous adipose lipid storage causing decreased fatty acid levels 4. Antidiabetic, but risk of important side effects such as weight gain 1. Biguanides may lead to some weight reduction 2. Sodium-­glucose cotransporter 2 inhibitors may cause modest weight reduction 3. Probably indirect effects on adipose tissue 1. Antilipolytic causing improved lipid profile but no change in body weight 2. May cause glucose intolerance 1. Enhances catecholamine-­induced lipolysis 2. Long-­term effects on body weight and metabolism are unknown 1. Decreased appetite. Increased energy expenditure 2. Not effective in common obesity 1. Neutralizing adipose inflammation 2. No clear effect on diabetes or insulin sensitivity 3. Body weight neutral

Surgical removal of adipose tissue Activation of brown adipose tissue Glucagon-­like peptides

Thiazolidinediones Other antidiabetic agents

Nicotinic acids Endurance training Leptin Antibodies against TNFα

The exception to this rule is adiponectin, which is one of the few adipokines negatively regulated by increased fat mass. As mentioned, the role of different locally released metabolites is not discussed herein, but it is interesting to note that several of them (e.g., lactate, succinate, and beta-­hydroxybutyrate) have been shown to bind and activate G-­protein coupled receptors, which are expressed on both adipocytes and immune cells.94 This implies that the number of “adipokines” could be substantially higher than what has so far been appreciated. Moreover, the levels of several amino acids (e.g., glutamine, branched-­ chain amino acids) and their derivatives (e.g., kynurenic acid) are altered in the obese state and impact adipose tissue function via both epigenetic (see immunometabolism section) and receptor-­mediated pathways. This area of research is therefore still very active, and novel insights are reported continuously.

ADIPOSE TISSUE AS A THERAPEUTIC TARGET Bearing in mind the multiple actions of adipose tissue, numerous site-­ directed therapeutic modalities are possible and have been tested in animal models. Those tried in human obesity and its comorbidities will be discussed and are summarized in Table 23.5. The most obvious approach is to decrease fat mass by means of reduced energy intake. Different diets have been publicly available since Banting published a formula based on reduced intake of carbohydrates and starch in 1863.95 A weight reduction of 5% or more usually leads to metabolic improvements, which could be further boosted in combination with enhanced physical activity. However, no diet is known to exert long-­ term effects on obesity remission in the general population.95 In fact, often the end result is higher body weight compared with the weight before starting the diet (“yo-­yo” effect). In contrast, bariatric surgery leads to long-­term reduction in fat mass and is accompanied by positive effects on diabetes incidence/remission, mortality, and several

other obesity complications.96 There are different surgical procedures. Earlier techniques are technically easier but result in less marked weight loss. The most common current operation procedures, such as gastric bypass and sleeve gastrectomy, are much more effective in terms of weight loss and are also sufficiently safe.97 After an initial and marked weight loss, body weight increases gradually to a new long-­term steady state, which is approximately 25% below the preoperative body weight. The mechanism behind the beneficial effects of body weight loss are not well understood but are likely to be multifaceted. Improved insulin sensitivity is linked more closely to diminished fat cell size than reduced fat mass per se.98 In theory, surgical removal of adipose tissue is an attractive antiobesity therapy. However, large amounts adipose tissue cannot be safely removed, due to immediate and long-­term risks of severe complications. Therefore, selective pernicious adipose areas that are relatively easy to remove have been taken out since the turn of the last century. The major omentum has been removed in connection with bariatric surgery.99 Positive effects were reported in connection to gastric binding but with gastric bypass. The reason for this difference might be the stronger effect on weight reduction of the latter surgical procedure. Liposuction is often used for cosmetic surgery, but removal of approximately 10 kg of abdominal SAT in obesity had no effect on the cardiometabolic risk profile, on either a short-­or long-­term basis.100 KEY POINTS  • So far bariatric surgery is the only proven effective means of reducing body weight for a long time in subjects with common forms of obesity. Even modest reductions in body weight have a positive effect on metabolic obesity complications. The ameliorating effect of weight reduction is not likely to be due to diminished fat mass per se. Instead, the reduced fat cell volume is likely to play such a role.

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Pharmacotherapy in Obesity The approved antiobesity medications are covered in another chapter. Herein only adipose-­specific therapies are discussed in some detail. Although many pharmacological agents have been tried, usually as a means to suppress appetite, most have been withdrawn from the market or are no longer used due to side effects.101 These agents act centrally, and are thus not important for fat cell–mediated actions. Glucagon-­like peptide 1 receptor agonists have been shown to induce long-­term, and in some cases marked, body weight reduction. Although receptors for the hormone are present in human fat cells and cause metabolic effects, they cannot be linked to effects on body weight. Biguanides may also reduce body weight, but the effect is usually modest (circa 3%). Although metabolic/endocrine effects on human adipose tissue are described, they are not important for the effect on body weight, and they are not approved for weight reduction Activation of BAT through stimulation with beta-­3 adrenoceptor agonists has been tested clinically.102 This issue was thoroughly discussed earlier. TZDs are enhancers of insulin sensitivity and have been used to treated type 2 diabetes in obese/overweight patients.103 Much of their antidiabetic effect is caused by activation of the nuclear receptor PPARγ in fat cells. This leads to retention of lipids, predominantly in SAT, and consequent lowering of circulating fatty acid levels, which, in turn, improves insulin action/secretion. However, these agents currently have limited use due to side effects such as weight gain (due to hyperplastic expansion of SAT), fluid retention, osteoporosis, and (at least for older agents) adverse cardiac events. Nicotinic acid and its derivatives have long been used to treat hypertriglyceridemia, which often accompanies obesity.104 One mechanism is through inhibition of fat cell lipolysis so that fewer fatty acids are delivered to the liver for synthesis of very low density lipoproteins. These drugs are body weight–neutral but may cause glucose intolerance, and they have been withdrawn from the market due to side effects. The effects of exercise training on human adipose tissue function has been studied for many years.105 If not compensated by increased energy intake, some body weight loss can be expected. Several exercise effects on adipose endocrine and metabolic functions, as well as on inflammation, have been reported. Most promising from a therapeutic point of view is that endurance training induces a long-­term increase in the lipolytic effect of catecholamines. However, long-­term effects of enhanced physical activity alone on body weight and metabolic profile are not documented. As regards endocrine function and inflammation, only leptin and TNFα have been intensely investigated in humans to date.90,106 These therapies were discussed thoroughly earlier. KEY POINTS  • In theory, a number of fat cell–specific regulatory factors such as adipocyte differentiation, beta oxidation, lipolysis, and the endocrine functions are potential pharmaceutical targets for combatting obesity and dysregulated metabolism. So far, this therapeutic area is not very advanced; the only important exception hitherto are leptin and thiazolidinediones.

Summary Other than bariatric surgery, no alternative adipose-­targeted therapies have so far demonstrated clear-­cut and long-­term effectiveness in the treatment of obesity and its comorbidities in the general population.

It is possible, though, that agents and their analogs derived from the gastrointestinal tract can be used as fruitful alternatives to bariatric surgery. Some important targets according to mouse models, such as adiponectin and hormone-­sensitive lipase, have not yet been tested in humans. In the future, gene therapy might be useful. For example, several long noncoding RNAs are fat cell–specific and functional, and could be useful tools to improve fat cell function.107

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47. Everett BM, Donath MY, Pradhan AD, et al. Anti-­inflammatory therapy with canakinumab for the prevention and management of diabetes. J Am Coll Cardiol. 2018;71:2392–2401. 48. Donath MY, Dinarello CA, Mandrup-­Poulsen T. Targeting innate immune mediators in type 1 and type 2 diabetes. Nat Rev Immunol. 2019;19:734–746. 49. Shimobayashi M, Albert V, Woelnerhanssen B, et al. Insulin resistance causes inflammation in adipose tissue. J Clin Invest. 2018;128:1538–1550. 50. Crewe C, An YA, Scherer PE. The ominous triad of adipose tissue dysfunction: inflammation, fibrosis, and impaired angiogenesis. J Clin Invest. 2017;127:74–82. 51. Petrus P, Lecoutre S, Dollet L, et al. Glutamine links obesity to inflammation in human white adipose tissue. Cell Metab. 2020;31:375–390 e11. 52. Morigny P, Boucher J, Arner P, et al. Lipid and glucose metabolism in white adipocytes: pathways, dysfunction and therapeutics. Nat Rev Endocrinol. 2021;17:276–295. 53. Luo L, Liu M. Adipose tissue in control of metabolism. J Endocrinol. 2016;231:R77–R99. 54. Thiam AR, Beller M. The why, when and how of lipid droplet diversity. J Cell Sci. 2017;130:315–324. 55. White U, Ravussin E. Dynamics of adipose tissue turnover in human metabolic health and disease. Diabetologia. 2019;62:17–23. 56. Chouchani ET, Kajimura S. Metabolic adaptation and maladaptation in adipose tissue. Nat Metab. 2019;1:189–200. 57. Karpe F, Pinnick KE. Biology of upper-­body and lower-­body adipose tissue–link to whole-­body phenotypes. Nat Rev Endocrinol. 2015;11:90–100. 58. Oikonomou EK, Antoniades C. The role of adipose tissue in cardiovascular health and disease. Nat Rev Cardiol. 2019;16:83–99. 59. Walker GE, Marzullo P, Ricotti R, et al. The pathophysiology of abdominal adipose tissue depots in health and disease. Horm Mol Biol Clin Investig. 2014;19:57–74. 60. Santosa S, Jensen MD. The sexual dimorphism of lipid kinetics in humans. Front Endocrinol (Lausanne). 2015;6:103. 61. Jensen MD. Visceral fat: culprit or canary? Endocrinol Metab Clin North Am. 2020;49:229–237. 62. Spalding KL, Bernard S, Naslund E, et al. Impact of fat mass and distribution on lipid turnover in human adipose tissue. Nat Commun. 2017;8:15253. 63. Arner P, Bernard S, Appelsved L, et al. Adipose lipid turnover and long-­ term changes in body weight. Nat Med. 2019;25:1385–1389. 64. McQuaid SE, Hodson L, Neville MJ, et al. Downregulation of adipose tissue fatty acid trafficking in obesity: a driver for ectopic fat deposition? Diabetes. 2011;60:47–55. 65. Hames KC, Koutsari C, Santosa S, et al. Adipose tissue fatty acid storage factors: effects of depot, sex and fat cell size. Int J Obes. 2015;39:884– 887. 66. Andrade AC. Lipoprotein lipase: a general review. Insights Enzyme Res. 2018;2:1–14. 67. Zechner R, Madeo F, Kratky D. Cytosolic lipolysis and lipophagy: two sides of the same coin. Nat Rev Mol Cell Biol. 2017;18:671–684. 68. Arner P, Langin D. Lipolysis in lipid turnover, cancer cachexia, and obesity-­induced insulin resistance. Trends Endocrinol Metab. 2014;25:255–262. 69. Nielsen TS, Jessen N, Jorgensen JO, et al. Dissecting adipose tissue lipolysis: molecular regulation and implications for metabolic disease. J Mol Endocrinol. 2014;52:R199–R222. 70. Stenkula KG, Erlanson-­Albertsson C. Adipose cell size: importance in health and disease. Am J Physiol Regul Integr Comp Physiol. 2018;315:R284–R295. 71. Morigny P, Houssier M, Mouisel E, et al. Adipocyte lipolysis and insulin resistance. Biochimie. 2016;125:259–266. 72. Arner P, Andersson DP, Backdahl J, et al. Weight gain and impaired glucose metabolism in women are predicted by inefficient subcutaneous fat cell lipolysis. Cell Metab. 2018;28:45–54 e3. 73. Goodpaster BH, Sparks LM. Metabolic flexibility in health and disease. Cell Metab. 2017;25:1027–1036. 74. Rabinowitz JD, Enerback S. Lactate: the ugly duckling of energy metabolism. Nat Metab. 2020;2:566–571.

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75. Mann JP, Savage DB. What lipodystrophies teach us about the metabolic syndrome. J Clin Invest. 2019;129:4009–4021. 76. Rosen ED, Spiegelman BM. What we talk about when we talk about fat. Cell. 2014;156:20–44. 77. Cinti S. UCP1 protein: the molecular hub of adipose organ plasticity. Biochimie. 2017;134:71–76. 78. Cannon B, de Jong JMA, Fischer AW, et al. Human brown adipose tissue: classical brown rather than brite/beige? Exp Physiol. 2020;105:1191– 1200. 79. Zoico E, Rubele S, De Caro A, et al. Brown and beige adipose tissue and aging. Front Endocrinol. 2019;10:368. 80. Baskin AS, Linderman JD, Brychta RJ, et al. Regulation of human adipose tissue activation, gallbladder size, and bile acid metabolism by a beta3-­adrenergic receptor agonist. Diabetes. 2018;67:2113–2125. 81. Choi SS, Kim ES, Jung JE, et al. PPARgamma antagonist gleevec improves insulin sensitivity and promotes the browning of white adipose tissue. Diabetes. 2016;65:829–839. 82. Gustafson B, Hammarstedt A, Hedjazifar S, et al. BMP4 and BMP antagonists regulate human white and beige adipogenesis. Diabetes. 2015;64:1670–1681. 83. Hoffmann JM, Grunberg JR, Church C, et al. BMP4 gene therapy in mature mice reduces bat activation but protects from obesity by browning subcutaneous adipose tissue. Cell Rep. 2017;20:1038–1049. 84. Maurer S, Harms M, Boucher J. The colorful versatility of adipocytes: white-­to-­brown transdifferentiation and its therapeutic potential in man. FEBS J. 2020;288:3628–3646. 85. Farr OM, Gavrieli A, Mantzoros CS. Leptin applications in 2015: what have we learned about leptin and obesity? Curr Opin Endocrinol Diabetes Obes. 2015;22:353–359. 86. Sollier C, Vatier C, Capel E, et al. Lipodystrophic syndromes: from diagnosis to treatment. Ann Endocrinol (Paris). 2020;81:51–60. 87. Hebebrand J, Milos G, Wabitsch M, et al. Clinical trials required to assess potential benefits and side effects of treatment of patients with anorexia nervosa with recombinant human leptin. Front Psychol. 2019;10:769. 88. Kim EY, Kim WK, Oh KJ, et al. Recent advances in proteomic studies of adipose tissues and adipocytes. Int J Mol Sci. 2015;16:4581–4599. 89. Scheja L, Heeren J. The endocrine function of adipose tissues in health and cardiometabolic disease. Nat Rev Endocrinol. 2019;15:507–524. 90. van Andel M, Heijboer AC, Drent ML. Adiponectin and its isoforms in pathophysiology. Adv Clin Chem. 2018;85:115–147. 91. Funcke JB, Scherer PE. Beyond adiponectin and leptin: adipose tissue-­derived mediators of inter-­organ communication. J Lipid Res. 2019;60:1648–1684.

92. Audrito V, Messana VG, Deaglio S. NAMPT and NAPRT: two metabolic enzymes with key roles in inflammation. Front Oncol. 2020;10:358. 93. Icer MA, Gezmen-­Karadag M. The multiple functions and mechanisms of osteopontin. Clin Biochem. 2018;59:17–24. 94. Husted AS, Trauelsen M, Rudenko O, et al. GPCR-­mediated signaling of metabolites. Cell Metab. 2017;25:777–796. 95. Dombrowski SU, Knittle K, Avenell A, et al. Long term maintenance of weight loss with non-­surgical interventions in obese adults: systematic review and meta-­analyses of randomised controlled trials. BMJ. 2014;348:g2646. 96. Xia Q, Campbell JA, Ahmad H, et al. Bariatric surgery is a cost-­saving treatment for obesity-­A comprehensive meta-­analysis and updated systematic review of health economic evaluations of bariatric surgery. Obes Rev. 2020;21:e12932. 97. Chang SH, Stoll CR, Song J, et al. The effectiveness and risks of bariatric surgery: an updated systematic review and meta-­analysis, 2003-­2012. JAMA Surg. 2014;149s:275–287. 98. Andersson DP, Eriksson Hogling D, Thorell A, et al. Changes in subcutaneous fat cell volume and insulin sensitivity after weight loss. Diabetes Care. 2014;37:1831–1836. 99. Lee Y, Pedziwiatr M, Major P, et al. The effect of omentectomy added to bariatric surgery on metabolic outcomes: a systematic review and meta-­analysis of randomized controlled trials. Surg Obes Relat Dis. 2018;14:1766–1782. 100. Wu S, Coombs DM, Gurunian R. Liposuction: concepts, safety, and techniques in body-­contouring surgery. Cleve Clin J Med. 2020;87:367– 375. 101. May M, Schindler C, Engeli S. Modern pharmacological treatment of obese patients. Ther Adv Endocrinol Metab. 2020;11:2042018819897527. 102. Trayhurn P. Brown adipose tissue-­a therapeutic target in obesity? Front Physiol. 2018;9:1672. 103. Soccio RE, Chen ER, Lazar MA. Thiazolidinediones and the promise of insulin sensitization in type 2 diabetes. Cell Metab. 2014;20: 573–591. 104. Garg A, Sharma A, Krishnamoorthy P, et al. Role of niacin in current clinical practice: a systematic review. Am J Med. 2017;130:173–187. 105. Giolo De Carvalho F, Sparks LM. Targeting white adipose tissue with exercise or bariatric surgery as therapeutic strategies in obesity. Biology (Basel). 2019;15:16. 106. Paz-­Filho G, Mastronardi CA, Licinio J. Leptin treatment: facts and expectations. Metabolism. 2015;64:146–156. 107. Squillaro T, Peluso G, Galderisi U, et al. Long non-­coding RNAs in regulation of adipogenesis and adipose tissue function. Elife. 2020;9:e, 1–15.

24 Lipodystrophy Syndromes Angeliki M. Angelidi, Michael A. Tsoukas, and Christos S. Mantzoros

OUTLINE Lipodystrophies: Definition and Diagnosis, 379 Pathophysiology of Lipodystrophy, 380 Classifications of Lipodystrophies and Their Clinical Manifestations, 381 Generalized Lipodystrophies, 381 Other Mutations Associated With a Congenital Generalized ­Lipodystrophy–Like Phenotype, 383 Partial Lipodystrophies, 384 Localized Lipodystrophies, 389 Mechanisms Responsible for Severe Insulin Resistance, 389 Fat Redistribution and Fat Metabolism, 389 Adipocytokines, 389 Inflammation, 390

Mitochondrial Stress, Oxidative Stress, and the Endoplasmic Reticulum, 390 Other Mechanisms, 390 Treatment of Syndromes of Lipodystrophies, 390 Lifestyle Modification, 390 Management of Insulin Resistance, 391 Management of Dyslipidemia, 392 Management of Human Immunodeficiency Virus–Infected Patients With Highly Active Antiretroviral Therapy–Induced Metabolic Syndrome, 393 Management of Cosmetic Appearance, 394 Adipokines in Lipodystrophy, 394 Future Perspectives, 395 Adiponectin, 395

  KEY POINTS  • Lipodystrophy syndromes are a heterogeneous group of disorders characterized by complete or partial lack of adipose tissue in certain areas of the body, with excess of adipose tissue elsewhere. • The etiology of lipodystrophy may be either a congenital or acquired and involves a loss of mature, functional adipocytes associated with failure of adipogenesis, adipocyte apoptosis, or storage of triglycerides. • Congenital lipodystrophy is caused by abnormal gene activations and signaling that impair adipocyte differentiation, whereas acquired lipodystrophy is most commonly observed in patients with human immunodeficiency virus who are receiving highly active antiretroviral therapy. • Metabolic complications, such as insulin resistance, dyslipidemia, hepatic steatosis, endovascular inflammation, and oxidative stress, are associated with lipodystrophy syndromes and constitute major comorbidities of these syndromes. • Current treatments such as metformin, thiazolidinediones, growth hormone replacement, growth hormone–releasing hormone analogs, and US Food and Drug Administration–approved recombinant leptin therapy may help with clinical features and associated metabolic complications of l­ipodystrophy.

LIPODYSTROPHIES: DEFINITION AND DIAGNOSIS Lipodystrophy syndromes are a diverse group of rare clinical disorders, the central feature of which is either a congenital or acquired complete or partial lack of adipose tissue (lipoatrophy) and/or a combination of lack of adipose tissue in certain body areas, with excess of adipose tissue (lipohypertrophy) elsewhere.1 Despite progress in

identifying the genetic basis and pathways of the disease, lipodystrophy is often underestimated or misdiagnosed because of its heterogeneity and rarity. According to the most recent multi-­society practice guidelines, diagnosis of a lipodystrophy disorder is based on patient’s history, clinical examination, metabolic state, and body composition.2 Metabolic disorders, and especially a severe form of the metabolic syndrome, are also implicated in this abnormal deposition of adipose tissue that cannot be stored in the appropriate subcutaneous depots.3 Abnormalities such as insulin resistance and its associated clinical features are present in nearly all varieties of lipodystrophies.4 In addition, patients with lipodystrophies also manifest a group of unique features such as hypertriglyceridemia and severe mixed dyslipidemia, progressive hepatic steatosis, progression to poorly controlled diabetes, and an increased metabolic rate (Fig. 24.1).5 Severe dysmetabolic situations associated with lipodystrophy syndromes are responsible for the increased risk of severe comorbidities and mortality in these patients.6 No firm diagnostic criteria currently exist, and a lipodystrophy disorder is usually recognized clinically, often based on both patients’ and doctors’ perceptions and assessment. A meticulous physical examination is required and can be further supported by anthropometry, dual energy x-­ray absorptiometry (DEXA), computed tomography (CT), and whole-­body magnetic resonance imaging (MRI). Anthropometry, or measurement of skin folds and limb circumference, is an easy, affordable, and practical way to estimate fat loss and fat redistribution. However, its reliability is heavily dependent on the consistency and the skill of the examiner(s), and it may have poor sensitivity, especially during early stages of the disease. Objective measurement of facial lipoatrophy also poses a challenge. Serial photographs (with the patient’s consent) have been used to document and compare the facial wasting over time.

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DEXA, CT, and MRI scans are additional modalities that have been developed to allow direct quantification of fat within specific tissues and/or body mass, as well as fat distribution. Although all these techniques are accurate and noninvasive, they are also expensive, have limited availability, and are not cost-­effective for use in everyday clinical practice. Their use, therefore, has been limited to the research field.7

In the past few years, ultrasound has emerged as a promising alternative quantitative tool to assess body fat changes. Although it demonstrates good accuracy and accessibility, more studies are needed to elucidate its value and to comparatively evaluate ultrasound with other imaging modalities. Standardization of ultrasound techniques and analyses of cost-­effectiveness will also be essential for its potential wider clinical applications.8 Table 24.1 summarizes the main advantages and disadvantages of the aforementioned body composition and adipose tissue imaging techniques. Laboratory testing and imaging are valuable tools for physicians to support their diagnosis and determine the type and severity of lipodystrophy. Patients with lipodystrophy tend to have low serum leptin levels. It should be noted that no defined serum leptin level threshold can be used to confirm or exclude the diagnosis of lipodystrophy.2,9,10 Genetic testing is the gold standard and may assist in clinical diagnosis confirmation.11 However, because of limited resources and DNA sequencing availability, the current recommendations suggest that genetic testing should only be considered in suspected familial lipodystrophies and at-­risk family members.2 It should be underlined that negative genetic tests cannot rule out the presence of a genetic lipodystrophy syndrome.

PATHOPHYSIOLOGY OF LIPODYSTROPHY

Fig. 24.1  Congenital lipodystrophy.

In previous decades, the study of mechanisms underlying lipodystrophy has attracted significant attention, to a certain extent owing to the interest of the scientific community in obesity research. It is now recognized that white adipose tissue is not an inert storage depot organ but an active endocrine organ that plays a critical role in regulating energy homeostasis and mediating inflammation.12 Because there are common features in the etiopathogenesis of lipodystrophy and obesity, lessons learned from studies of the lipodystrophies may provide essential information not only for the management of these rather rare cases, but also for obesity research and management. Much of our knowledge regarding the mechanisms underlying the pathogenesis and manifestations of lipodystrophies has been obtained

TABLE 24.1  Advantages and Disadvantages of Different Body Composition and Adipose Tissue

Imaging Techniques Ultrasound Advantages No radiation exposure Safe for repeated measurements Satisfactory reliability Easy to operate Portable and easily available Low cost Disadvantages Low accuracy Interpretation is more difficult, subjective, and affected by excessive edema Experience-­related No validated procedure or clinical protocols

Dual Energy X-­Ray Absorptiometry Computed Tomography

Magnetic Resonance Imaging

Low radiation exposure Well tolerated for repeated measures High accuracy and precision Information on total and regional percentage of fat Information on fat and fat-­free mass Moderate cost

High accuracy and precision Information on specific body fat compartments Assessment of subcutaneous and visceral fat and fat-­free mass

No radiation exposure Safe for repeated measurements High accuracy and precision Information on specific body fat compartments Body mass composition differentiation, assessment of subcutaneous and visceral fat

Moderate cost Not portable Radiation exposure (even low) Hard to distinguish subcutaneous and visceral fat Interpretation is affected by fat-­free mass hydration status and body thickness Experience-­related Variability among different versions and manufacturers

High cost Not portable Limited availability Radiation exposure Need for specific software (for fat-­free mass) and technical skill for image analysis

High cost Not portable Limited availability Requires specific software Long scan duration

CHAPTER 24  Lipodystrophy Syndromes through mouse studies and as a result of human genome sequencing. It is now understood that patients with lipodystrophy have primarily a loss of mature, functional adipocytes, as opposed to an absence of lipids in otherwise normal adipocytes.13,14,15 The underlying defects could be associated with failure of adipogenesis, adipocyte apoptosis, or a failure to store triglycerides in existing adipocytes because of ineffective lipogenesis or excessive lipolysis.16 Differentiation of adipocytes is controlled by a variety of mitogenic and adipogenic gene activations and signaling pathways. Abnormal development and differentiation of preadipocytes and adipocytes, as well as increased adipocyte apoptosis, may lead to the development of a particular lipodystrophy (Fig 24.2).16 Furthermore, the overall alterations in adiposity may result in the metabolic derangements and subsequent complications observed in patients with lipodystrophies.17,18,19 In this chapter, we will discuss the classifications of different types of lipodystrophies, their distinctive clinical presentations, our current understanding of the underlying mechanisms, and the recommended treatment modalities.

CLASSIFICATIONS OF LIPODYSTROPHIES AND THEIR CLINICAL MANIFESTATIONS

active but not mechanically important adipose tissue.10,23,30,31 Other somatic abnormalities that contribute to the abnormal appearance are acanthosis nigricans, a protuberant abdomen associated with hepatomegaly, and/or splenomegaly, hernias, prominent musculature, and acromegaloid features.24,27

Mesenchymal stem cells Pro-adipogenic Wnt signaling Committed pre-adipocyte

Adipocyte

The classification of lipodystrophies is based on distinct clinical presentation, unique patterns of adipose tissue deficiency (namely, generalized or partial lipodystrophy), and manner of acquisition (inherited and acquired forms). Thus, lipodystrophies are categorized into four major categories: congenital generalized lipodystrophy (CGL), acquired generalized lipodystrophy (AGL), familial partial lipodystrophy (FPL), and acquired partial lipodystrophy (APL). However, lipodystrophy syndromes include additional subtypes such as the progeroid syndromes, and it is likely that this classification may become inadequate as more genetic causes and pathophysiological mechanisms associated with lipodystrophy are identified.11

Mature adipocyte

Generalized Lipodystrophies

Apoptotic adipocyte material

Generalized lipodystrophy encompasses rare but clinically striking disorders that may be congenital (Berardinelli–Seip syndrome)20,21 or acquired (Lawrence syndrome).

Congenital Generalized Lipodystrophy. CGL, or Berardinelli-­ Seip congenital lipodystrophy (BSCL), was initially described in 1954 by Berardinelli in Brazil in two children, and 5 years later by Seip in Norway in three other patients.20,21 It is a rare syndrome that inherited in an autosomal recessive manner and is observed mainly in cases of parental consanguinity or in isolated communities, probably because of “founder effect.”22 It is characterized by near-­complete absence of body fat that may spare the supportive adipose tissue in the palms, soles, joints, and the area under the scalp, depending on the underlying genetic cause.23 CGL is also associated with the early onset and progressive development of several metabolic disorders and comorbidities.6,24 To date, it has been reported in approximately 300 patients of various ethnic backgrounds.24-­29 Babies with CGL are noted to have an abnormal appearance owing to absence of body fat that usually arises at birth or during the first year of life, whereas some specific subtypes of CGL may become prevalent during childhood or later.11 Adipose tissue is absent from not only subcutaneous, but also from intraabdominal sites, and the resultant prominent musculature causes a striking recognizable phenotype. MRI of the abdomen shows complete absence of intraabdominal, retroperitoneal, and subcutaneous fat, but a prominent fatty liver, as well as the presence of fat in certain anatomic sites such as the orbits, palms, and soles. Thus, this genetic defect results in poor development of metabolically

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Pre-adipocyte differentiation • Adipogenic stimuli: insulin, gIucocorticoids, IGF-1, prostaglandins • BSCL-2 (seipin) • AKT2 • PPARγ/RXRα and C/EBP β/δ Lipogenic gene activation • FAS • AGPAT • PTRF • ACC • DGAT

Apoptosis • ZMPSTE24 • LMNA

Fig. 24.2  Differentiation of adipocytes, demonstrating gene activation and signaling pathways, in relation to lipodystrophies: lipodystrophy genes and their associated protein products are shown in bold. Mesenchymal stem cells have the pluripotent ability to differentiate into osteocytes, myocytes, chondrocytes, stromal cells, or adipocytes. Wnt signal transduction pathways and other transcription factors lead to the development of a committed preadipocyte. Subsequently, adipogenic stimuli, such as insulin, glucocorticoids, IGF-­1, and prostaglandins, initiate cell changes that lead to differentiation into an adipocyte. Current data suggest that the BSCL-­2 gene, coding for seipin, and the AKT-­2 gene play a role in adipocyte differentiation, and their mutations are involved in congenital generalized lipodystrophy (CGL) type 2 and familial partial lipodsytrophy (FPL) type 4, respectively. CCATT–enhancer-­binding protein (C-­EBP) β/δ transcription factors stimulate factors PPARу, C-­EBPα, and sterol regulatory element–binding protein (SREBP) 1c, which are upregulated in this process; PPARу mutations are associated with FPL3, and intracellular lipid accumulation in an adipocyte is dependent on substrate availability. In response to a variety of lipogenic signals, the adipocyte matures as the size of the lipid droplets increases. Fatty acid synthase (FAS), acetyl coenzyme A carboxylase, and diacylglycerol acyltransferase (DGAT) expression are upregulated, which is necessary for the biosynthesis of phospholipids and triglycerides. AGPAT2 is responsible for acylating phosphatidic acid to synthesize triglycerides and phospholipids, and its mutation is involved in CGL1. PTRF regulates caveolae intracellularly, and its functional loss results in CGL4. Apoptosis of adipocytes is influenced by LMNA, encoding for nuclear lamina proteins, and the ZMPSTE24 gene product is required for posttranslational lamin A and C processing. Mutations in these genes lead to mandibuloacral dysplasia via premature nuclear disruption.

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Congenital generalized muscular weakness and cervical spine instability have also been reported in occasional cases.31 Many females have polycystic ovaries and may also present with enlarged clitoris, hirsutism, and absence of regular menstrual cycles or oligomenorrhea. Only a few affected women have had successful pregnancies, whereas affected men have normal fertility. Other rare manifestations of CGL include postpubertal focal lytic lesions in the long bones,32-­34 mild mental retardation,28 and hypertrophic cardiomyopathy.35-­37 The basal metabolic rate may also be increased. Although patients may have accelerated linear growth and advanced bone age during their childhood, they normally have normal or reduced heights as adults. Although both sexes are affected at similar rates, the multiple metabolic features associated with this syndrome tend to be more severe and develop earlier in girls. Children with CGL tend to have voracious appetites and accelerated growth; precocious menarche and/or pubarche, signaling early puberty, are rarely observed. Insulin resistance has been noted at an early age and may be present even at birth. Although diabetes is infrequently seen in infancy, its development is common during adolescence or young adulthood. It is rarely ketotic and is usually refractory to insulin therapy. Diabetic complications of nephropathy, retinopathy, acute pancreatitis, and hepatic steatosis are typically present and are a major cause of morbidity in affected individuals. Dyslipidemia poses a therapeutic dilemma as well. Hypertriglyceridemia is characterized by increased concentrations of very low-­density lipoproteins and chylomicrons, whereas serum high-­ density lipoprotein (HDL) is usually low. Individuals with CGL may develop severe hypertriglyceridemia even from the first months of disease that may lead to acute pancreatitis.38 Severely elevated triglycerides may not only provoke acute pancreatitis, but are also frequently related to the development of fatty liver disease. This commonly progresses to cirrhosis, which in many cases may be fatal. Serum adipocytokines, the hormones produced by adipose tissue (such as leptin and adiponectin), circulate in extremely low levels in CGL.10 Patients with CGL usually demonstrate low leptin levels (∼1.0 ng/mL), regardless of age or gender.39 At least four molecularly distinct forms of congenital lipodystrophy have been defined, with mutations in AGPAT2 and BSCL2 responsible for 95% of reported cases of CGL. Type 1 CGL (CGL1) is caused by AGPAT2 gene mutation, as first described in 1999. This gene has been mapped to chromosome 9q34.40,41 It encodes the enzyme 1-­ acylglycerol-­ 3-­ phosphate O-­acyltransferase 2 (AGPAT2), a lysophosphatidic acid acyltransferase isoform consisting of 278 amino acids localized within the endoplasmic reticulum (ER) membrane. It is highly expressed in white adipocytes42 and catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, a key intermediate in the biosynthesis of triacylglyceride and glycerophospholipids.43 AGPAT2 deficiency leads to a reduction of triglycerides inside the adipose organ and impaired signaling of critical elements engaged in adipogenesis such as PI3K/AKT and peroxisome proliferator-­activated receptor gamma (PPARγ).44 Only metabolically important adipose tissue (e.g., intraabdominal, intermuscular, subcutaneous, bone marrow) and not mechanically important adipose tissue (e.g., soles, palms, scalp, periarticular) are markedly reduced in patients with type 1 CGL. Forty-­two mutations of the AGPAT2 gene have been identified in 150 individuals, and the number of the related variants has increased over time.45,46 Two more novel mutations of AGPAT2 were recently observed in Italian individuals with CGL1.46 Genetic testing of the first case revealed a point mutation (c.430C>T) in exon 3 of the AGPAT2 gene. The patient, a 7-­year-­old boy, demonstrated clinical characteristics of CGL (namely lipoatrophy sparing the palms and soles, and muscle hypertrophy), as well as severe hypertriglyceridemia, elevated liver enzymes, and hepatic steatosis. The other case had a homozygous AGPAT2 missense variant

(c.475C>G, resulting in p. Arg159Cys) in exon 3. The 53-­year-­old female patient was diagnosed with lipoatrophic diabetes, bilateral proliferative retinopathy, diabetic and hypertensive nephropathy, hyperphagia, eruptive xanthomatosis, liver steatosis, and splenomegaly. The patient displayed signs of acromegaly (marked prognathism and enlarged hands and feet), muscle hypertrophy, phlebomegaly, acanthosis nigricans, and umbilical hernia. Type 2 CGL (CGL2) is associated with BSCL2 gene mutations including base substitutions, insertions, and deletions, events that can introduce a frameshift and/or a premature stop codon. This gene is located on chromosome 11q13 and encodes a 398–amino-­acid protein named seipin, as a tribute to Martin Seip.47 The BSCL2 gene mutation has been found in patients of European and Middle Eastern origins, as well as those from South America and southern Asia. At least 36 genetic mutations of BSCL2 have been identified in 167 individuals.45 Seipin is expressed diffusely in many tissues but predominantly in testis and brain47; its function in humans is largely unknown but includes adipocyte differentiation and lipid droplet formation.48 According to a study performed in yeast, seipin is important for lipid droplet morphology and perhaps assembly.26 Several studies indicate that BSCL2 expression is critical for normal adipogenesis in vitro, as cells lacking BSCL2 failed to induce expression of key lipogenic transcription factors (PPARγ and CCAAT/enhancer binding protein alpha [C/EBP-­α]), as well as enzymes (AGPAT2, DGAT2, and lipin 1).49-­51 BSCL2 mutations are usually related to a more predominant adipose tissue loss than in CGL1.26 CGL attributed to BSCL2 mutation appears to have a more severe disease phenotype than that owing to AGPAT2 mutation, with a higher incidence of premature death and a lower prevalence of partial and/or delayed onset of lipodystrophy.28 Furthermore, some patients with BSCL2 mutations present with cardiomyopathy even from a young age35 and have a higher prevalence of intellectual impairment.27,28 Compared with patients with CGL1, CGL2 patients have a more pronounced absence of adipose tissue. In addition to the loss of metabolically active fat (subcutaneous regions, intermuscular regions, bone marrow, intraabdominal and intrathoracic regions), CGL2 patients also lack mechanical fat (orbital regions, palms, soles, and joints).23 Both type 3 and type 4 are rarely observed and demonstrate some discrete clinical characteristics that are not present in type 1 or 2. Pathogenic variants of the caveolin 1 (CAV1) gene cause type 3 CGL, whereas variants in the polymerase I and transcript release factor (PTRF) gene are responsible for the development of type 4.52,53 Type 3 CGL (CGL3) is caused by CAV1 gene mutations and was first described in 2008.52 CAV1 is located on chromosome 7q31, and its end product, caveolin 1, is a highly conserved 22-­kD protein. The latter is a crucial component of plasma membrane microdomains known as caveolae. These domains have important roles in regulating signaling pathways and processes such as cell migration, polarization, and proliferation. Caveolae also promote insulin signaling and inhibit PKA signaling.54 Caveolin 1 has been identified as a major binder of fatty acids on the plasma membranes, translocating them to lipid droplets. CAV1 knockout mice are resistant to diet-­induced obesity and exhibit severe hypertriglyceridemia, high postprandial free fatty acid (FFA) levels, reduced levels of both high and total molecular weight adiponectin, and increased risk of diabetes mellitus, atherosclerosis, and cardiovascular disease.54-­56 Mutated CAV1 may induce lipodystrophy by interfering with lipid handling, lipid droplet formation, and adipocyte differentiation26,57 and is phenotypically classified as type CGL3. A mutation has been identified in one individual with CGL24,52,58 who had a homozygous nonsense mutation of CAV1, probably as a result of a consanguineous union. The patient with CAV1 mutation (CGL3) had clinical features similar to those of patients with CGL1 and CGL2, whereas the

CHAPTER 24  Lipodystrophy Syndromes degree of her lipodystrophy was intermediate between these two phenotypes. The patient had also developed diabetes mellitus in adolescence and presented some distinctive features, including hypomagnesemia, hypocalcemia (probably owing to vitamin D resistance), functional megaoesophagus, short stature, and decreased bone density. The patient still preserved both mechanical adipose tissue and bone marrow fat, whereas the metabolically active adipose tissue was absent. Three distinct mutations of CAV1 gene (one in exon 2 and two in exon 3) were identified in five individuals. Two of the three mutations were nonsense mutations (c.112G>T, p.Glu38 and c.424C>T, p.Gln142*), and the third was a frameshift mutation (c.479_480delTT, p.Phe160fs*).45 In 2009, Hayashi et al. reported for the first time a new type of CGL linked with mutations in the PTRF, gene, also known as CAVIN1.53 Type 4 CGL (CGL4) is caused by homozygous or compound heterozygous mutations in the PTRF gene, which is located at 17q21.2, and its product Cavin-­1 (PTRF) is involved in biogenesis of caveolae, regulating caveolin 1 and 3 expression.53 It should be mentioned that caveolin 3 is most prevalent in muscle tissue, including cardiac myocytes.59 It consists of 151 amino acids and is involved in signal transduction on the myocardial cell membrane and regulation of ion channel function in caveolae.60 Cavin-­1 is a peripheral membrane protein localized on the inside surface of caveolae that supports the stabilization and assembly of the membrane structure, probably through interaction with the cellular cytoskeleton.53,61 Cavin-­1 is present in adipocytes and regulates their differentiation and the expandability of adipose tissue.61,62 In type 4 CGL, adipose tissue loss may occur progressively during infancy and is significant in several areas of the body, including the face. To date, 34 patients with 14 different mutations of the CAVIN1 gene have been reported in the literature. Frameshift mutations were found to be most common, followed by nonsense and missense mutations.45 Clinical features include generalized lipodystrophy in association with congenital distal myopathy, muscular hypertrophy, osteopenia, distal metaphyseal deformation, atlantoaxial instability, pyloric stenosis, gastrointestinal dysmotility, hepatosplenomegaly, and acromegaloid characteristics31,53,63 Other findings are hypertriglyceridemia, insulin resistance, and elevated serum creatine kinase concentration. These patients exhibit a high risk of QT interval prolongation, cardiomyopathy, cardiac fibrosis, life-­threatening cardiac arrhythmias such as exercise-­induced ventricular tachycardia, catecholaminergic polymorphic ventricular arrhythmia, and sudden death.59,63-­66 One potential link between type 4 CGL and these serious cardiac complications is the impact of CAVIN1 on the expression pattern of caveolin-­3 in cardiac myocytes.60,67,68 KEY POINTS • Congenital generalized lipodystrophy (CGL) is a rare heterogeneous autosomal recessive syndrome observed mainly in cases of parental consanguinity. It is characterized by near-­complete absence of body fat that may spare the supportive adipose tissue, as well as extreme muscularity, present at birth or soon thereafter. Patients with CGL develop profound hypoleptinaemia, and they are predisposed to metabolic disorders. Four distinct subtypes of congenital lipodystrophy have been defined, with AGPAT2 and BSCL2 mutations being the most frequent. Metreleptin is approved for use in patients with CGL. EPIDEMIOLOGY: ETIOLOGY Congenital generalized lipodystrophy (CGL) is a rare heterogeneous autosomal recessive syndrome observed mainly in cases of consanguinity and in isolated communities. Approximately 300 patients with CGL have been described. Four molecularly distinct subtypes of congenital lipodystrophy have been defined, with AGPAT2 and BSCL2 mutations being the most frequent.

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PATHOPHYSIOLOGY The pathophysiology of congenital generalized lipodystrophy may include impaired signaling of PI3K/AKT and PPARγ, affecting adipogenesis and lipid storage; defects in adipogenesis and expression of several enzymes (AGPAT2, DGAT2, and lipin 1) and lipogenic transcription factors (PPARγ, C/EBP-­α); impaired lipid droplet biogenesis, accumulation, and metabolism; and defects in adipocyte differentiation, adipose tissue expandability, caveolae formation, and stability.

MAIN CLINICAL CHARACTERISTICS Congenital generalized lipodystrophy (CGL) is characterized by near-­complete absence of body fat that may spare the supportive adipose tissue, as well as extreme muscularity present at birth or soon thereafter. Patients with CGL develop profound hypoleptinaemia and are predisposed to metabolic disorders.

Other Mutations Associated With a Congenital Generalized Lipodystrophy–Like Phenotype There are several patients with the CGL phenotype who do not have mutations in any of the four genes known to be mutated in CGL, which suggests that novel CGL-­related genes have yet to be identified. PPARγ plays an important role in adipogenesis and lipid synthesis. Mutations in the PPARγ gene are involved in the pathogenesis of FPL; however, biallelic PPARγ pathogenic variants may be associated with generalized lipodystrophy (recessive). A female patient has been reported with compound heterozygous mutations (c.413_416delAATG; p.Glu138Valfs*168 and c.490C>T; p.R164W) and a similar CGL phenotype. The individual showed the hallmark features of early onset of CGL with some facial adipose tissue. Moreover, the patient developed hypertriglyceridemia, eruptive xanthomata, acanthosis nigricans, insulin-­resistant diabetes, end-­stage renal disease, hyperparathyroidism, irregular menstruation, prominent veins, and marked hepatosplenomegaly. She has also experienced several episodes of pancreatitis and secondary bone fractures, and her plasma leptin levels are barely detectable.69 The LMNA gene encodes the nuclear envelope proteins lamin A and lamin C. Pathogenic variants of LMNA lead to the production of abnormal lamins and disrupt the interaction between chromatin and the nuclear lamina, which may result in apoptosis and premature adipocyte death. However, the pathogenic mechanisms by which these variants modify adipocyte function are still not fully understood. Mutations in LMNA are associated with the development of FPL and progeroid syndrome.70,71 However, a heterozygous lamin A/C (LMNA) p.T10I mutation was reported to be associated with near total loss of adipose tissue developing in early childhood, diabetes mellitus, insulin resistance, acanthosis nigricans, hypertriglyceridemia, hepatomegaly, and progeroid features.72,73 Thus, it has been proposed that this should also be included as a potential CGL subtype.73 Recently, a novel mutation in the LMNA gene and a subsequent autosomal recessive lipodystrophy syndrome was reported in two sisters aged 17 and 19 years old. Both individuals had the homozygous mutation p.Arg545his in LMNA, probably as a result of a consanguineous union. The patients had developed near-­generalized loss of subcutaneous adipose, severe hypertriglyceridemia, hepatic steatosis, hepatomegaly, diabetes mellitus, short stature, intellectual disability, thin lips with perioral pigmentation, dry skin, bilateral clinodactyly of the toes, joint contractures, dystrophic nails, bilateral cataracts, and genitourinary disorders such as uterine fibroids, ovarian cysts, and hydrosalpinx.74 Another CGL-­related gene variant involve mutation of the proto-­ oncogene encoding the cFOS protein. Sequence analyses performed in

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a White female patient with the typical appearance of CGL and insulin resistance identified a novel homozygous point mutation (c.–439, T>A) in the C-­FOS promoter. No relevant mutations of the known pathogenic genes were identified. Her parents were healthy, not consanguineous, and did not carry this mutation. The patient died at the age of 8 years; the primary cause of death was hyper acute varicella infection. However, by the age of 5 years, the patient had also developed acanthosis nigricans, insulin resistance, prediabetes, severe hypercholesterolemia, mild hypertriglyceridemia, lipoatrophy and marked muscularity associated with complete loss of subcutaneous adipose tissue, hypertrichosis, and progressive hepatosplenomegaly. It has been proposed that diminished C-­FOS expression potentially interferes with other genes essential for initiation of adipocyte differentiation or maturation of preadipocytes, and thus might play a role in CGL development.75

Acquired Generalized Lipodystrophy. The acquired syndrome of total lipoatrophy, also known as Lawrence syndrome, is similar to that of the congenital disorder, except that it develops in a previously healthy individual over days to weeks, often after a nonspecific febrile illness. The syndrome is very rare, has a later onset compared with CGL, and is more prevalent in females than males, with a ratio of 3:1. It commonly develops during childhood and adolescence, predominantly in White patients.24,76 In addition to the generalized loss of fat that has an active metabolic function, as seen in CGL, fat loss in AGL also occurs in palms, soles, and genital areas. In some cases, facial lipoatrophy is not initially present but develops gradually. However, retroorbital and bone marrow fat may be preserved.58 The median time to develop diabetes after loss of fat tissue is approximately 4 years.58 Diabetic ketoacidosis has been reported, and severe hypertriglyceridemia, hepatic steatosis, acanthosis nigricans, menstrual irregularities, and polycystic ovary syndrome (PCOS) are also common findings.58 Patients with AGL also have markedly reduced adiponectin levels and moderately reduced leptin levels.10 Several autoimmune diseases and inflammatory conditions have shown a temporal relationship with AGL. These include juvenile-­onset dermatomyositis (JDM), rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, Sjögren syndrome, and panniculitis.58,77 JDM shows a particularly strong correlation with lipodystrophy; 8% to 40% of patients with JDM develop acquired lipodystrophy.78-­81 The chronicity and severity of JDM, as well as the high frequency of calcinosis, have been shown to predict the onset of lipodystrophy.78 AGL following autoimmune diseases is also termed AGL type 2 or the autoimmune disease variety. Patients with type 2 AGL tend to be older compared with patients with other AGL types.58 Recently a new heterozygous missense mutation c.29C>T (p.T10I) in the LMNA gene was described in a 17-­year-­old African-­American female patient with JDM and AGL. The patient had progressive subcutaneous adipose loss that started in the extremities and progressed to the entire body, including the face. Other characteristics include severe hypertriglyceridemia (>10,000 mg/dL), hepatosplenomegaly, nonalcoholic steatohepatitis, acute pancreatitis, diabetes, acute diabetic ketoacidosis, PCOS, ovarian cysts, bilateral renal cysts resulting in nephromegaly, micrognathia, bilateral parotid enlargement, atrial septal defect, and moderate aortic stenosis.82 Panniculitis is another inflammatory condition that frequently heralds the onset of AGL. It is estimated to be present in approximately 25% of affected patients.58,83 Panniculitis manifests as subcutaneous inflammatory nodules that show a mixed infiltrate of lymphocytes and mononucleated macrophages in adipose tissue. The course of AGL is frequently protracted in patients with panniculitis and linked to less fat

loss, lower prevalence of diabetes, and less severe metabolic disorders (e.g., mild hypertriglyceridemia).76,79 The panniculitis variety is also known as AGL type 1. Up to 50% of AGL patients have no apparent history of autoimmune disease or panniculitis, however. These lipodystrophies are known as AGL type 3 or the idiopathic type. Of note, according to several recent studies, AGL is associated with antiprogrammed cell death 1 (anti-­PD1) therapy and may result in severe metabolic complications. Evidence suggests that PLIN1 autoantibodies may be a possible cause of AGL development in these patients.84 Anti-­PD1 therapy is an advanced anticancer therapy with substantial and significant antitumor activity that targets several types of cancers. As a result, anti-­PD1 has come to be used more frequently over time. Thus, physicians should be aware of this potential adverse event, as early recognition is of paramount importance.85-­87 The pathogenesis of AGL is unknown. The autoimmune-­mediated destruction of adipocytes or preadipocytes has been hypothesized to be the underlying mechanism. Autoantibodies against adipocyte membranes may also impair fat uptake and adipocyte differentiation.77,78,88 Several antibodies have been found to be present in AGL, but no causative relationship has been established.78 Cytokines, including tumor necrosis factor alpha (TNF-­α) and interleukin 1 (IL-­1), are also likely to play important roles in the immunopathogenesis of lipodystrophy. They can potentially lead to lipodystrophy by inhibiting adipogenesis89 or increasing receptor-­mediated apoptosis of adipocytes and preadipocytes.90 Other studies in patients with AGL have documented an association between complement activation and adipocyte destruction.91 Chronic hepatitis with autoimmune features and low serum complement 4 levels is also reported in some patients with AGL, suggesting involvement of the classical complement pathway causing loss of fat.92 However, it is still not clear whether this association is characteristic of lipodystrophy or rather an outcome of the autoimmune process itself. EPIDEMIOLOGY Acquired generalized lipodystrophy is a rare syndrome (∼80 patients) that develops in a previously healthy individual.

PATHOPHYSIOLOGY The pathophysiology of acquired generalized lipodystrophy is not fully clarified, although several autoimmune diseases and inflammatory conditions have been shown to have a temporal relationship with this condition.

MAIN CLINICAL CHARACTERISTICS Patients present with generalized loss of fat and marked metabolic disorders, including severe hypertriglyceridemia, nonalcoholic steatohepatitis, acute pancreatitis, acanthosis nigricans, diabetes mellitus, and polycystic ovary syndrome.

Partial Lipodystrophies Partial lipodystrophy is characterized by selective regional fat loss. It is often associated with hypertrophy of adipose tissue in nonatrophic areas and is subclassified into inherited and acquired forms.

Familial Partial Lipodystrophies. Several syndromes have been described according to distinctive clinical features or underlying pathogenetic mechanisms. Most are inherited in an autosomal dominant fashion, and the patients are born with normal fat distribution but notice local fat loss, usually during puberty. FPLs exhibit phenotypic heterogeneity that has not yet been fully delineated.

CHAPTER 24  Lipodystrophy Syndromes

Familial Partial Lipodystrophy Type 1 or Köbberling-­ Type Lipodystrophy. Also known as Köbberling-­type lipodystrophy, FPL type 1 (FPL1) was first reported by Köbberling and colleagues in 1971. In comparison to other forms of FPL, the loss of adipose tissue is restricted to the extremities. The distribution of fat on the face and neck is normal or increased, frequently in association with significant central obesity. The hallmark anthropomorphic feature of this syndrome includes a palpable “ledge” formed between the normal and lipodystrophic areas and high triceps-­to-­forearm and abdomen-­to-­thigh skinfold ratios.93 Metabolic syndrome, especially hypertriglyceridemia, is common in FPL1. This correlates with a high incidence of pancreatitis and premature coronary artery disease. Leptin concentrations are low and correspond to the body mass index (BMI) and the level of fat loss of individual patients.93 Mostly women have been diagnosed with FPL1 to date, as they are more severely affected than men. Diagnosis of affected men is difficult given the normal muscular physique and very mild clinical presentation that does not allow early detection. FPL1 tends to have a childhood onset, with metabolic complications occurring during adulthood. Acanthosis nigricans is present on the neck, axillae, and groin but is usually mild. The genetic defect associated with FPL1 is currently unknown, and no LMNA or PPARγ mutations have been identified. It appears that this syndrome may be familial for some patients but may also occur spontaneously. However, according to recent studies, a significant number of cases of FPL1 follow polygenic inheritance.93-­95

Familial Partial Lipodystrophy Type 2 or Dunnigan-­ Variety (Face-­Sparing) Lipodystrophy. Also known as Dunnigan variety

(face-­sparing) lipodystrophy, FPL type 2 (FPL2) is an autosomal dominant condition found mostly, but not exclusively, in subjects of northern European descent. More than 300 cases have been reported, but the true prevalence of this syndrome is thought to be much higher. Currently known cases are associated with variants in exon 8 of the LMNA gene.11 It is characterized by gradual loss of almost all subcutaneous fat from the extremities, commencing at puberty. This gives rise to the characteristic phenotype of “increased muscularity” in the arms and legs. Variable and progressive loss of fat from the anterior abdomen and chest occurs later. Excess fat may subsequently accumulate in the face and neck and in the intraabdominal region, resulting in a Cushingoid appearance.24 Affected females tend to have more recognizable phenotypes. Although questions of gender differences have been raised, anthropometric measures and MRI data demonstrated that both affected men and women have similar patterns of fat loss. In comparison to the affected men, women may have more severe hypoleptinemia and metabolic sequelae (insulin resistance), and they may also have higher prevalence of diabetes and atherosclerotic vascular disease, as well as higher serum triglycerides and lower high-­density lipoproteins. The prevalence of hypertension and fasting serum insulin concentrations are similar in men and women.58,83 The prevalence of diabetes is not related to age, menopausal status, or family history of type 2 diabetes.88 Patients with FPL2 are more prone to develop PCOS, infertility, and gestational diabetes. The prevalence of gestational diabetes and miscarriage is significantly higher in these patients than in women with similar BMI and PCOS.96 The first gene identified as containing mutations associated with FPL2, named LMNA, is located on chromosome 1q21-­22. As previously mentioned, LMNA encodes lamins A and C, which are essential components of the nuclear lamina and provide structural integrity for the nuclear envelope. Most FPL2-­associated mutations in LMNA are missense mutations within the 3′ end of the gene,97 whereas the LMNA mutations leading to the R482W and R482Q amino acid

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substitutions seem to be the most common pathogenic variants.98,99 The mutant gene products may disrupt interaction with chromatin or other nuclear lamina proteins, resulting in apoptosis and premature death of adipocytes.100 The accumulation of prelamin A may also impair adipogenesis by interfering with the key adipocyte transcription factors/regulators, including sterol response element-­binding protein 1 (SREBP-­1) and PPARγ.100-­103 It is interesting to note that there is no difference in the levels of lamin A and lamin C expression in different adipose depots, even though the fat loss associated with FPL2 is regionally selective, suggesting that the downstream effects of LMNA mutations are differentially regulated in different areas of the body.96 According to recent findings, there is an anthropometric, clinical, and biochemical phenotypic heterogeneity among individuals with FPL2 based on LMNA mutation site and gender.104 Females tend to have a more severe FPL2 phenotype, likely because of increased levels of proinflammatory cytokines or glucocorticoid receptor GRβ overexpression and LMNA mutations affecting both lamins A and C.104 It has also been suggested that the percentage of fat in the lower limb is the most reliable anthropometric measure for FPL2 diagnosis in women. Females with coexisting metabolic disorders and a negligible lower limb fat content (G; p.Ile354Val) in PPARγ2 is responsible for a decrease (i.e., ∼ 30%–35%) in receptor transcriptional activity and for the development of FPL3 in an Italian family. This finding suggests that PPARγ mutations that affect transcriptional activity to a lesser extent than haploinsufficiency may be also responsible for FPL3 development.111

Familial Partial Lipodystrophy Caused by PLIN1 Mutation, or Familial Partial Lipodystrophy 4. Also known as FPL4, FPL caused

by PLIN1 mutation has been characterized phenotypically as loss of subcutaneous fat from the extremities. FPL4 is associated with mutation in the PLIN1 gene coding for perilipin 1, which is a required component of lipid droplet membranes and is essential for lipolysis and lipid storage. Six patients with this mutation had histologically small adipocytes with increased macrophage infiltration and abundant fibrosis.112 Recently, renal involvement has been described for the first time as a clinical manifestation in a patient with FPL4. In more detail, a novel heterozygous frameshift mutation (c.1201_1202insT) was described in a 15-­year-­old Chinese female with insulin-­resistant diabetes, hypertriglyceridemia, nonalcoholic steatohepatitis, and proteinuria. 113

Familial Partial Lipodystrophy Caused by CIDEC Mutation, or Familial Partial Lipodystrophy 5. FPL5 is a rare autosomal

recessive condition that appears in early childhood and it is caused by pathogenic variants in the CIDEC gene. The CIDEC gene is located on chromosome 3 (3p25.3) and participates in adipocyte differentiation.

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It also encodes the CIDEC protein, which is an essential regulator of glucose and lipid metabolism and insulin sensitivity. Pathogenic variants of CIDEC are hypothesized to prevent lipid droplets from storing fat and cause defects in adipocyte differentiation.114,115 FPL5 is characterized by partial lipodystrophy and muscular hypertrophy, but no accumulation of adipose tissue, acanthosis nigricans, metabolic abnormalities, severe insulin-­resistant diabetes, and hepatic steatosis. Recently, two single nucleotide polymorphisms (rs2479 or C allele at rs1053239) were associated with abnormal fasting glucose levels and hypertriglyceridemia. Patients with an A allele at rs2479 or a C allele at rs1053239 tend to rapidly develop high blood pressure and benefit from angiotensin II–targeted treatment.116

Familial Partial Lipodystrophy Caused by LIPE Mutation, or Familial Partial Lipodystrophy 6. FPL6 is an autosomal recessive

syndrome, and only a few cases have been reported to date. It appears in adulthood and is caused by mutations in the LIPE gene (lipase E, hormone-­sensitive type). It is associated with impaired lipolysis, which may result simultaneously in lipomatosis and partial adipose loss. It is also characterized by insulin resistance, diabetes, hypertriglyceridemia, and hepatic steatosis.117 Individuals with FPL6 may exhibit multiple symmetric lipomatosis, accumulation of adipose tissue in the neck, axillae, supraclavicular area, and back, partial adipose tissue loss from the lower extremities, and in some cases distal symmetric muscular dystrophy.117,118 Familial Partial Lipodystrophy Caused by AKT2 Mutation. FPL caused by AKT2 mutation has been reported in a single family by George and associates.119 It is inherited in an autosomal dominant fashion and manifests as severe insulin resistance and partial lipodystrophy confined to the extremities.119 Although the specific mutations in AKT2 has not yet been identified, and this conditions is not included in earlier FPL classification systems,98 recent studies consider it to be another FPL syndrome.2,73 AKT, also known as protein kinase B, is a serine/threonine protein kinase and plays multiple roles in cell signaling, cell growth, and glycogen synthesis, as well as in insulin-­stimulated glucose transport.120 Lipodystrophy in patients with AKT2 mutations is thought to be attributed to reduced adipocyte differentiation and dysfunctional postreceptor insulin signaling.

face, dental abnormalities), cutaneous changes (restrictive dermatopathy, skin atrophy, alopecia, and mottled cutaneous pigmentation), and lipodystrophy. Although MAD is present at birth, dysmorphic manifestations and progeroid features become more prominent with time, and the full clinical phenotype is recognizable during the early school years. The patients have normal intelligence,123 and their serum leptin concentration can be low or normal. Hyperinsulinemia, insulin resistance, impaired glucose tolerance, diabetes mellitus, and hyperlipidemia have been reported in some patients. There are two distinctive phenotypes of MAD: type A involves the loss of subcutaneous fat from the extremities and trunk but normal or excessive deposition of fat in the face and neck, and type B is characterized by more generalized loss of subcutaneous fat and premature renal failure. MAD type A (MADA) is also considered to be as a result of mutations of the LMNA gene that result in accumulation of prelamin A and lead to alterations of nuclear architecture and chromatin defects. It remains unclear how different mutations in the same gene lead to a variety of phenotypes. Patients with MAD type B (MADB) have been reported to carry compound heterozygous mutations in ZMPSTE24, the gene encoding an endoprotease, zinc metalloprotease, which is located on chromosome 1q34. The enzyme is important in posttranslational processing of prelamin A to mature lamin A. Recently, eight related Surinamese individuals of African descent with MADB were described. All of these individuals, however, shared the same homozygous founder mutation in the ZMPSTE24 gene, resulting in a missense variant c.1196A>G, p.(Tyr399Cys).124 As in MADA, the accumulation of farnesylated prelamin A is proposed to be responsible for the phenotype.123 Only eight other patients with ZMPSTE24 mutations are known, the majority from Italy.125 Focal segmental glomerulosclerosis and calcified skin nodules have been reported in patients with ZMPSTE24 deficiency.126 EPIDEMIOLOGY: ETIOLOGY The majority of familial partial lipodystrophy (FPL) syndromes are inherited in an autosomal dominant pattern and are characterized by selective regional fat loss. More than 500 patients with FPL have been reported. Six main FPL types have been described that are caused by mutations in LMNA, PPARγ, PLIN1, CIDEC, and LIPE.

Familial Partial Lipodystrophy Caused by CAV1 Mutation.

Heterozygous CAV1 mutation has also been identified as a rare cause of partial lipodystrophy.121 Two cases with different frameshift CAV1 mutations have been reported. Both patients were described to have partial lipodystrophy with subcutaneous fat loss in the face and upper body, micrognathia, and congenital cataracts. One case also exhibited abnormal neurologic findings. Diabetes, hypertriglyceridemia, and recurrent pancreatitis were reported in both cases.121

PATHOPHYSIOLOGY The different types of familial partial lipodystrophy syndrome exhibit phenotypic heterogeneity and distinct genetic causes. The underlying pathophysiological mechanisms are not fully delineated and may involve disrupted adipogenesis, impaired differentiation of adipocytes, defects in lipid storage and lipolysis, increased adipocyte apoptosis, and premature adipocyte death.

Familial Partial Lipodystrophy Caused by PCYT1A Mutation.

Another gene that has been reported to be associated with FPL is the phosphate cytidylyltransferase 1 alpha (PCYT1A) gene. PCYT1A is the rate-­limiting enzyme in the Kennedy pathway, participating in de novo phosphatidylcholine synthesis. Biallelic pathogenic loss-­of-­function mutations in PCYT1A have been reported to be associated with a severe form of partial lipodystrophy, significant insulin resistance and diabetes, severe nonalcoholic fatty liver disease, and low HDL cholesterol levels in two unrelated female patients.122 Because of the phenotypes and characteristics of these patients, it has been suggested that this mutation could be considered a potential cause of CGL.73

Mandibuloacral

Dysplasia–Associated

Lipodystrophy.

Mandibuloacral dysplasia (MAD) is an extremely rare autosomal recessive progeroid syndrome that has been reported in approximately 40 cases. MAD is characterized by postnatal growth retardation, craniofacial and skeletal abnormalities (mandibular and clavicular hypoplasia, delayed closure of the cranial sutures, acroosteolysis, joint contractures, birdlike

MAIN CLINICAL CHARACTERISTICS Patients are born with normal fat distribution, with the local fat loss occurring later in life, usually during puberty. Muscular hypertrophy and cardiometabolic disorders are common.

Autoinflammatory Syndromes. Several autoinflammatory syndromes may also be associated with lipodystrophic states. A rare autosomal recessive, autoinflammatory syndrome presenting with joint contractures, microcytic anemia, muscle atrophy, and panniculitis-induced lipodystrophy (JMP) in childhood has recently been reported in three individual patients from Japan and two families from Mexico and Portugal.127 Additional clinical features of JMP include hepatosplenomegaly, intermittent fever, calcification of the basal ganglia, and hypergammaglobulinemia. Sequencing of .

l

CHAPTER 24  Lipodystrophy Syndromes candidate genes involved in immune system dysfunction led to the discovery of a loss-­of-­function mutation of the proteasome subunit beta-­type 8 (PSMB-­8) gene on chromosome 6. PSMB8 encodes b5i, a catalytic subunit of immunoproteasomes, which mediates proteolysis and generates MHC class 1 molecules. Mutations may result in adipose tissue lymphocytic infiltration and loss of surrounding fat tissue. CANDLE (chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature) is another newly described syndrome causing partial lipodystrophy that has so far been reported in six patients and is likely inherited in an autosomal recessive fashion.128,129 Infants present with annular violaceous plaques and recurrent fevers, with eventual loss of adipose tissue from the upper limbs and face.130 Other associated clinical characteristics include hepatosplenomegaly, anemia, eyelid swelling, and calcifications of the basal ganglia. The molecular mechanism of this syndrome is yet to be determined. It has been proposed that physicians should include this syndrome in their differential diagnosis when treating individuals with early-­onset systemic inflammatory disease and skin manifestations.131

Other Syndromes and Progeroid Disorders With a Component of Lipodystrophy. Multiple other syndromes are also linked to

lipodystrophy, and several have been identified as laminopathies, including Hutchinson–Gilford progeria syndrome (a very rare and uniformly fatal segmental progeroid syndrome with progressive and generalized fat loss), restrictive dermopathy, progeria-­ associated arthropathy, and atypical progeroid syndrome.15 The majority of these syndromes are associated with de novo LMNA mutations, yielding a MADA-­type lipodystrophy. Werner syndrome (short stature, birdlike appearance of the face, early onset of aging processes, and progeroid features) has been linked to homozygous mutations in RECQL2, which encodes a DNA helicase. In contrast, the genetic basis and inheritance patterns have yet to be clarified for the following syndromes: Cockayne syndrome (short stature, photosensitivity, hearing loss, premature aging, and progeroid appearance), carbohydrate-­ deficient glycoprotein syndrome (nonprogressive ataxia associated with cerebellar hypoplasia, stable mental retardation, variable peripheral neuropathy, and strabismus), SHORT syndrome (S–short stature; H–hyperextensibility of joints and/or hernia (inguinal); O–ocular depression; R–Rieger anomaly; T–teething delay), and ectodermal dysplasia in association with generalized lipodystrophy acral renal ectodermal dysplasia lipoatrophic diabetes (AREDYLD) syndrome.

Acquired Partial Lipodystrophy (Barraquer–Simons Syndrome).

First reported in 1885 by Mitchell, acquired partial lipodystrophy (APL) was further characterized by Barraquer Roviralta in 1907. There have been approximately 250 cases reported in the English-­language literature.24 Patients with APL are primarily of European descent; however, cases have also been reported in Asian Indian, Vietnamese, and Samoan populations. The disease shows a female dominance, with a 4:1 ratio, and most patients have clinical manifestations in early puberty or early adulthood, usually before 15 years of age. Female patients normally have regular menses and intact fertility. The characteristic fat loss progresses in a “cephalocaudal” fashion, with fat loss appearing first in the face and spreading to the upper part of the body. Fat under the umbilicus is rarely affected. Many patients develop excess fat accumulation over the lower abdomen, gluteal region, thighs, and calves. Breasts may lose fat and consist of firm glandular tissue only.58 In contrast to other types of lipodystrophies, acanthosis nigricans, hirsutism, and hypertrichosis are rare. Insulin resistance is uncommon, and the prevalence of diabetes is much reduced compared to other types of lipodystrophies. The prevalence of the metabolic syndrome is also significantly lower in patients with APL. Hepatomegaly

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is common among patients with APL. In the series of case reports by Misra and Garg, 35% of APL patients had hypertriglyceridemia, and a third had low concentrations of HDL.58 Serum leptin levels were normal in the majority of patients.10 A strong association between APL and membranoproliferative glomerulonephritis (MPGN) type 2 has been proposed. The spectrum of presentation ranges from acute glomerulonephritis, hematuria, nocturia, urinary casts, albuminuria, and nephritic syndrome to chronic glomerulonephritis and uremia.121,132 The serum C3 complement levels are usually low, with the presence of C3 nephritic factor,121 which blocks degradation of the enzyme C3 convertase. Patients with low C3 levels tend to have an earlier onset of lipodystrophy than those with normal serum C3 levels. The median time interval between the onset of lipodystrophy and the development of MPGN is approximately 5 to 10 years but could be as long as 20 years.121,133 Similar to AGL, APL is also frequently seen in the context of autoimmunity or infections.77 The most frequently cited infection preceding APL is measles. The low C3 levels may also render APL patients susceptible to recurrent pyogenic infections, particularly Neisseria.134 Systemic lupus erythematosus and dermatomyositis/polymyositis are the autoimmune diseases that are most frequently associated with APL.58 The precise mechanisms leading to adipose-­tissue atrophy in APL remain unclear. C3 nephritic factor has been shown to induce lysis of adipocytes expressing factor D (adipsin).58 According to recent findings, C3 hypocomplementemia (and to a lower extent low serum C4 complement levels) and autoimmunity are associated with Barraquer–Simons syndrome (BSS). Furthermore, C3NeF, as well as other autoantibodies (such as anti-­C3, anti-­P, and anti-­FB) against components of the C3 convertase of the alternative pathway, may also be present in several patients with BSS.135 Rare variants in the LMNB2 gene encoding lamin B were reported in five APL patients, but half of those variants were also reported in normal controls.136 In a study by Guallar and coworkers, PPARγ gene downregulation and mitochondrial toxicity were observed in a patient with APL, suggesting that impaired adipogenesis and adipocyte metabolism may also underlie the pathogenesis of APL.137 KEY POINTS • Acquired partial lipodystrophy (APL) is more frequent in females, and most patients have clinical manifestations in early puberty or early adulthood. The etiology of APL is still uncertain, although an association with autoimmune-­ mediated loss of adipose tissue has been suggested. Patients with APL may demonstrate circulating autoantibody (C3 nephritic factor) and low complement component 3 levels. The progressive loss of adipose tissue follows a “cephalocaudal” trend, starting from the face and spreading to the upper part of the body, while fat may be preserved or accumulated over the lower abdomen, gluteal region, thighs, and calves. Membranoproliferative glomerulonephritis or autoimmune disease may exist. Associated metabolic syndrome, insulin resistance, and related complications are not common.

EPIDEMIOLOGY Acquired partial lipodystrophy is more common in females than in males; approximately 250 patients have been reported.

ETIOLOGY: PATHOPHYSIOLOGY Although the etiology of acquired partial lipodystrophy (APL) is still uncertain, a mechanism involving autoimmune-­mediated loss of adipose tissue has been suggested. Patients with APL may demonstrate circulating autoantibody (C3 nephritic factor) and low complement component 3 levels.

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MAIN CLINICAL CHARACTERISTICS Most patients have clinical manifestations in early puberty or early adulthood. The progressive loss of adipose tissue follows a “cephalocaudal” trend, starting from the face and spreading to the upper part of the body, while fat may be preserved or accumulated over the lower abdomen, gluteal region, thighs, and calves. Membranoproliferative glomerulonephritis or autoimmune disease may exist. There is a low prevalence of metabolic syndrome, insulin resistance, and related complications.

Human Immunodeficiency Virus–Associated Lipodystrophy associated Syndrome. Human immunodeficiency virus (HIV)-­ lipodystrophy syndrome (HALS) is currently the most common form of partial lipodystrophy. First reported in 1998, HALS mainly develops in patients infected with HIV who are receiving highly active antiretroviral therapy (HAART). Importantly, it is independent of fat loss caused by either the HIV infection itself or by cancers or opportunistic infections. The pattern of fat loss is also different. HALS patients may experience lipoatrophy, lipohypertrophy, or a combination of both, namely mixed lipodystrophy.138,139 Cases of lipodystrophy have also been reported in HAART-­naïve patients. The prevalence of HALS increases with increased duration of exposure to HAART and is reported to be present in up to 50% of patients on antiviral treatment for more than 1 year, thus affecting more than 100,000 patients in the United States. HALS usually manifests as peripheral fat wasting that involves the face, arms, legs, and buttocks. The generalized depletion of subcutaneous fat in HALS is distinct from HIV-­related wasting, which is associated with advanced acquired immunodeficiency syndrome and loss of other tissues, such as muscle mass. Accumulation of fat is frequently seen in the dorsocervical area (“buffalo-­hump” pads), abdomen, occasionally in the breasts of both men and women, in the suprapubic area, under the axillae, and over the anterior aspect of the neck. Lipomatosis manifests in a small percentage of patients. Facial fat loss can be quite severe, sometimes resulting in an emaciated appearance. Most studies have shown an increase in central fat over the first 6 months after the initiation of antiretroviral therapy that subsequently levels off. Most HIV-­infected patients with lipodystrophy are otherwise relatively healthy, but dyslipidemia, especially hypertriglyceridemia, is common among HIV-­ infected patients receiving HAART. HIV viremia has been linked to decreased plasma concentrations of total, low-­density lipoprotein (LDL), and HDL cholesterol, and at later stages elevated triglyceride levels. HAART has been shown to cause a worsening lipid profile, with increased plasma triglycerides, increased total and LDL cholesterol, and decreased HDL cholesterol, which can be further accompanied by increases in small, dense LDL particles, lipoprotein (a), and apolipoproteins B, C-­III, E, and H.140 HAART-­ associated dyslipidemia is associated with accelerated atherosclerosis and signs of endothelial dysfunction.141-­144 Seen in 35% of patients, insulin resistance and frank diabetes mellitus are more prevalent in HIV subjects with lipodystrophy,145 but acanthosis nigricans seems to be extremely rare. These metabolic disorders are predictive of the development of metabolic syndrome, whereas their prevalence may vary among HIV-­infected individuals, according to findings from different studies.146,147 The underlying mechanisms related to the development of the metabolic syndrome and the different subtypes of lipodystrophy in HIV-­infected population have not been fully elucidated, and seem to involve a multifactorial process.148 The pathophysiology of metabolic syndrome in HIV patients may include HIV per se, duration of antiretroviral drug use, and chronic inflammation.149 Moreover, findings suggest that variants in candidate genes associated with lipid metabolism and adipogenesis may be predictors of these metabolic and anthropometric disorders.150,151

Hepatic steatosis may also develop. Both leptin and adiponectin levels are decreased in patients with HALS. The reduction of leptin levels correlates with decreased subcutaneous fat mass,152 whereas decreased adiponectin levels are more closely associated with intraabdominal fat accumulation.153 Fat atrophy and fat deposition appear to be associated with different risk factors in HALS. Low baseline fat mass and increased disease severity are associated with a higher incidence of fat atrophy.154 Epidemiology studies have also shown that coinfection with hepatitis C virus can increase the chance of fat atrophy in HIV-­infected individuals.155 On the other hand, older age, female sex, high baseline body fat, increased HIV viral load, low CD4 count, and longer duration of HAART are associated with a higher risk for fat accumulation in HIV patients.156,157 The frequency and manifestations of lipodystrophy also differ with respect to the drugs used. Nucleoside reverse transcriptase inhibitors (NRTIs), particularly zidovudine and stavudine, are commonly associated with morphologic changes, particularly fat loss from the extremities, whereas protease inhibitors (PIs) are more frequently linked to hypertriglyceridemia, insulin resistance, and localized fat accumulation.158 Interestingly, fat loss worsens with ongoing HAART therapy but does not reverse on its discontinuation. Because PIs and NRTIs are usually given in combination as part of HAART, the individual effects of each drug on phenotype are not clear. The mechanism behind HALS is complex and is currently not completely understood.159 HAART has been widely accepted as playing a central role in the development of lipodystrophy, but accumulating evidence indicates that the HIV virus per se, as well as host immune responses, also contribute to the development of HALS. NRTIs have been shown to suppress adipogenesis either through mitochondrial toxicity (by inhibiting mitochondrial DNA polymerase gamma) or by induction of genes that inhibit adipogenesis. In vitro studies suggest that zalcitabine, didanosine, and stavudine have the strongest effects in a reducing order of magnitude, whereas tenofovir and lamivudine show minimal or no mitochondrial toxicity.138 Combinations of drugs can act synergistically and lead to mitochondrial depletion. In addition, zidovudine, emtricitabine, and abacavir can also impair cell proliferation and increase lactate and lipid production. NRTIs may also contribute to insulin resistance by altering the levels of IL-­6, TNF-­α, and adiponectin.158 PIs can lead to adipose tissue changes through several potential mechanisms: (1) impairment of adipocyte differentiation by downregulation of the expression of master adipogenic transcription factors, such as C/ EBP-­α and C/EBP-­β, PPARγ, and SREBP-­1;13 (2) increase of adipocyte apoptosis, leading to a reduction in cell numbers; and (3) decrease of lipid accumulation in adipocytes through reactive oxygen species (ROS) production and increased macrophage recruitment.160 Therapy with PIs has also been implicated in the causation of metabolic abnormalities by inhibiting glucose transport 4 (GLUT4)-­mediated glucose transport; by suppressing insulin signaling; and through activation of lipolysis, induction of IL-­6 and TNF-­α, reduction in gene expression, and secretion of adiponectin, as well as proteasome dysfunction. According to a study, ritonavir significantly affected the expression of 389 genes involved in adipocyte differentiation, glucose metabolism, adiponectin secretion, and triglyceride accumulation. In detail, genes responsible for adipocyte triglyceride accumulation (namely CFD, CIDEC, and PPARγ) and glucose transport genes (including GLUT4 and ADIPOQ) were downregulated. Moreover, the expression of PPARγ regulatory genes CEBPA and liver-­X-­receptor α was also reduced, whereas IL-­6 was increased.161 Lopinavir, ritonavir, saquinavir, and nelfinavir are the worst offenders. The newer PI, atazanavir, has a much milder effect. Indinavir does not have much effect on cell viability or lipogenesis but inhibits glucose uptake to a greater extent than the other PIs.158 Nonnucleoside reverse transcriptase inhibitors (NNRTIs), including efavirenz and nevirapine, appear to have more favorable safety

CHAPTER 24  Lipodystrophy Syndromes profiles in terms of lipodystrophy complications. Although an in vitro study showed that efavirenz may interfere with adipogenesis by reducing the expression of SREBP-­1, a key adipogenic transcription factor,162 and a prospective randomized trial suggested that efavirenz could have greater potential for causing lipoatrophy than the combination of lopinavir plus ritonavir,163 the results from several clinical studies imply that the potential role of efavirenz in the development of lipodystrophy is minimal, and that it may depend on the NRTIs that form the backbone of the regimen.164 Lipoatrophy is lowest with the NRTI-­sparing regimen of lopinavir and efavirenz, but this combination led to worsening dyslipidemia.165 Additionally, in vitro data have shown that efavirenz may increase production of ROS, reduce lipid content in mature adipocytes, and inhibit mitochondrial activity.162,166 There is also increasing evidence that HIV-­1 infection itself, regardless of HAART, may induce inflammatory and proapoptotic pathways in adipose tissue and thus contribute to lipoatrophy. In vitro experiments have demonstrated that the HIV-­1 viral protein R may act as a corepressor of PPARγ-­mediated gene transcription, inhibiting adipocyte differentiation.167 This could be either occurring through the direct HIV-­1 infection of cells in adipose tissue or may be mediated by HIV-­1–encoded proteins.168 Genetic background may also influence the degree of HALS. A single nucleotide polymorphism in the resistin gene has been associated with increased risk for developing limb fat loss, dyslipidemia, and insulin resistance in patients on HAART therapy.169 Additionally, two single nucleotide polymorphisms in the adiponectin receptor gene ADIPOR2 (rs11061925 and rs929434) have been found to be associated with biochemical parameters, such as the levels of triglycerides, total cholesterol, glucose and adiponectin, in HIV-­infected men receiving HAART.170 Inflammatory cytokines, including interferon alpha (IFN-­α), TNF-­ α, IFN-­γ, monocyte chemoattractant protein-­1 (MCP-­1), IL-­1, IL-­6, and IL-­12, may also contribute to or mediate the clinical manifestations of this syndrome,152,168 and this remains an active area of research. EPIDEMIOLOGY: ETIOLOGY Human immunodeficiency virus (HIV)-­associated lipodystrophy syndrome (HALS) is currently the most common form of partial lipodystrophy and develops in patients infected with HIV who are receiving highly active antiretroviral therapy. Approximately 40% of HIV patients receiving protease inhibitor therapy for more than 1 year develop HALS.

PATHOPHYSIOLOGY The pathophysiological mechanism behind human immunodeficiency virus– associated lipodystrophy syndrome is complex and not fully elucidated. It may involve impaired adipogenesis and expression of adipogenic transcription factors (such as PPARγ, SREBP-­1, C/EBP-­α, and C/EBP-­β), as well as defects in adipocyte differentiation and apoptosis.

MAIN CLINICAL CHARACTERISTICS Patients may experience lipoatrophy, lipohypertrophy, or a combination of both, namely mixed lipodystrophy. Human immunodeficiency virus–associated lipodystrophy usually manifests as peripheral fat wasting involving the face, arms, legs, and buttocks, and excess fat deposition in the neck (“buffalo-­hump” pads) and abdomen. Of note, nucleoside reverse transcriptase inhibitors are commonly associated with morphologic changes, particularly fat loss from the extremities, whereas protease inhibitors are more frequently linked to hypertriglyceridemia, insulin resistance, and localized fat accumulation. Patients may experience metabolic disorders, such as severe insulin resistance, diabetes mellitus, dyslipidemia, and liver steatosis.

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Localized Lipodystrophies Localized lipodystrophies are characterized by loss of subcutaneous adipose tissue from small areas or from small parts of an extremity, but insulin resistance or metabolic abnormalities do not usually develop in these patients. Drug-­induced lipoatrophy at the site of insulin injection was a frequent complication before the availability of purified human insulin but is rather uncommon today. Localized hypertrophy is still frequently seen in patients who use the same injection site too often. Other rare causes of localized lipodystrophy as a result of repeated pressure in specific areas, to panniculitis, or as part of a rare syndrome called lipodystrophia centrifugalis abdominalis infantilis have been reported.171

MECHANISMS RESPONSIBLE FOR SEVERE INSULIN RESISTANCE As mentioned earlier, the mechanisms of insulin resistance in several syndromes associated with lipodystrophy are not fully understood, and it is likely that the etiology of lipodystrophies is multifactorial. Lipodystrophy may develop because of abnormal preadipocyte development, adipocyte dysfunction, or increased fat cell death. The study of lipodystrophy syndromes provides unique insights regarding the lack of adipocyte enlargement and adipose expansion and the inability of subcutaneous fat to store excess energy. The excess energy is deposited as ectopic fat with a subsequent decrease in adipokine levels, mechanisms that are associated with the development and severity of insulin resistance.4,16,172 Inflammation is thought to contribute to insulin resistance via impairment of lipolysis and adipocyte metabolism.173 TNF-­α reduces insulin receptor kinase activity, downregulating insulin receptor substrate (IRS)-­1 and GLUT-­4 phosphorylation. Furthermore, IL-­6 at elevated levels may induce triglyceride secretion from the liver and promote hepatic gluconeogenesis. The impaired secretion of adipokines, such as adiponectin and leptin, leads to abnormalities of insulin sensitivity. Finally, mitochondrial stress plays a key role in metabolic dysregulation. These mechanisms are further discussed in the following sections.

Fat Redistribution and Fat Metabolism Changes in fat distribution (reduced subcutaneous fat with or without increased visceral fat) may cause increased insulin resistance. The lack of adipose tissue can result in inadequate storage of and therefore increased levels of FFAs. Intracellular fatty acid accumulation can directly inhibit insulin-­mediated glucose transport in skeletal muscle,174,175 and excess FFAs can also lead to lipotoxicity by inducing ectopic fat accumulation in the liver and muscle, where the adipose tissue is considered to have more “pathogenic” potential. The deposition of fat in the pancreas can also impair the β cell response and further contribute to insulin resistance.176 Moreover, because the fat tissue at different depots shows different degrees of metabolic activity, such as lipolysis and inflammation, lipodystrophic states that are associated with higher volume of visceral fat and abdominal fat are likely to display a higher degree of insulin resistance.176

Adipocytokines Alterations of adipocytokine levels can affect metabolic homeostasis and insulin resistance. Leptin and adiponectin are two of the most abundant adipocytokines produced by adipocytes, and their levels decrease in the lipodystrophic states.10 Serum adiponectin levels correlate positively with insulin sensitivity177,178 and are upregulated by PPARγ agonists.179 Adiponectin acts by reducing hepatic gluconeogenesis (mainly via adiponectin receptor 2 and activation of AMPK phosphorylation) and increasing fatty acid oxidation in muscle (mainly via

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adiponectin receptor 1).180 An animal study also demonstrated that adiponectin may also act in the hypothalamus (via adiponectin receptor 1) to activate insulin and leptin signaling pathways, thus promoting reduction of food intake.181 Antiinflammatory effects of adiponectin have also been shown in various animal models of liver inflammation81 and suggested by several observational studies in humans.182 In earlier animal models, it was shown that increased expression of adiponectin correlates with improved insulin sensitivity, with adiponectin decreasing insulin resistance by decreasing the triglyceride content of liver and muscle.183,184 Although adiponectin or leptin alone partially improves insulin resistance in mouse models of lipodystrophy,185,186 the combined administration of physiologic doses of both fully normalizes insulin sensitivity.183 In patients with lipodystrophy, adiponectin levels are low in certain subsets of the disease, particularly in many patients with CGL1 and HALS,187-­189 and implicate hypoadiponectemia in the development of associated insulin resistance, hypertriglyceridemia, and fat redistribution.190,191 Serum leptin levels reflect the overall amount of adipose tissue in the body and are positively correlated with adiposity.176 In addition to regulating food intake and increasing energy expenditure, leptin also plays an important role in the regulation of glucose homeostasis, possibly independently of its weight-­reducing effects.192 Aside from its actions in the central nervous system (CNS), leptin may exert its insulin-­sensitizing effects peripherally by decreasing gluconeogenesis in the liver and adipose tissue, by exerting its lipolytic activity, and/ or by increasing glucose utilization in skeletal muscle.192 Leptin may also prevent the “lipotoxic” effects of intramyocellular lipid accumulation by activating fatty acid oxidation in skeletal muscle.192,193 It has been shown that leptin levels are decreased in a significant proportion of patients with HALS175 and that leptin administration improves metabolic manifestations of HALS in humans in the short and long term.194-­196 In generalized lipodystrophy, leptin concentrations are very low to undetectable, while in partial lipodystrophy they may vary from normal to low.10 Leptin levels depend on several factors such as age, sex hormones, and fat mass. Moreover, serum leptin concentration may assist physicians in identifying and treating lipodystrophy disorders.5,197 Leptin levels per se, however, should not be considered as a reliable and sufficient index to confirm or exclude lipodystrophy diagnosis.2

Inflammation Inflammation in adipose tissue is likely to contribute to increased insulin resistance in the lipodystrophic state. Altered innate immunity and chronic inflammation appear to be strongly associated with insulin resistance in obesity and type 2 diabetes.198 Inflammatory adipocytokines, such as TNF-­α, IL-­6, IL-­8, macrophage inflammatory protein (MIP)-­1α and 1β, monocyte chemotactic protein-­ 1 (MCP-­ 1; also known as CCL-­2), plasminogen activator inhibitor-­1 (PAI-­1), angiotensinogen, retinol-­binding protein-­4 (RBP-­4), and others have been implicated as the key regulators of insulin sensitivity.179,199 The expression of several of these proteins, including TNF-­α, IL-­6, and IL-­8, as well as macrophage markers (CD68, ITGAM, EMR1, ADAM8) and chemokines (MCP-­1 and CCL-­3), is increased in the subcutaneous tissue of patients with HALS.200 In a small study of HALS, plasma PAI-­1 was also found to be elevated, although its expression level in adipose tissue was not.201 Accumulating evidence supports an association between inflammation and insulin resistance. TNF-­α mediates insulin resistance via reduction of insulin receptor kinase activity, induction of lipolysis, and downregulation of GLUT-­4.202,203 It may also induce apoptosis of adipocytes.203 The impact of IL-­6 on insulin resistance is less clear, but the majority of the evidence indicates that chronic elevation of IL-­6

promotes hepatic insulin resistance and impedes differentiation of adipose tissue,204 whereas acute elevation of IL-­6 levels after exercise may promote improved glucose and lipid metabolism.204 MCP-­1 has been shown to induce insulin resistance by downregulation of GLUT-­ 4, beta-­adrenergic receptors, and PPARγ in mice. It is also associated with increased levels of atherosclerosis. Two pathways, the NF-­κB pathway and the c-­Jun NH2-­terminal pathway, are essential for mediating insulin resistance. Pharmacologic inhibition of the pathways has resulted in improved insulin resistance.179,199

Mitochondrial Stress, Oxidative Stress, and the Endoplasmic Reticulum Mitochondrial defects have been considered as a central factor in NRTI-­ induced lipodystrophy. Mitochondrial dysfunction will lead to oxidative phosphorylation defects and ROS accumulation. Observational studies have shown that clinical conditions associated with increased ROS levels are also associated with increased insulin resistance.174 Oxidative stress may also trigger β cell apoptosis and may contribute further to insulin resistance.205,206 Angiotensin receptor blockers can attenuate oxidative stress and prevent further progression of insulin resistance.207 The ER plays an important role in regulating lipid, glucose, cholesterol, and protein metabolism. Stress on the ER luminal environment may generate an increased load of unfolded or misfolded proteins and may lead to adipocyte apoptosis, inflammation, and insulin resistance.174 Seipinopathy has recently been identified as an ER stress– associated disease208 and may contribute to insulin resistance in patients with CGL2.

Other Mechanisms In addition to the mechanisms mentioned earlier, HAART may also contribute to insulin resistance by directly blocking glucose uptake (PIs) or by reducing phosphorylation of a key step in postreceptor insulin signaling (indinavir).209,210 As mentioned, CGL2/BSCL2 is a severe form of lipodystrophy that is caused by loss-­of-­function mutations of the BSCL2/seipin gene and is accompanied by several metabolic disorders, including extreme insulin resistance and hepatic steatosis. Recent findings support that seipin downregulates glycerol-­ 3-­phosphate acyltransferases (GPATs). The latter are probably also responsible for the defective adipogenesis. Furthermore, GPAT3 deficiency is associated with significant improvement in insulin sensitivity and hepatic steatosis.211,212 Finally, patients with severe lipodystrophic states who have voracious appetites, supposedly because of leptin insufficiency, are theoretically more prone to these insults because overnutrition may aggravate central obesity and has been associated with higher levels of inflammation and ROS load.

TREATMENT OF SYNDROMES OF LIPODYSTROPHIES Lifestyle modification, hypoglycemics, and lipid-­lowering agents are generally required to treat metabolic disturbances associated with lipodystrophies. However, they commonly yield limited results. The development of new drugs targeted to specifically reverse insulin resistance is expected with great anticipation, including the potential use of adipocytokines such as leptin in certain subsets of patients with lipodystrophic syndrome.

Lifestyle Modification There are limited data on the effectiveness of diet and nutrition support on body composition and metabolic abnormalities in patients

CHAPTER 24  Lipodystrophy Syndromes

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with lipodystrophy; however, a balanced macronutrient intake is recommended.2,213 The general clinical recommendations have been to follow the standard dietary advice regarding management of dyslipidemia, obesity, insulin resistance, and impaired glucose tolerance, with the goal of attaining ideal body weight. Supplementation with dietary fiber and fish oil containing high doses of omega-­3 fatty acids should be encouraged.214,215 For patients with severe hypertriglyceridemia, an extremely low-­fat diet (preferably 200 units daily), concentrated forms of insulin such as U-­500 regular insulin should be considered. The pharmacokinetics of long-­acting insulins may be affected when they are injected into lipodystrophic areas. Their long duration of action requires the presence of subcutaneous adipose tissue.2,73,235,236 The new antidiabetic drug classes, such as sodium-­glucose transporter inhibitors and glucagon-­like peptide-­1 receptor agonists, display significant renal and cardiovascular benefits, with improvements in cardiovascular outcomes and all-­cause mortality, especially in high-­risk diabetes patients.237,238 However, their efficacy and safety in patients with lipodystrophy syndromes have not been systematically confirmed. Bariatric surgery is the most effective possible weight-­loss therapeutic strategy and also demonstrates weight loss–independent effects on glycemic control and metabolism. 239,240 A number of studies have been conducted to explore the impact of Roux-­en-­Y gastric bypass (RYGB) in patients with FPL. RYGB led to significant weight loss and metabolic improvements.241-­243 However, long-­term controlled studies are needed to examine the health outcomes, as well as the overall impact and safety of this intervention, in these specific subpopulations.

Metformin. Metformin, which acts by inhibiting gluconeogenesis in the liver and increasing peripheral glucose utilization, has shown efficacy in improving insulin sensitivity in patients with lipodystrophies.221 It may also potentially improve fat redistribution in HALS, as indicated by a randomized controlled trial.222 Other studies have cast doubt on the potential usefulness of metformin, however, by suggesting that metformin may lead to no changes in waist-­to-­hip ratio and may possibly cause further loss of limb fat.222-­224 Moreover, metformin, particularly in combination with exercise training, may be useful in HIV-­infected patients with significant lipohypertrophy and minimal lipoatrophy. Metformin, in combination with lifestyle modifications, is considered as first-­line pharmacotherapy to treat individuals with insulin resistance and diabetes. Furthermore, the US Food and Drug Administration (FDA) has approved metformin usage in pediatric patients 10 years of age and older, noting its efficacy and safety in this subpopulation.2,73

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Management of Dyslipidemia The treatment of dyslipidemia in lipodystrophy should follow the same guidelines as in the general population. Lifestyle modification should be emphasized and attempted first. If ineffective, a change of antiretroviral treatment may be considered initially, followed by starting lipid-­lowering medications in high-­risk HALS patients. Lipodystrophy-­related dyslipidemia can be difficult to treat, and multiple agents may be necessary to lower lipids to target ranges.220 Extra caution may be necessary, given the potential drug–drug interactions among lipid-­lowering agents and HIV-­specific treatments.

Statins. Statins, such as 3-­ hydroxy-­ 3-­ methylglutaryl coenzyme A reductase inhibitors, are normally used as first-­line agents for hypercholesterolemia, especially for patients who have fasting triglyceride levels less than 500 mg/dL. Statins have antiinflammatory, antithrombotic, and endothelial effects that contribute to their overall beneficial effects on reducing mortality from cardiovascular disease. Extensive studies on the use of statins to treat the hyperlipidemia associated with HAART have demonstrated efficacy in lowering total and LDL cholesterol and triglycerides. Specifically, rosuvastatin and pravastatin have been shown to decrease total LDL cholesterol levels.227,244 Among the statins, pravastatin may also increase subcutaneous and limb fat.245 However, caution must be exercised in cases of coadministration of statins and HAART. Coadministration of PIs normally results in increased levels of statins, except for pravastatin. Therefore, simvastatin and lovastatin should be avoided in HIV-­infected patients receiving PIs, whereas atorvastatin should be used with caution. A higher dose of pravastatin may be necessary to achieve optimal lipid-­lowering activity in HALS,246,247 and coadministration of statins with efavirenz reduces atorvastatin, simvastatin, and pravastatin serum concentrations, meaning that statins need to be administered at higher doses.248 Tailoring statin therapy to the HAART therapy regimen is thus very important.

Fibrates and Omega-­3 Fatty Acids. Fibrates and long-­chain omega-­3 fatty acids have been extensively used to treat severe hypertriglyceridemia, but they have not been formally studied in patients with lipodystrophy. However, both medications should be used in patients with elevated triglycerides, especially when triglycerides are higher than 500 mg/dL and may be considered for levels greater than 200 mg/dL.2 Combination therapy with fibrates and statins may be required to treat dyslipidemia to achieve lipid goals and should be used concomitantly with lifestyle modification. However, caution regarding their use is advised, given the increased risk of hepatotoxicity and myopathy, especially in the presence of known myositis or muscular dystrophy, and in patients with renal insufficiency. Fibrates are normally well tolerated and efficacious in the context of HALS.140 Head-­to-­head comparisons of the efficacy of fenofibrate versus gemfibrozil in HALS are lacking. Omega-­3 fatty acids have also proved to be effective in reducing triglycerides in HALS.249 They are generally well tolerated, but their use may be associated with increased LDL levels.140,250 In children and adolescents with lipodystrophy and very high levels of triglycerides, when lifestyle interventions are not sufficient, the use of lipid-­lowering drugs should be considered. Fibrates are commonly used in children with severe hypertriglyceridemia who are at high risk of pancreatitis.251 Omega-­3 fatty acids may also prove beneficial.251,252. Fibrates alone or in combination with statins can effectively improve the lipid profile. However, data are limited in children, and physicians should pay attention to potential adverse effects.252,253

Nicotinic Acid. Nicotinic acid, or niacin, is effective for hypertriglyceridemia, but its use may be limited by adverse effects including

flushing, rashes, pruritus, and exacerbation of insulin resistance and hyperuricemia. Extended-­release niacin preparations are generally better tolerated in HIV lipodystrophy. The side effects can be controlled with daily aspirin intake.140 Acipimox, a long-­acting niacin analog, may also improve triglycerides and improve insulin sensitivity.254 In 2016, after reviewing the data of large cardiovascular outcome studies (HPS2-­THRIVE, AIM-­HIGH, and ACCORD), the FDA concluded that, when niacin extended-­release tablets and fenofibric acid delayed-­release capsules are coadministered with statins, the risks outweigh the benefits, and this combination is not recommended.255-­258

Other Lipid-­Lowering Agents and Novel Therapies. Ezetimibe, a cholesterol absorption inhibitor, might be useful in the treatment of statin-­ intolerant patients or in severe dyslipidemia associated with lipodystrophies.140 Studies have shown that ezetimibe can provide incremental reduction in LDL cholesterol levels when combined with a statin, but a recent study suggested that the combination treatment may not provide additional reduction in cardiovascular risk.259 Tetradecylthioacetic acid, cholestin, and L-­carnitine have also shown efficacy in controlling dyslipidemia via unknown mechanisms,140 but their use in HALS remains limited. Treating extreme hypertriglyceridemia in lipodystrophy patients with conventional dietary and drug therapy may prove to be challenging. Novel lipid-­lowering agents directed towards lowering triglyceride levels have been developed, and several potential therapeutic agents are under examination. Genome-­wide association studies have uncovered potential targets in multiple circulating factors that may play a vital role in the regulation of lipid metabolism, hepatic steatosis, and insulin sensitivity. Furthermore, clinical pharmacology promotes the development of novel and promising therapeutic strategies, such as the antisense oligonucleotide-­based therapies.260 Volanesorsen is a second-­ generation antisense oligonucleotide agent targeted to human apolipoprotein CIII (apoCIII) mRNA. ApoC-­III is an important regulator of plasma triglyceride concentration. According to the results of phase III clinical trials, volanesorsen reduced triglycerides significantly compared to baseline.261,262 Volanesorsen has been approved to treat patients with familial chylomicronemia syndrome.263 The BROADEN study is an ongoing randomized, double-­blind, placebo-­controlled trial that enrolled patients with hypertriglyceridemia and FPL. The study aims to evaluate the safety and efficacy of volanesorsen in this subpopulation, and it is expected to be completed in the last quarter of 2021. Moreover, inhibition of angiopoietin-­like protein 3 and 4 affects lipid metabolism and is associated with low serum triglycerides.264-­267 Trials involving ANPL-­3 inhibition either in the form of monoclonal antibodies or antisense oligonucleotides are ongoing and may result in approval of novel treatments for hyperlipidemia. 268,269 Patients with hypercholesterolemia who do not achieve sufficient LDL reduction despite receiving maximally tolerated first-­line lipid-­ lowering treatment (namely statin drugs) may be prescribed additional therapy with ezetimibe or proprotein convertase subtilisin/kexin type 9 antibody inhibitor (PCSK9i).270,271 Two PCSK9is have been approved in the United States and the European Union as adjuncts to dietary changes and maximal-­tolerated statin therapy. Evolocumab (at a dose of 140 mg biweekly or 420 mg monthly) and alirocumab (75 mg/150 mg every 2 weeks or 300 mg monthly) are human monoclonal antibodies against PCSK9 that are administered subcutaneously. Both drugs are highly potent in reducing LDL (up to approximately 60% in addition to statin monotherapy) and the risk of major cardiovascular events, particularly in high-­risk patients, in phase III studies.272,273 Another promising approach that is being examined for PCSK9 inhibition is inclisiran. Inclisiran is a chemically synthesized small

CHAPTER 24  Lipodystrophy Syndromes interfering RNA molecule that reduces PCSK9 synthesis.274 The ORION program includes several trials that examine the safety and efficacy of inclisiran. According to the findings of phase III placebo-­ controlled, double-­blind, randomized clinical trials, ORION-­10 and ORION-­11, inclisiran at a dose of 300 mg every 6 months reduced LDL levels by approximately 50%.275 It should be noted that inclisiran, because of its long duration of action, can be administered subcutaneously twice yearly, unlike conventional monoclonal PCSK9i. However, no available data exist regarding the efficacy and safety of these drugs in patients with lipodystrophy. Bempedoic acid is a novel, once-­daily, oral agent for treating hypercholesterolemia. It is an inhibitor of adenosine triphosphate citrate lyase, which is involved in the cholesterol synthesis pathway in the liver.276 Bempedoic acid monotherapy was associated with LDL reduction up to 16.5% (vs. placebo) in patients with heterozygous familial hypercholesterolemia and/or atherosclerotic cardiovascular disease and up to 21.4% in patients with statin intolerance after 12 weeks of treatment.277,278 Moreover, bempedoic acid administration, when added to maximally tolerated statin therapy, may reduce mean LDL by 18% (vs. placebo) in patients with heterozygous familial hypercholesterolemia and/or atherosclerotic cardiovascular disease and by 24% (vs. placebo) in patients with a history of statin intolerance.279 A metaanalysis of seven randomized clinical trials has shown that bempedoic acid is an important option for patients at high cardiovascular risk and for those with statin intolerance, accompanied by an acceptable safety profile.280 In 2020, bempedoic acid either as a monotherapy or as a combination with ezetimibe received approval (both in the United States and in the European Union) for use in patients with established atherosclerotic cardiovascular disease who require additional cholesterol lowering or in patients with heterozygous familial hypercholesterolemia.281 Among the potential adverse events, elevated liver enzymes, tendon rupture, hyperuricemi,a and gouty attacks have been described.281 By the end of 2022, the randomized, double-­ blind, placebo-­ controlled, CLEAR Outcomes study is expected to be completed. The purpose of this phase III study is to examine the effects of bempedoic acid on major cardiovascular events in statin intolerant patients with or at high risk for cardiovascular disease (NCT02993406). Finally, plasmapheresis is an option in patients with extreme hypertriglyceridemia associated with significant complications such as acute or relapsing pancreatitis who are unresponsive to conventional therapeutic agents. Plasmapheresis has been found to be effective; however, it must be repeated frequently, and long-­term data are not available.282,283

Management of Human Immunodeficiency Virus–Infected Patients With Highly Active Antiretroviral Therapy–Induced Metabolic Syndrome

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Treatment with high doses of GH (2–6 mg/day) has shown to effectively reduce visceral fat and improve lipid parameters.287-­289 However, its application is hampered by significant side effects, including fluid retention, arthralgia, myalgia, carpal tunnel syndrome, and worsening glucose control. There is also the theoretical concern that long-­term GH therapy may increase the risk for cancer. In addition, high-­dose GH replacement may lead to a further decrease in peripheral subcutaneous fat,290 which is undesirable in this patient population from a cosmetic point of view. The effect of GH generally diminishes after discontinuation of treatment; however, improvement in facial lipoatrophy for up to 6 months after cessation of treatment has been reported.291 Increasing evidence demonstrates that physiologic GH replacement, with doses as low as 2 to 6 mcg/kg/day, can also effectively raise circulating IGF-­1 levels and may provide similar benefits in visceral fat reduction but fewer adverse effects.291-­294 Although low-­dose GH treatment did not result in worsened insulin sensitivity,285 this potential side effect and the lack of durability with GH treatment limit its clinical application. GH therapy is not currently FDA-­approved for treatment of HALS. GHRH, the hormone that regulates GH secretion by the pituitary gland, has recently emerged as an alternative treatment option for HALS. GHRH augments endogenous GH pulsatility and may preserve the negative feedback of IGF-­1 on the pituitary gland.285 Initial recombinant forms used, such as Sermorelin or GHRH 1-­29 (Geref), have been shown to increase lean body mass, decrease abdominal visceral fat, and decrease truncal fat without altering glucose levels.286,295,296 Tesamorelin (Egrifta), a GHRH analog, is the first FDA-­approved medication for the treatment of HALS. Two large phase III clinical trials were designed to evaluate the efficacy and safety of tesamorelin, demonstrating evidence of benefit without affecting glycemic control; patients receiving tesamorelin experienced an average visceral fat reduction of 11% at 6 months, and 18% by 12 months, while preserving insulin sensitivity.297 Trunk fat, waist circumference, and waist-­to-­hip ratio all significantly improved, and patients reported improved feelings about body image.295 Treatment with tesamorelin is also associated with improvement of triglycerides and total cholesterol levels,297 but its effect on HDL seems to be variable297,298 and needs to be confirmed by long-­term controlled studies. Adverse events are usually limited to arthralgia, myalgia, and local site irritation.295 The current indication for tesamorelin, which is administered by subcutaneous injection of 1 to 2 mg/day in single or divided doses, is HIV-­infected patients without active malignancy who are distressed by moderate to severe abdominal fat accumulation. Therapy should not be continued beyond 6 months if there is no treatment response. Similar to other interventions, such as lifestyle modification or metformin, the effect of GHRH analogs ceases to exist upon discontinuation, and while analogs are generally well-­ tolerated, long-­term effects of treatment remain to be elucidated.

Growth Hormone and Growth Hormone–Releasing Hormone Analogs. Growth hormone (GH) replacement has been proposed

Human Immunodeficiency Virus–Associated Lipodystrophy Syndrome–Specific Treatment. In addition to the above-­mentioned

to have a promising role in HALS treatment, in that this group of patients is prone to GH deficiency (GHD). It has been observed that basal GH concentrations, overnight GH secretion, and pulse amplitude are reduced in patients with HIV lipodystrophy,284 although normal pulse frequency is maintained.285 Relative GHD also appears to be common in HALS, as evidenced by decreased response to a standardized GH–releasing hormone (GHRH) arginine stimulation test.286 Although the causative relationship between GHD and metabolic abnormalities is not well understood, interventions that normalize GH concentrations have demonstrated efficacy in improving metabolic abnormalities associated with visceral fat accumulation in the HIV-­infected population.285

treatment options, HALS-­specific options may also be valuable in the treatment of these patients.

Modification of Highly Active Antiretroviral Therapy. Interrup­ tion of antiretroviral therapy is associated with increased mortality and opportunistic infections. Therefore, it should be avoided. Instead, a careful evaluation for cardiovascular risk factors should be conducted before antiretroviral therapy is initiated, and the drug with the fewest metabolic implications and comparable efficacy should be selected. Once lipoatrophy or metabolic complications develop, switching from the offending drug to another agent may be an important strategy. However, the reversal is normally slow and gradual.

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In the case of lipoatrophy, the treatment regimen with two thymidine analogs should generally be avoided. Switching to abacavir or tenofovir may partially restore subcutaneous fat.299 Switching from a PI to an NNRTI or abacavir has not shown any beneficial effects in terms of improvement of lipoatrophy. Regarding metabolic abnormalities, switching from a PI to nevirapine or abacavir has generally resulted in improved total cholesterol and triglycerides, whereas switching to efavirenz has generated less consistent results.300 Newer-­generation PIs (e.g., atazanavir, darunavir, and saquinavir) are associated with favorable lipid profiles. However, because antiretroviral regimens containing PIs normally require low-­dose ritonavir for its boosting effect, the impact of switching from one ritonavir-­boosted PI to another may be modest.220

Uridine. Uridine is a pyridine precursor that reverses mitochondrial toxicity. It may have clinical value in treating HALS induced by pyrimidines such as zalcitabine and stavudine. Uridine can also reverse the cell depletion and lactic acidosis seen with zidovudine and lamivudine combination. It has no effect on lipoatrophy caused by didanosine, a purine analog. Uridine is generally well-­tolerated.158

Management of Cosmetic Appearance Autologous fat transplant and implantation of synthetic bulking agents have been used for the cosmetic correction of facial lipoatrophy and appear to be associated with improvements in quality of life. However, long-­term, well-­designed studies are needed to assess their efficacy and safety.301 Occasionally, the fat harvested from HIV-­infected patients with buffalo hump can cause hypertrophies in the transplanted sites (cheeks) and cause a disfiguring “hamster” appearance. The intradermal injections of synthetic agents (polylactic acid or New-­Fill) have been shown to result in a more durable increase in total cutaneous thickness persisting up to 48 weeks. The efficacy and the safety of surgical treatment of HIV-­associated facial lipoatrophy using dermal fillers were assessed in a clinical trial. Four different dermal fillers (poly-­L-­lactic acid, calcium hydroxylapatite, polyacrylamide, and autologous fat) were examined. According to the results of the study, autologous fat transfer and permanent fillers achieved superior long-­term durability for HIV-­ associated facial lipoatrophy, with high rates of facial volume restoration and patient satisfaction.302 Recently, a 10-­ year follow-­ up study demonstrated that highly purified liquid-­injectable silicone was an effective long-­term therapy for HIV-­associated lipoatrophy. A microdroplet serial puncture technique was used, and small volumes of the product were injected at monthly or greater intervals to achieve the desired outcome. The most common adverse events that the patients experienced were excess fibroplasia of mild severity that could be treated and, to a lesser extent, severe acute inflammatory reaction presenting as facial edema without warmth or tenderness.303

Adipokines in Lipodystrophy

Leptin. A relatively large proportion of patients with lipodystrophy are found to have low leptin levels, including 20% to 30% of HALS patients153 and the vast majority of patients with generalized lipodystrophy. Apart from its central and peripheral effects on satiety and glucose metabolism,304,305 leptin has been associated with increased skeletal muscle lipoprotein lipase activity,304 which may partially explain its ability to preserve lean tissue during weight loss. The mechanisms through which leptin exerts its role remain under intensive investigation. Animal studies indicate that leptin acts mainly at the hypothalamus, particularly at neurons containing pro-­opiomelanocortin and neuropeptide Y, to regulate food intake and fuel partitioning.305 Functional MRI studies in humans confirm

that leptin mediates its “adipostatic” effect through hypothalamic and other brain areas that are important in emotional and cognitive control.306,307 Although animal and human studies indicate that leptin may also work peripherally (i.e., in the liver, muscle, and white adipose tissue) to affect lipid metabolism,308,309 other studies suggest that these effects are largely mediated by the CNS.310 Long-­term leptin treatment may also attenuate β cell function and decrease glucose-­induced insulin secretion,305,311 but whether these effects are independent from reduction of insulin resistance remains to be seen. Leptin Treatment in Generalized Lipodystrophy. The admini­ stration of recombinant leptin (r-­metHuLeptin or metreleptin) has been tested in the treatment of congenital and acquired non–HIV-­ related lipodystrophies and has shown amelioration of metabolic abnormalities.304,312 Several small, open-­ label studies show that subcutaneous injection of leptin (0.04–0.08 mg/kg/day) in patients with severe generalized lipodystrophy results in significant and sustained weight loss, with decreased fat and lean body mass. The weight loss is associated with a decrease in appetite, calorie intake, and resting energy expenditure. If excessive weight loss occurs, dosing should be decreased.313-­316 Metreleptin treatment has been associated with a significant reduction in hemoglobin A1c (HbA1c) levels, a reduction in fasting plasma glucose, and improvements in insulin sensitivity. Moreover, after metreleptin initiation, some patients were also able to discontinue insulin therapy.317-­319 Furthermore, metreleptin improved gonadotropin secretion, leading to the normal progression of female puberty and normalized menstrual cycles.320-­322 Hypertriglyceridemia, usually refractory to traditional lipid-­ lowering agents, is commonly responsive to leptin treatment.314 The liver volume decreases with leptin treatment, most likely as a result of decreased intrahepatic lipid content.323,324 Transaminases and hepatocellular injuries that are associated with nonalcoholic steatohepatitis are also reduced.323 In comparison with patients with generalized lipodystrophies, patients with familial partial lipodystrophy may have a less dramatic response to leptin treatment.325,326 Because all these studies are uncontrolled and open label, it remains to be proven beyond any doubt whether the beneficial effects observed are r-­metHuLeptin–specific. Metreleptin has been approved in Japan as a leptin therapy for the treatment of lipodystrophy for several years.327 Despite its declining use for metabolic disorders associated with partial lipodystrophy, the FDA advisory panel in 2014 suggested approval of metreleptin administration in adult and pediatric patients with generalized lipodystrophy. This announcement thus paved the way for a new modality in the treatment of this disorder, probably in the context of risk evaluation and mitigation strategy, given that the above uncontrolled studies have also demonstrated several potentially serious side effects. In individuals with generalized lipodystrophy, metreleptin in combination with dietary recommendations is the first-­line treatment strategy for the disease-­related metabolic and endocrine comorbidities. Metreleptin use may also be considered in children to prevent these disorders.2

Leptin Treatment in Partial Lipodystrophy. Some findings suggest that leptin replacement therapy may be beneficial in a subset of patients with severe partial lipodystrophy. Metreleptin appeared more effective in partial lipodystrophy patients with significant hypoleptinemia and severe metabolic disorders (such as HbA1c >8%, triglycerides >500 mg/dL).5,197 Currently, metreleptin usage, both in CPL and APL, is off-­label in the United States.

Leptin Treatment in Human Immunodeficiency Virus– Associated Lipodystrophy Syndrome. In addition to generalized lipodystrophies, we have reported a modest effect of leptin therapy on

CHAPTER 24  Lipodystrophy Syndromes metabolic abnormalities in a randomized, placebo-­controlled study in patients with HALS. Compared with placebo, r-­metHuLeptin therapy administered at 0.02 mg/kg twice a day decreases body weight mass and truncal fat mass but not peripheral fat or lean body mass and improves fasting insulin levels, insulin resistance, and levels of HDL.194 Leptin treatment was associated with a 15% decrease in central fat mass, as well as significant improvements in glucose levels, insulin sensitivity, and fasting insulin, despite having no effects on LDL and triglycerides.194 Other studies of longer duration confirmed these results, demonstrating a 32% decrease in visceral fat, increased hepatic insulin sensitivity, and improved dyslipidemia with metreleptin treatment.195 The improvements in lipids and abdominal fat were comparable to those reported with metformin and thiazolidinediones in HALS patients and provide an advantage over GH replacement because leptin replacement has not been observed to induce glucose intolerance. Although no direct comparisons are available, the decrease of visceral adipose tissue and the lipid-­lowering effect of leptin are either comparable or better than those reported for GHRH analogs.293,297 Thus, recombinant human leptin holds promise as an agent that could improve HIV-­associated lipoatrophy and features of the associated metabolic syndrome. Future randomized, placebo-­controlled trials of adequate duration are needed to fully quantitate efficacy and clarify the side-­effect profiles of metreleptin in HALS.

Clinical Use of Leptin: Benefits and Risks. Replacement of leptin may provide additional benefits beyond ameliorating metabolic abnormalities, which may include alleviation of the glomerular injury in humans with lipodystrophies321,328 and improvement of pituitary-­ gonadal function in lipodystrophic patients with severe leptin deficiency.322,329 It remains inconclusive, however, whether recombinant leptin therapy will affect such systems and/or bone density in the long term, given the uncontrolled nature of prior studies. Small human studies suggest that long-­term treatment may not affect bone density in patients with generalized lipodystrophies with regular menses indicating normoleptinemia.315,321,330 Metreleptin combined with lifestyle modification is currently indicated for adults and pediatric patients with generalized lipodystrophy (congenital or acquired). The effectiveness and safety of metrelpetin for the treatment of partial lipodystrophy and liver disease, including nonalcoholic steatohepatitis, have not been established. Additionally, metreleptin is not indicated for patients with HIV-­related lipodystrophy or metabolic disease (including hypertriglyceridemia and diabetes), without concurrent evidence of generalized lipodystrophy. The recommended daily dosage for patients with body weight 40 kg or under is 0.06 mg/kg/day at baseline, and may then be increased or decreased by 0.02 mg/kg to a maximum daily dose of 0.13 mg/kg. For patients with body weight more than 40 kg, the daily dosage for males is 2.5 mg/day, and then it may be increased or decreased by 1.25 to 2.5 mg/day to a maximum dose of 10 mg/day. The starting dose for females is 5 mg/day, which can be increased or decreased by 1.25 to 2.5 mg/day to a maximum amount of 10 mg/day. It should be noted that the effects of leptin do not appear to be sustained after the therapy is discontinued.206,210,331,332 Several patients experience side effects associated with metreleptin therapy. The most common and clinically significant adverse reactions are hypoglycemia (especially in insulin-­treated patients), headache, abdominal pain, and decreased weight.319 Other potential side effects of metreleptin treatment include deterioration of renal function328 and possible occurrence of T-­cell lymphomas. T-­cell lymphoma has been reported in individuals with AGL, independent of treatment with metreleptin. Thus, further investigation is required. Physicians

395

should evaluate the potential benefits and risks of metreleptin therapy in patients with significant hematologic abnormalities or AGL. Moreover, hypersensitivity reactions (including urticaria, anaphylaxis, generalized rash) and autoimmune disorder progression have been described in patients receiving metreleptin. In vivo, antimetreleptin antibodies with neutralizing activity have been identified. Individuals with loss of efficacy or severe infections during metreleptin treatment should be tested for neutralizing antibodies. The consequences of this neutralizing antibody response to leptin are not well characterized and may include inhibition of endogenous leptin activity and loss of metreleptin efficacy. However, potential risks are unclear and should be weighed against benefits.320,332,334

FUTURE PERSPECTIVES Adiponectin Adiponectin, as well as its receptors AdipoR1 and AdipoR2, are attractive future targets for drug development, given that levels of this hormone are lower in patients with HALS.188 Based on the fact that adiponectin is decreased in lipodystrophic states and evidence from mouse studies showing that adiponectin administration can improve insulin sensitivity, dyslipidemia, sustained weight loss without reducing food intake, and production of proinflammatory cytokines,335,336 adiponectin analog administration in replacement doses may prove to be an effective future treatment option. Because of its complex molecular structure, synthetic adiponectin is not yet available for therapy in humans. However, increasing adiponectin levels by pharmacologic means is likely to become a valuable addition to our armamentarium as a treatment option for lipodystrophy. Medications that increase endogenous levels of adiponectin in lipodystrophy, such as pioglitazone or INT-­131, a selective PPARγ modulator currently in development, may prove useful as therapeutic possibilities, because they increase circulating adiponectin levels. Further research is needed to determine the efficacy and long-­term durability of symptomatic improvement of lipodystrophy and its associated metabolic disorders, as well as short-­and long-­term morbidity and mortality. For your free Expert Consult eBook with bibliographic citations, as well as the ability to take notes, highlight important content, search the full text, and more, visit http://www.ExpertConsult.Inkling.com.

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CHAPTER 24  Lipodystrophy Syndromes 329. Oral EA, Ruiz E, Andewelt A, et al. Effect of leptin replacement on pituitary hormone regulation in patients with severe lipodystrophy. J Clin Endocrinol Metab. 2002;87:3110–3117. 330. Simha V, Zerwekh JE, Sakhaee K, et al. Effect of subcutaneous leptin replacement therapy on bone metabolism in patients with generalized lipodystrophy. J Clin Endocrinol Metab. 2002;87: 4942–4945. 331. Myalept safely and effectively. https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/125390s004lbl.pdf 332. Drug label information. https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=c986f93b-­855d-­4ef0-­b620-­5d41a0513e48#S1

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25 Lipoprotein Metabolism and the Treatment of Lipid Disorders Mason W. Freeman, Geoffrey Walford, and Janet Lo

OUTLINE Lipoprotein Metabolism, 404 Lipoproteins, 404 Metabolism of Lipids and Lipoproteins, 408 Disorders of Lipid Metabolism in Patients with Diabetes, 409 Type 1 Diabetes, 409 Type 2 Diabetes, 410 Genetic Basis of Lipid Disorders, 410 Monogenic Low-­Density Lipoprotein Disorders, 410

Monogenic High-­Density Lipoprotein Disorders, 412 Polygenic Lipid Disorders, 413 Diagnosis of Lipid Disorders, 413 Management/Treatment, 413 Assessing the Need to Treat Patients With Hyperlipidemia, 414 Dietary and Drug Treatment of Lipid Disorders, 415 Outcome Studies of Pharmacologic Therapy for Hyperlipidemia, 416

  Atherosclerosis is a chronic inflammatory disease of large-­and medium-­sized arteries that results from the deposition of lipids and lipoproteins in the intima of these vessels.1,2 This process is a major cause of morbidity and mortality in the developed world, and the toll is particularly high in the diabetic population, accounting for approximately two thirds to three quarters of all diabetic deaths.3 When combined with significant morbidity arising from ischemic injury and loss of limb perfusion, the impact of atherosclerosis on human health is enormous.4 Improvements in the care of patients with and without diabetes, including the use of intensive lipid-­lowering therapy, has led to significant reductions in mortality from cardiovascular (CV) disease in recent years.5 This chapter will review basic principles of lipoprotein metabolism, focusing on the impact diabetes has on these lipid pathways. Data that address the value of lipid-­altering treatments in reducing the burden of atherosclerosis in patients with and without diabetes will be presented. Finally, an approach to the management of patients with dyslipidemia will be outlined.

LIPOPROTEIN METABOLISM Lipoproteins The clinically reported serum triglyceride and cholesterol values represent the sum of the cholesterol and triglyceride contents of the circulating lipoproteins present in a volume of blood (expressed either as mg/dL or mmol/L). While knowledge of these total lipid values is very helpful in assessing CV risk, it is the distribution of cholesterol among the different lipoprotein fractions that is critical to any sophisticated understanding of atherosclerosis risk. Lipoproteins are structured to solve the problem of transporting highly hydrophobic lipids, such as cholesterol ester and triglyceride, in the aqueous environment of the blood. Because the bulk of lipoproteins are synthesized in the liver or gut, they must be moved via the bloodstream from these sites of synthesis to tissues where lipids will be taken up and utilized. By complexing neutral lipids with both proteins and more polar lipids such as unesterified cholesterol and phospholipids, the lipoprotein structure accomplishes this task. The more hydrophobic lipids are sequestered

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on the inside of the spherical lipoprotein, whereas more polar lipids and proteins decorate the outer surface (Fig. 25.1). In addition to solving the biophysical transport problem, the lipoprotein structure also enables particles to utilize a class of proteins known as apoproteins (Table 52.1) that serve as targeting molecules which interact with lipid-­ modifying enzymes (Table 25.2) or receptors and transporters (Table 25.3) critical for the normal functioning of the disparate lipoprotein classes. Complete loss of function mutations in the genes encoding apoproteins or the enzymes and receptors with which apoproteins interact (see Tables 25.1 to 25.3) are uncommon, but they can have a very significant influence on the circulating levels of lipoproteins in individuals with and without diabetes. As lipids play fundamental roles in a host of other cellular processes, mutations in lipid enzymes, receptors, transporters, and apoproteins can have profound effects on human biology beyond serum lipoproteins and CV disease, some of which are cited in Tables 25.1 to 25.3. The nomenclature for most lipoprotein particles derives from the method by which they were originally identified and separated, that is, density gradient ultracentrifugation. For a brief period after the introduction of gel electrophoresis, lipoproteins were also classified by their migration properties on these gels, giving rise to alternative names such as alpha and beta lipoproteins. This period coincided with the identification of many of the clinical syndromes caused by abnormalities of lipid metabolism, so the names for some of these disorders still carry the signature of the electrophoresis era (e.g., hypoalphalipoproteinemia indicates low levels of the alpha-­migrating or high-­density lipoprotein [HDL]). Gel electrophoresis is rarely used in clinical laboratories today for standard lipoprotein measurements, though it may be used in specialty lipid labs to further subdivide classes of lipoproteins. As a consequence, the current medical literature has largely returned to the use of the density nomenclature to describe and characterize disorders of lipoprotein metabolism. Although lipoproteins constitute a continuous spectrum of density within the plasma, they are commonly divided into five major classes (see Fig. 25.1). These are: (1) chylomicrons; (2) very low-­density lipoproteins (VLDLs); (3) intermediate-­density lipoproteins (IDLs); (4)

CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders

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Fig. 25.1 The upper panel of the figure lists the density range that encompasses each lipoprotein class and the major tissue in which the lipoprotein is synthesized. The bottom panel is a graphic representation of a generic lipoprotein, emphasizing the spherical shape of the particle, the use of amphipathic apoproteins and polar lipids on the particle surface that interact with cells and the blood environment, and the sequestration of nonpolar lipids in the core of the lipoprotein. All lipoproteins share these general features, but they differ in size, lipid composition, and in the specific apoproteins embedded.

TABLE 25.1  Apolipoproteins of the Serum Lipoproteins Apoprotein Ref

Major Lipoprotein

Functional Role (If Known)

Human Disease Association

AI239 AII240 AIV241 AV242 B100243

HDL HDL HDL HDL, VLDL LDL

LCAT activation Structural protein, ? function Fat absorption, LCAT activation TG metabolism VLDL and LDL synthesis

B48243 CI244 CII245

Chylomicron, chylo remnant HDL, VLDL, chylomicron Chylomicron, VLDL

CIII246 D247

Chylomicron, VLDL HDL

E248

Chylomicron remnant, IDL, HDL

Intestinal fat absorption TG-­rich lipoprotein uptake Lipoprotein lipase activation (LPL inhibition) Lipoprotein lipase inhibition ? Activator of LCAT, neuronal function, cytoprotection LDL receptor and LRP binding

Cataracts, low HDL levels, CHD No defect identified in a deficient subject ? Altered plasma lipid and glucose levels Hypertriglyceridemia Hypobetalipoproteinemia (low LDL levels), familial defective apo B (high LDL levels) Hypobetalipoproteinemia (low LDL levels) Unknown Chylomicron syndrome, pancreatitis

J (clusterin)249 M250

HDL HDL

Complement, chaperone, oxidative stress ? Cholesterol transport

Hypertriglyceridemia Unknown Familial dysbetalipoproteinemia (type III), isoforms linked to Alzheimer risk Unknown Unknown

Modified from Genest J, Libby P, and Gotto AMJ. Lipoprotein disorders and cardiovascular disease. In: Zipes DP, Libby P, Bonow RO et al., eds, Braunwald’s Heart Disease: A Textbook of Cardiovascular Medicine 7th ed. Philadelphia: Elsevier Saunders; 2005; pp. 1013–1033. HDL, High-­density lipoprotein; LCAT, lecithin cholesterol acyltransferase; CHD, coronary heart disease; VLDL, very low-­density lipoprotein; LDL, low-­ density lipoprotein; LPL, lipoprotein lipase; TG, triglyceride; LRP, low-­density lipoprotein receptor–related protein; IDL, intermediate-­density lipoproteins.

low-­density lipoproteins (LDLs); and (5) HDLs. Within these classes, further subdivision of lipoproteins can be made. In diabetics, a higher proportion of LDL is carried in a denser, smaller diameter subfraction that is called small dense LDL (sdLDL). HDLs are commonly divided into HDL2 and HDL3 subclasses, but some investigators have adopted

a gel separation method to segregate HDL into a much large number of subtypes.6 In this chapter, the density classification terminology will be used for all lipoproteins other than chylomicrons. A standard lipid profile, consisting of measured total serum or plasma cholesterol and triglyceride levels, combined with a measured HDL-­cholesterol

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TABLE 25.2  Lipid Transporters, Receptors, and Regulatory Proteins Protein Ref

Function

Major Tissue Location

Human Disease Association

ABCA143 ABCA3251 ABCA4252 ABCA12253,254 ABCG1255 ABCG5256 ABCG8256 Apo E-­R2/(LRP8)257 CD36258 CETP259 HM74/GPR109A260 LDL-­R36 LOX-­1261 LRP1262

Transporter of phosphatidylcholine and cholesterol Transporter of lung surfactant Transporter of N-­retinylidene-­phosphatidylethanolamine Transporter of ceramide esters Transporter of cholesterol Transporter with G8 of sterols Transporter with G5 of sterols Apo E receptor, reelin receptor Fatty acid translocase, scavenger receptor Cholesterol transfer in plasma Receptor for nicotinic acid Receptor for LDL Scavenger receptor for oxidized LDL Endocytosis of many ligands, including chylomicron remnants Endocytosis receptor, multiple ligands, including lipoproteins Lipid transfer in assembly of VLDL Cellular cholesterol trafficking Cellular cholesterol trafficking Phospholipid transfer protein LDL-­R regulating protein Scavenger receptor, multiple ligands Scavenger receptor, CE transfer Apo E receptor, reelin receptor

Ubiquitous (macrophages, liver, and gut) Alveolar type II cells Retinal pigment epithelial cells Keratinocyte Macrophages Enterocyte Enterocyte Brain, testis Macrophages, skeletal muscle, platelets Liver, adipose Liver Ubiquitous (liver, adrenal) Endothelium, smooth muscle Liver, brain, placenta

Tangier disease (low or absent HDL) Neonatal respiratory failure Stargardt’s macular degeneration Harlequin and lamellar ichthyosis Unknown Sitosterolemia Sitosterolemia Unknown ? Hypertriglyceridemia Hyperalphalipoproteinemia (high HDL) Unknown Familial hypercholesterolemia (high LDL) Unknown Unknown

Kidney, brain

Donnai–Barrow and facio-­ oculoacoustico-­renal syndromes Abetalipoproteinemia Niemann–Pick C disease Unknown Unknown High and low levels of LDL Unknown Unknown Cerebellar hypoplasia

LRP2/megalin263 MTP264 NPC1265 NPC1L1266 PLTP267 PCSK9268 SR-­A269 SR-­B1270 VLDL-­R257

Liver Brain, liver Hepatocyte, enterocyte Ubiquitous, liver Liver Macrophages Liver, adrenal Brain, heart, adipose, muscle

Modified from Genest J, Libby P, and Gotto AMJ. Lipoprotein disorders and cardiovascular disease In: Zipes DP, Libby P, Bonow RO et al., eds, Braunwald’s Heart Disease: A Textbook of Cardiovascular Medicine 7th ed. Philadelphia: Elsevier Saunders, 2005; pp. 1013–1033. ABC, ATP-­binding cassette; ABCA, a class of ABC transporter, ABCG, G class of transporter; ACAT, acyl cholesterol acyl transferase; Apo E-­R, apo E receptor; CETP, cholesterol ester transfer protein; LDL-­R, LDL receptor; LOX-­1, lectin-­like oxidized LDL receptors; LRP, LDL-­R–related protein; MTP, microsomal triglyceride transfer protein, NPCILI, Niemann–Pick C1 like protein; PLTP phospholipid transfer protein; PCSK9, proprotein convertase, subtilisin/kexin type 9; SR-­A, scavenger receptor A; SR-­BI, scavenger receptor B1; VLDL-­R, VLDL receptor; HDL, high-­density lipoprotein; LDL, low-­ density lipoprotein; VLDL, very low-­density lipoprotein; CE, cholesterol ester.

TABLE 25.3  Lipid Metabolism Enzymes Protein Ref

Major Function

Major Tissue Location

ACAT144 ACAT244 Acid lysosomal lipase272 Cholesterol 7-­α hydroxylase273 Cholesterol 27-­hydroxylase273 DGAT1274 DGAT2274 Endothelial lipase275 Hepatic lipase276 HMG CoA reductase277,278 Hormone-­sensitive lipase279 LCAT280

Cellular cholesterol esterification Cellular cholesterol esterification Hydrolysis of CE Synthesis of bile acids Synthesis of bile acids TG synthesis TG synthesis Hydrolysis of PL and TG Hydrolysis of TG Cholesterol synthesis Hydrolysis of intracellular TG Cholesterol esterification (HDL)

Macrophages, steroidogenic tissues Liver, intestine Macrophage, adrenal, liver Liver Liver Intestine, liver, adipose, mammary Liver, adipose, mammary Liver, lung, kidney, endothelium Liver Ubiquitous, liver Adipose Plasma, bound to HDL

Lipoprotein lipase281,282

Hydrolysis of TG

Muscle, adipose

Condition Caused by Altered Levels or Activity Unknown Unknown CE storage disease, Wolman syndrome Cholestatic liver disease Cerebrotendinous xanthomatosis Unknown Unknown ? Metabolic syndrome Elevated lipids, xanthomas, early atherosclerosis Unknown Unknown Anemia, low HDL, renal failure, Fish eye disease (corneal opacities) Chylomicronemia syndrome

ACAT, Acyl cholesterol acyl transferase; CE, cholesterol ester; DGAT, diacyl glycerol acyl transferase; ER, endoplasmic reticulum; HMG, 3-­hydroxy-­3-­methylglutaryl; LCAT, lecithin cholesterol acyl transferase; PL, phospholipids; TG, triglyceride; ACAT, acyl cholesterol acyl transferase; CE, cholesterol ester; DGAT, diacyl glycerol acyl transferase; HDL, high-­density lipoprotein.

CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders level, is adequate to determine the relevant lipoprotein profile of most patients. Occasionally, specialty lipid assays are required to further characterize abnormal blood lipid values. A brief description of each of the five major lipoprotein classes is summarized in the following paragraphs. Readers interested in a more comprehensive introduction to lipoprotein metabolism should consult the lipid sections of The Online Metabolic and Molecular Bases of Inherited Disease. 8th ed.7

Chylomicrons. Chylomicrons derive from dietary fat and carry triglycerides throughout the body. Chylomicrons are generated in enterocytes in a process that appears to depend on lipin 2/3 phosphatidic acid phosphatase enzyme activity.8 The major structural protein of chylomicrons is apolipoprotein (apo) B48, a protein that is produced from apo B100 RNA by a unique editing process employed by gut cells that generates a protein that is 48% of the length of apo B 100.9 Chylomicrons also contain other apoproteins, including members of the apo C and apo A family. These proteins can redistribute to other lipoprotein classes within the blood as chylomicrons transfer their lipid content to cells and interacting lipoproteins. Chylomicrons have the lowest density of all lipoproteins and will float to the top of a plasma specimen left in a refrigerator overnight, forming a cream-­like layer. Owing to its large size, the chylomicron does not readily enter the artery wall and is therefore thought not to be atherogenic, but the atherosclerotic role of the triglyceride-­depleted chylomicron remnant remains controversial. Triglyceride makes up most of the chylomicron and is removed by the action of an enzyme that is bound to the surface of endothelial cells, lipoprotein lipase (LPL). Patients deficient in this enzyme or its apoprotein cofactor (apo CII) have very high serum triglyceride levels and are at increased risk for developing acute pancreatitis. Inasmuch as insulin is also a critical cofactor for LPL activity, the majority of patients presenting with hypertriglyceridemic pancreatitis have poorly controlled diabetes as a major contributing factor to their delayed chylomicron clearance. Very Low-­Density Lipoproteins. VLDLs are also triglyceride-­rich, but their triglyceride content is lower and their cholesterol content higher than those of chylomicrons. The protein composition of VLDLs also differs from chylomicrons in that the major structural protein is full-­length apo B (apo B100) as opposed to the truncated apo B48 form. Like chylomicrons, VLDLs are substrates for LPL-­mediated triglyceride removal. The function of VLDL and chylomicrons is to carry triglycerides synthesized in the liver and intestines to capillary beds in adipose tissue and muscle, where they are hydrolyzed to provide fatty acids that can be oxidized to produce adenosine triphosphate (ATP) for energy production. Alternatively, if not needed for energy production, they can be reesterified to glycerol and stored as fat. After removal of their triglyceride, VLDL remnants (called IDLs) can be further metabolized to LDL. VLDLs serve as acceptors of cholesterol transferred from HDL, accounting in part for the inverse relation between HDL cholesterol and VLDL triglyceride. This transfer process is mediated by an enzyme called cholesterol ester transfer protein (CETP). The most common lipid abnormality seen in patients with type 2 diabetes is an elevated VLDL level, the causes of which will be discussed in the section on diabetic lipid abnormalities.

Low-­Density Lipoproteins. LDLs are the major carriers of cholesterol in humans, responsible for supplying cholesterol to tissues with the highest sterol demands. LDLs are also the lipoproteins most clearly implicated in causing atherogenic plaque formation.10 Circulating LDL levels can be increased in persons who consume large amounts of dietary saturated fat and/or cholesterol.11 LDL levels are also elevated in those who have genetic defects that affect LDL receptor function

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(e.g., familial hypercholesterolemia [FH], proprotein convertase subtilisin kexin 9 (PCSK9) mutations, autosomal recessive hypercholesterolemia) or the structure of LDL’s apoprotein, apo B, or who have polygenic disorders affecting LDL metabolism.12 Circulating LDLs are taken up by the endothelium lining the artery wall, traverse it, and are deposited and trapped in the arterial intima. There, they may undergo oxidation or other biochemical modification, be taken up by macrophages, and stimulate atherogenesis. The association of total serum cholesterol with coronary heart disease (CHD) is predominantly a reflection of the strong correlation of LDL-­cholesterol levels with total cholesterol levels in most humans. Studies in patients with diabetes have produced somewhat conflicting data on the prevalence of increased LDL levels, as defined by general population norms, but the preponderance of the evidence indicates that diabetics have LDL levels that are similar to those seen in well-­matched, nondiabetic control populations. Given the greater risk of developing coronary artery disease (CAD) in individuals with diabetes, however, the National Cholesterol Education Program guidelines defined LDL levels above 100 mg/dL as being undesirable in those with diabetes. When judged by this stringent criterion, elevated LDL levels are extremely common in patients with diabetes. In addition, the LDL in individuals with diabetes is frequently smaller, denser, and more readily oxidized––all properties that are associated with a higher degree of atherogenicity.13,14

High-­Density Lipoproteins. HDLs are the smallest of the lipoproteins; despite this, they carry a variety of apoproteins, including members of the apo A and C families. HDLs, unlike the other lipoproteins, are believed to function in humans primarily to return lipids from peripheral tissues to the liver and gut for excretion, rather than moving lipids from the gut organs to the periphery.15 This perspective on HDL function is an oversimplification, as cell culture experiments indicate that HDL can donate lipids as well as pick them up; moreover, other components of blood in addition to HDL may be involved in the reverse transport process.16-­18 It has proven to be quite difficult to quantitate the net movement of cholesterol carried in HDL in and out of peripheral tissues in whole animals. Further complicating the issue is the fact that many of the standard laboratory animals used in metabolic studies carry most of their serum cholesterol in HDL rather than LDL, diminishing the relevance of studying their lipoprotein metabolism as a surrogate for understanding that of the LDL-­rich human. Nevertheless, a considerable body of evidence suggests that HDL functions to protect tissues from unwanted accumulation of cholesterol. The mechanisms by which HDL may protect against atherosclerosis include its involvement in the reverse cholesterol transport pathway just described, as well as through contributions arising from its carriage of proteins that appear to have anti-inflammatory and antioxidant properties.19,20 The unesterified cholesterol from tissues that is transferred to HDL is esterified by the action of lecithin cholesterol acyltransferase (LCAT) and stored in the central core of HDL. This esterified cholesterol can be transferred back to lower-­density lipoproteins by the action of cholesterol ester transfer protein, or it may be removed at the liver by the action of a plasma membrane receptor called scavenger receptor B-­1. A particularly effective reverse transport system is thought to explain, at least in part, the association of elevated HDL-­cholesterol levels with a reduced risk of developing CHD. To date, however, therapies directed at raising HDL-­cholesterol levels have failed to improve CV outcomes unless LDL cholesterol was also substantially lowered. In addition, studies of some genetic variants that appear to control HDL levels have been shown not to predict atherosclerosis risk, unlike genetic variants that control LDL levels, calling into question a causal role for HDL in impeding atherosclerosis.21 Thus, the inverse association of HDL-­cholesterol levels with atherosclerotic vascular disease risk, while

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epidemiologically well established, does not ensure that manipulations of HDL-­cholesterol by therapeutic agents or lifestyle modifications will confer a CV benefit via that lipoprotein change. Exercise increases HDL levels; obesity, hypertriglyceridemia, and smoking lower them. Patients with diabetes, having higher body mass indices and hypertriglyceridemia, typically have lower HDL cholesterol levels than do nondiabetics, contributing further to the atherogenic lipid profile of the diabetic.

Metabolism of Lipids and Lipoproteins The synthesis of lipoproteins in the liver and gut is a complex process regulated by hormonal and transcriptional networks that are affected by dietary intake of both lipid and carbohydrate. The disorders of carbohydrate and lipid metabolism caused by diabetes have both direct and indirect effects on lipoprotein metabolism. A brief summary of normal lipoprotein metabolism will be presented in this section, followed by a description of the impact of diabetes on these processes in the next section. Following ingestion of a meal containing fat, the triacylglycerols (TAGs) are broken down by pancreatic triacylglycerol lipase to yield sn-­2-­monoacylglycerol and fatty acids. Ingested cholesterol is solubilized by bile salts into micellar structures, a step that is disrupted by bile acid resin binders. The lipids then transverse the enterocyte brush border membrane and are reassembled in the intestinal cell into a triglyceride-­rich lipoprotein, the chylomicron. The role of transport proteins in the passage across the enterocyte membrane remains disputed, with evidence for both a passive diffusion and an active transport mechanism.22,23 For cholesterol, a second transport step translocates the sterol into intracellular pools that can be accessed for lipoprotein packaging.24 Niemann-­Pick C1-­like 1 protein appears to be required for this second step to occur and is the target of the cholesterol absorption inhibitor ezetimibe.25,26 The internalized cholesterol is moved to the endoplasmic reticulum (ER), where the monoacylglycerol and fatty acids are also carried by transport proteins. The latter are recombined into TAG and bound by microsomal triglyceride transport protein (MTP). A second pathway of triglyceride synthesis uses acylation of glycerol-­3-­phosphate to phosphatidic acid, dephosphorylation to diacylglycerol (DAG), and then acylation of DAG to TAG. A high-­density, protein-­rich particle synthesized in the enterocyte, containing apo B48 and apo AIV, is then packaged with the absorbed lipids to generate the chylomicron.27 This is then transported by vesicles to the basolateral membrane for exocytosis into the mesenteric lymph. The lipoproteins in the lymph enter the blood circulation via discharge from the thoracic duct. Two ABCG half transporters, ABCG5 and G8, contribute to net cholesterol absorption in the gut through their ability to resecrete sterol that has been absorbed by the enterocyte back into the gut lumen, thereby reducing net sterol uptake.28 The total amount of diet-­derived TAG that is delivered to the circulation varies and is influenced by multiple factors, including the fat content of the diet, the amount of phosphatidylcholine in the intestinal lumen, and the expression level of apo AIV in the enterocyte. Overall, humans have a remarkable ability to absorb large quantities of both cholesterol and fat; 35% to 60% of ingested cholesterol is absorbed, with the amount inversely correlated with hepatic cholesterol synthesis. Up to 600 g of fat is absorbed with 95% efficiency. In individuals with normal lipid metabolism, the ingested fat is cleared from the enterocyte in less than 14 hours, forming the basis for measuring serum lipids after a fast of this duration. Circulating chylomicrons are subsequently cleared from the plasma by the action of endothelial-­bound LPL, which cleaves the TAG, enabling the liberated glycerol and free fatty acids to move into the adjacent adipose or muscle tissue. The resulting chylomicron remnant particle

can be further metabolized by lipases and appears to be retained in the hepatic space of Disse, where it can bind to heparan sulfate proteoglycans and acquire additional apo E. The acquisition of the apo E enables the remnant to be cleared efficiently by either the LRP1 or LDL receptor, but patients with diabetes have accumulation of remnants and slower lipoprotein hepatic clearance.29,30 The other major triglyceride-­rich lipoprotein, VLDL, is synthesized in the liver and contains apo B100 rather than apo B48 as its major structural protein.31 As with chylomicrons, VLDLs rely on MTP to combine lipids with the apo B protein to produce a nascent, lipid-­poor lipoprotein.32,33 This VLDL can be secreted from the liver or further lipidated to produce a mature VLDL. Individuals lacking MTP activity develop abetalipoproteinemia, a disorder characterized by erythrocyte acanthocytosis, anemia, steatorrhea, spinocerebellar degeneration, and fat-­soluble vitamin deficiency. Acquisition of lipid by the mature VLDL appears to depend on triglycerides that are stored in the cytosol. Increased fatty acid delivery to the liver, from dietary sources or that released by lipase activity in peripheral tissues, stimulates VLDL production. After secretion of the mature VLDL from the liver, its triglycerides are initially removed by the action of LPL, and the resulting IDL can be further catabolized by hepatic lipase to produce mature, cholesterol-­rich LDL.

Low-­Density Lipoprotein Metabolism. There is still some dispute about whether mature LDL can be secreted from the liver rather than always being derived from the progressive removal of triglyceride from VLDL/IDL precursors.34 However, the preponderance of data indicates that little if any LDL is directly secreted from the liver. Each LDL particle contains a single apo B molecule, which dictates that individuals heterozygous for apo B mutants will have two populations of LDL: one that carries a wild-­type protein and one with a mutant protein. LDLs are utilized throughout the body as a source of exogenous cholesterol and are taken up by LDL receptors present at the plasma membrane. While LDL receptors are ubiquitous, the majority are expressed by the liver. The LDL receptor binds to both apo E– and apo B–containing lipoproteins. As the affinity of the receptor is higher for apo E than for apo B, apo E–containing lipoproteins, such as IDL, can compete with LDL for uptake by the LDL receptor. However, there are other receptors that can bind apo E–containing lipoproteins, permitting the clearance of the latter, but not that of LDL when LDL receptor activity is lost or diminished. Thus, mutations in the LDL receptor result in elevated LDL levels in the plasma, causing the autosomal codominant disorder FH, which is among the most common of Mendelian genetic disorders (see Genetics section later).35 The elucidation of the cholesterol homeostatic mechanisms controlled by the LDL receptor constituted one of the major advances in our understanding of human physiology and disease.36 The internalization of LDL by the LDL receptor leads to trafficking of the internalized lipoprotein to the endosome, where, after fusion with lysosomes, the sterol in the lipoprotein is released and shuttled to the ER. A sterol-­sensing system in the ER regulates the movement of a membrane-­bound transcription factor sterol response element binding protein (SREBP) from the ER to the nucleus.37-­42 SREBP works in concert with other transcriptional regulators to influence the expression of many of the cholesterol synthesis, metabolizing, and transport molecules, particularly in the liver. This elegant homeostatic system enables cells to reduce their de novo cholesterol synthesis when adequate sterol supplies are present and to increase synthesis and LDL receptor expression when cellular demands require more cholesterol.

High-­ Density Lipoprotein Metabolism. Although the relationship between elevated levels of HDL and lower rates of cardiovascular disease was recognized more than 65 years ago, a mechanism by

CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders which HDL causally protects against CHD remains to be elucidated. HDL are initially produced as lipid-­poor phospholipid discs containing apolipoprotein AI. Following interaction with phospholipid/ cholesterol transporters, primarily ABCA1, the nascent HDL particle acquires unesterified cholesterol from cellular stores.43 For macrophages in particular, this cholesterol transport process constitutes a cholesterol efflux pathway that appears to be critical to the cell’s ability to unload excess cholesterol acquired by phagocytosis of dead cells and by the unregulated uptake of lipoproteins by scavenger receptor pathways.15 Cholesterol is stored in an intracellular lipid pool, having been esterified at its 3’OH position by the activity of acyl coenzyme A (CoA):cholesterol O-­acyltransferase (ACAT).44 A neutral cholesterol hydrolase cleaves the fatty acid from cholesterol ester to convert it back to free (unesterified) cholesterol when cellular signals call for the use or export of the sterol.45 This free cholesterol can be shuttled to the plasma membrane, where it can be transported across by the action of ABCA1. The interaction of lipid-­poor nascent HDL with ABCA1 appears to involve direct binding of apo AI to the transporter, but the actual lipid transfer process is poorly understood.46-­50 Once the unesterified cholesterol transfers to the nascent HDL, it can be reesterified by the action of LCAT and centralized in the lipid core of the growing HDL particle. This HDL particle can now interact with a second ABC transporter, ABCG1, and additional cholesterol can be acquired.49 The role of ABCA1 is well established, as loss of its activity leads to Tangier disease (see Genetics section) and the absence or near absence of HDL in the plasma. Once the HDL has acquired sufficient cholesterol and esterified it, it can donate this lipid back to lower-­density lipoproteins in the plasma, such as VLDL, through the activity of CETP. Alternatively, HDL can interact with a cellular receptor, SR-­ BI, to selectively transfer the cholesterol ester to cells, including the liver, where it could be excreted in the bile as cholesterol or one of its bile acid derivatives.51 The protein component of HDL is cleared via the kidney, a process that appears to depend on the HDL being lipid depleted. The pathway just described, moving cholesterol from cellular storage pools to HDL and ultimately to the liver, is termed the reverse cholesterol transport pathway. The activity of this pathway has been postulated to explain at least in part the link between HDL levels and lower CHD risk. However, HDL also carries antioxidants and a host of proteins linked to complement and inflammation pathways that suggest it may be playing a more general role as a scavenger of molecules that stimulate inflammation. The complexity of its function has made targeting increases in HDL levels as a therapeutic objective very challenging. Both animal and human data have clearly established that simply raising HDL cholesterol levels does not ensure that a benefit on the atherosclerotic process will ensue. KEY POINTS • Lipoproteins vary in their lipid and protein composition forming a continuum of density from chylomicrons, which are the largest and least dense, to high density lipoproteins which are the smallest and most dense. They serve various biological functions but primarily are involved in moving cholesterol and triglycerides between tissues via the bloodstream. Both excess numbers and the inability to generate sufficient numbers of specific lipoproteins are associated with adverse health consequences.

DISORDERS OF LIPID METABOLISM IN PATIENTS WITH DIABETES Type 1 Diabetes Studies dating back more than three decades documented that individuals with type 1 diabetes have increased rates of CHD.52-­54 This

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risk was estimated to be 5-­to 10-­fold or greater than that seen in age-­matched controls without diabetes. In the Diabetes UK cohort of 23,751 subjects who were diagnosed with insulin-­dependent diabetes prior to the age of 30 years, for example, those individuals in the study who were 20 to 29 years of age at its onset had a standardized mortality ratio for ischemic heart disease of 11.8 for men and 44.8 for women. For those 30 to 39 years of age, the ratios were 8.0 and 41.6 for men and women, respectively.55 More recent studies still show a significantly greater relative risk of atherosclerotic vascular disease in patients with type 1 diabetes, though the absolute risk has fallen substantially in that population in recent decades.5,56,57 Despite the long and consistent reproducibility of this risk association, the pathophysiology underlying that relationship is still poorly understood. Unlike type 2 diabetes, where obesity and insulin resistance lead to changes in the standard lipid profile that are considered atherogenic, the type 1 diabetic who has not developed nephropathy and whose glucose is well controlled will typically have a normal serum lipid profile.58 In the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications study, the mean LDL-­ cholesterol in the group that was given intensive glucose-­lowering treatment was 111 ± 29 mg/dL in women and 119 ± 31 in men; HDL-­ cholesterol levels averaged 63 ± 16 and 51 ± 14 for women and men, respectively. Serum triglyceride levels were also in the normal range, with women averaging 76 ± 37 mg/dL, and male values running moderately higher at 98 ± 67 mg/dL. These lipid values, which would be considered normal in most Western countries, did not differ significantly from those of the patients enrolled in the conventional glucose-­ lowering treatment group of the study. These findings make clear that the standard lipid profile provides few insights into the increased risk of CHD in type 1 diabetics.59 Surprisingly, epidemiologic risk-­factor assessment of the correlates of CHD in type 1 diabetes shows a weak association to the one metabolic risk that predominates in the type 1 population: i.e., hyperglycemia.60,61 It was therefore somewhat unexpected when a 17-­year follow-­up of the DCCT cohorts showed that the intensive treatment group had a 57% reduction in the risk of nonfatal myocardial infarction, stroke, or death from CV disease compared with the conventional treatment group (95% confidence interval [CI]: 0.21–0.88; P = 0.02).62 Other studies of the impact of glucose control on CV outcomes have produced results that suggest that strict control of HbA1c levels is less important in reducing atherosclerotic vascular events or, in some circumstances, may even be detrimental.63 Independent of the mechanism by which CV benefit is conferred, recent studies do suggest that current therapies are making a clear impact on reducing disease in type I diabetics.5 Miller et al. reported a significant improvement in overall life expectancy in type 1 diabetics who were diagnosed between 1965 and 1980 versus similarly aged type 1 diabetics whose diagnosis was made between 1950 and 1964.64 Although the data presented do not permit delineation of what accounts for this improvement, better care of ketoacidosis, renal disease, blood pressure and lipids are all strong contenders for contributors to the longevity improvement. With increasing numbers of patients experiencing the onset of type 2 diabetes in childhood or adolescence, it is now possible to compare outcomes of type 1 and type 2 diabetics in individuals whose diabetes was diagnosed early in life. Those with early-­onset type 1 diabetes fare significantly better than those with early-­onset type 2 diabetes, and a clear difference in CV death has been observed.65 Finally, a study of diabetes-­related complications, not specific as to type 1 or type 2, but likely predominantly representing type 2 diabetics, did find a substantial reduction in major complications of diabetes between 1990 and 2010, with a reduction in acute myocardial infarction showing the largest improvement.66

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Type 2 Diabetes There are alterations in multiple lipoprotein metabolism pathways in type 2 diabetics that lead to the typical abnormal lipid profile commonly seen. This profile includes hypertriglyceridemia secondary to high VLDL levels, as well as increased amounts of sdLDL and lower levels of HDL cholesterol. LDL-­cholesterol levels are typically not higher in diabetic populations when compared with well-­matched nondiabetic subjects. In an elderly Finish population, for example, approximately 30% of diabetic subjects had serum triglyceride levels greater than 200 mg/dL compared with 13% of nondiabetic subjects. HDL cholesterol levels were less than 35 mg/dL in about 25% of diabetics compared with 12% of nondiabetics. In this study, elevated LDL levels as defined by an LDL greater than 130 mg/dL were actually more prevalent in the nondiabetic population than in those with diabetes.67,68 The causes of classic diabetic dyslipidemia are only partly understood. Individuals with insulin resistance and type 2 diabetes overproduce mature VLDL in the liver. This is accompanied by a slower clearance of triglyceride-­rich lipoproteins via reduced activity of LPL. Coupled with decreased receptor uptake of the remnant and IDL particles that form after LPL partially depletes VLDL of its triglyceride, diabetes produces substantial elevations in serum triglyceride levels. Production of sdLDL appears to be tightly linked to the resulting hypertriglyceridemia insofar as CETP exchanges triglyceride from VLDL for cholesterol ester from LDL, producing a triglyceride-­enriched LDL.69 This lipoprotein appears to be a preferred substrate for hepatic lipase, which hydrolyzes the triglyceride to produce an LDL that is smaller, lower in cholesterol ester content, and higher in density than LDL produced in the absence of excess VLDL. The diabetic with increased sdLDL levels has a higher LDL particle number for an equivalent serum LDL-­cholesterol concentration than does an individual with normal LDL. This increased particle number, whether measured by nuclear magnetic resonance assays or serum apo B levels, is associated with an increased risk of CAD.14 Thus, the central metabolic change that accounts for much of the diabetic dyslipidemia is the overproduction of VLDL by the liver. The molecular mechanisms accounting for VLDL overproduction are not fully elucidated, but there is evidence that insulin activates a phosphatidylinositol-­3 kinase pathway that inhibits apo B secretion while activating a mitogen-­activated protein kinase (MAPK) that downregulates MTP expression.70,71 Insulin effects on transcriptional regulators of VLDL secretion may also be involved. In vivo metabolic studies suggest that the prevalence of hypertriglyceridemia and low HDL-­cholesterol levels is higher in diabetics who are insulin-­resistant as opposed to insulin-­deficient, accounting for the higher prevalence of these disorders in patients with type 2 versus type 1 diabetes. KEY POINTS • Normal body mass individuals with type I Diabetes commonly have a normal lipid profile with neither high levels of LDL or VLDL nor low levels of HDL. Patients with type 2 diabetes, particularly those with significant elevations in BMI, often have increased levels of VLDL (serum triglycerides are elevated) and lower levels of HDL cholesterol. The overall CV risk of those with type 2 diabetes is greater than those with type 2 diabetes, but both groups are higher risk than those without diabetes

GENETIC BASIS OF LIPID DISORDERS Although metabolic disorders associated with diabetes, hypothyroidism, and nephropathy can exacerbate or even cause hyperlipidemia (Table 25.4), a substantial number of dyslipidemic individuals have none of these problems. Individuals with the greatest deviation from normal

TABLE 25.4  Secondary Causes of Hyperlipidemia and Dyslipidemias Metabolic/hormonal

Hepatic Renal Dietary

Medications

Diabetes Hypothyroidism Lipodystrophy Polycystic ovarian disease Primary biliary cirrhosis Other forms of cirrhosis Chronic renal failure Nephrotic syndrome Alcohol Foods highly enriched in cholesterol and saturated fat Estrogens Glucocorticoids Anti-­HIV– treatments, especially protease inhibitors Oral androgens and anabolic steroids Thiazide diuretics Beta-­blockers Retinoic acid Antipsychotics

levels of lipoproteins often have a single gene defect that is responsible for their lipid disorder. The vast majority of hyperlipidemic patients, however, do not have a monogenic defect. Rather, they are most likely to have a polygenic disorder and an additional contribution from environmental factors (e.g., excessive saturated fat intake for high LDL levels; obesity or smoking for lower HDL levels). Currently, a genetic diagnosis is rarely required to enable appropriate lipid care to be given. Nevertheless, the identification of the causes of monogenic lipid disorders has been critical to our understanding of both normal and abnormal lipoprotein physiology. The lipid disorders with the greatest impact on developing CAD risk are those that increase the serum LDL level or reduce the HDL level, and these are briefly presented next.

Monogenic Low-­Density Lipoprotein Disorders The molecular basis of four monogenic disorders that primarily affect LDL levels have been characterized: (1) FH, (2) familial defective apo B, (3) autosomal recessive hypercholesterolemia, and (4) PCSK9 mutations. These disorders illustrate distinct mechanisms leading to elevated LDL levels.

Familial Hypercholesterolemia. FH is one of the most thoroughly studied and common genetic disorders of mankind. Recent analyses have suggested the prevalence of individuals carrying a mutation in one allele encoding the LDL receptor is approximately 1 in 300, considerably higher than the prior estimates of 1 in 500.72 These mutations results in defective clearance of LDL from the blood and a rise in serum total and LDL cholesterol levels. The elucidation of this defect and its associated cell biology led to insights into the homeostatic control of cholesterol metabolism that transformed the lipid field.35,36 Well over 1000 individual mutations in the LDL receptor have now been identified. The gene, located on chromosome 19 and spanning 45 kb, has 18 exons that encode a mature protein of 839 amino acids. The inheritance of the disorder is autosomal codominant. Heterozygous patients typically have LDL-­cholesterol levels in the 200 to 500 mg/dL range, and the rare homozygous FH patient usually has an LDL-­cholesterol well above 500 mg/dL. Heterozygous patients commonly have tendon xanthomas and premature CAD, whereas these are universal in the untreated homozygous individual. Mutations in the LDL receptor have

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CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders

TABLE 25.5  3-­Hydroxy-­3-­Methylglutaryl Coenzyme A Reductase Inhibitors (Statins): Key

Properties of Clinical Interest Statin

Atorvastatin

Fluvastatin

Lovastatin Pitavastatin Pravastatin Rosuvastatin Simvastatin

Dose (typical range for agent) LDL-­C reduction Cytochrome P450 metabolism Renal excretion of absorbed dose Mortality or CHD event benefit shown Generic available

10–80 mg 38%–54% 3A4 2% Yes Yes

20–80 mg 17%–33% 2C9 500 mg/dL), tendon xanthomas, and premature coronary atherosclerosis.84 The autosomal recessive mode of inheritance is an important differentiating factor between ARH and FH, as is greater LDL receptor activity, as measured in cultured fibroblasts taken from the patients. The defect in ARH patients appears to affect liver cholesterol metabolism disproportionately, and current data indicate that the ARH gene product likely serves as an adaptor protein required for LDL receptor internalization via clathrin-­coated pits.85,86 ARH patients do respond to HMG CoA reductase inhibitors, but this treatment is usually inadequate to control their markedly elevated LDL-­cholesterol levels, making them candidates for lomitapide and other combination lipid therapy.87

Proprotein Convertase Subtilisin Kexin 9 Mutations. PCSK9 is

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LDL-­cholesterol levels to fall approximately 30%. White Americans can carry a missense mutation that reduces LDL-­cholesterol by approximately 15%. Individuals with complete loss of PCSK9 activity have extraordinarily low levels of LDL-­cholesterol but appear to be otherwise normal. The mechanism by which PCSK9 regulates LDL receptor activity is still being elucidated, but it appears that the protease can bind to the receptor, targeting it for degradation in the lysosome. This action does not depend on the protein’s enzymatic activity, and exogenously administered PCSK9 can reduce LDL receptor expression and increase serum LDL-­cholesterol levels, while approaches that reduce the amount of circulating PCSK9 have the opposite effect. The mechanism by which PCSK9 works made it an extremely attractive target for pharmaceutical intervention, and multiple agents that inhibit this protein have been developed as LDL-­cholesterol–lowering therapies (see Diet and Drug Treatment section of this chapter). ANGPTL3 Mutations. ANGPLT3 is hepatically-­derived circulating protein that inhibits both LPL and endothelial lipase. Mutations in this gene were first discovered to confer low plasma lipid levels inherited in a recessive manner in animal models, and resequencing of ANGPLT3, ANGPLT4, and ANGPLT5 in the Dallas Heart Study identified multiple rare synonymous variants associated with low plasma triglyceride concentrations.90,91 Exome sequencing in 2010 identified mutations in ANGPTL3 as the cause of low LDL, HDL, and triglyceride levels in two siblings.92,93 Studies to date suggest that mutations in ANGPTL3 may cause a significant percentage of hypolipoproteinemia not explained by APOB mutations, particularly in the context of low total cholesterol and HDL levels. Properties of this protein have made it an attractive target for novel lipid-­lowering therapeutics, and a recent trial of a monoclonal antibody (evinacumab) targeting ANGPTL3 resulted in significant LDL lowering in an FH homozygous population.81

Monogenic High-­Density Lipoprotein Disorders Three distinct monogenic disorders cause markedly reduced levels of HDL in the plasma by different mechanisms. They are: (1) Tangier disease, (2) LCAT deficiency, and (3) apo AI mutations.

Tangier Disease. Tangier disease was first recognized in 1960 in a sibling pair living on Tangier Island in the Chesapeake Bay in Virginia. Enlarged, yellow-­orange tonsils and little or no circulating HDL cholesterol are the classic findings in the disorder. Subsequently, cases have been identified in which the presenting symptom was a peripheral neuropathy. Patients may also have hepatosplenomegaly. Serum LDL and total cholesterol levels are usually quite low, while serum triglyceride values are moderately elevated. Tangier disease is an autosomal recessive disorder. The cause of Tangier disease was identified by several groups following mapping of the gene defect to chromosome 9. Candidate gene analysis in the appropriate genetic interval identified mutations in an ATP-­binding cassette (ABC) transporter as the cause. The transporter, now called ABCA1 (ATP-­binding cassette, subfamily A, member 1), is a full-­length ABC transporter transmembrane protein that is predicted to span the plasma membrane 12 times. ABCA1 is a 2261–amino acid protein encoded by a gene spanning 50 exons. Approximately 100 mutations in the gene have been identified to date.50,94 ABCA1 mediates the efflux of cholesterol from cholesterol-­ enriched cells when stimulated by the major apoprotein of HDL, apo AI.95 This activity is lost in Tangier patients and is reduced by approximately half in carriers of one abnormal ABCA1 allele. The mechanism of the movement of cholesterol from inside the cell to outside the cell has not been established. Individuals with Tangier disease, as well as heterozygous carriers of ABCA1 mutations (a disorder called familial hypoalphalipoproteinemia), appear to have increased risk of premature coronary disease. There is no specific therapy for this disorder.

Lecithin Cholesterol Acyltransferase Deficiency. The esterification of free cholesterol in circulating lipoproteins is catalyzed by a plasma enzyme called lecithin cholesterol acyltransferase. Two clinically separable syndromes result from a deficiency of LCAT. Fish-­eye disease is due to a partial deficiency of LCAT, with patients presenting with dense corneal opacities and very low HDL-­cholesterol levels. Familial LCAT deficiency arises from a nearly completely absence of LCAT activity and produces a more severe syndrome characterized by corneal opacities, anemia, and proteinuric renal failure.96 The serum lipid and lipoprotein profile in the more severe disorder is characterized by normal or increased triglyceride levels, reduced LDL-­cholesterol values, and markedly diminished HDL levels. The gene encoding LCAT is located on chromosome 16 and is composed of six exons. Cleavage of a signal peptide of 24 amino acids converts the proenzyme from a 440– amino acid precursor to the final 416–amino acid glycoprotein that circulates in the plasma. When unesterified cholesterol from tissues is transferred to HDL, either by passive diffusion or by ABCA1-­mediated lipid transport, LCAT’s activity esterifies the transferred cholesterol, trapping it in the HDL core. Because cholesterol ester is more hydrophobic than unesterified cholesterol, it is energetically unfavorable for the cholesterol ester to transfer back to the cell of origin. These steps of cholesterol transfer and esterification are the initial events in the reverse cholesterol transport pathway whereby cholesterol is moved from peripheral tissues back to the liver. Apo AI is the major activator of LCAT activity, accounting for the predominant effect of the enzyme deficiency on HDL levels. LCAT does, however, contribute to esterification of cholesterol in lower-­density lipoproteins as well. Despite very low HDL levels, patients with either fish-­eye disease or familial LCAT deficiency do not seem to have a predilection for very early coronary atherosclerosis. The small number of patients with the disease, some of whom have been found to have CHD, makes it difficult to determine if the risk of coronary atherosclerosis is substantially altered by the enzyme deficiency. There is no specific treatment for LCAT deficiency. Corneal and kidney transplantation are performed in these patients to ameliorate their major clinical disabilities.

Apolipoprotein AI Mutations. Apo AI is the major structural protein of HDL. The gene encoding apo AI is located on chromosome 11 and comprises four exons. Following cleavage of the signal and prohormone sequences, a 243–amino acid mature protein is produced. The protein has multiple repeats of an amphipathic helical structure that enables it to interact with both lipid and aqueous environments. Mutations that cause profound alterations in apo AI structure or expression have been reported, though they are extremely rare.97-­100 The individuals carrying these mutations have virtually no circulating HDL and typically develop early CHD. There are, however, a number of case reports of apo AI mutations that are not associated with early CHD. Corneal opacities and xanthomas have been documented in many, but not all, individuals with major apo AI mutations. Most patients harboring these mutations have been found to be homozygous for the gene defect, usually as a result of consanguinity. Heterozygotes carrying these mutations commonly have half-­normal HDL-­cholesterol levels, although cases with greater reductions in HDL levels have been reported, suggesting that some mutations may exert a dominant negative effect. HDL typically contains four apo A molecules per particle (either four apo AI or two apo AI and two apo AII proteins), so a heterozygous individual carrying an expressed apo AI mutant would have at least one mutant apo AI on most HDL particles. The atherosclerosis of individuals with structural mutations in apo AI appears to be much more pronounced than that seen in patients with either LCAT deficiency or Tangier disease. No specific therapy is available for this disorder, but aggressive LDL lipid-­lowering therapy is justified.

CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders

Polygenic Lipid Disorders Approximately 50% of the total interindividual variation in serum lipid levels is attributed to heritable factors.101 Several GWAS studies have been conducted to identify genetic variants that contribute to population-­wide lipid variability. One of the early, large lipid GWA studies was conducted in approximately 10,000 individuals and identified a total of 31 genetic loci associated with HDL levels, 22 loci associated with LDL levels, 16 loci associated with triglyceride levels, and 39 loci associated with total cholesterol levels; these results accounted for approximately 25% to 30% of the genetic component of these lipid traits, suggesting many lipid associated genetic variants remain to be found.102 A main finding of lipid GWA studies has been that genes which confer monogenic lipid disorders also harbor commonly occurring genetic variants with more modest effects on lipid levels.103,104 The implications of genetic variants discovered in gene regions without known effect on lipid biology are less clear. Novel biology has been revealed through the functional characterization of genetic variants discovered in the SORT1 and TRIB1 genes, but more work is required to understand the effect of many variants discovered through GWA studies.105,106 Findings from GWA studies have also been used to test hypotheses within lipid biology. If genetic variants contribute to serum lipid levels, and serum lipid levels contribute to CV risk, then genetic variants for lipid levels should influence CV risk. In 2012, Voight and colleagues found no relationship between genetic variants associated with HDL levels and myocardial infarction.107 This experiment has contributed to the debate as to whether HDL directly contributes to, or is only a marker of, CV risk. In the same publication, a clear association between genetic variants for LDL levels and myocardial infarction was identified, providing further support for the causal relationship between LDL levels and CV risk. KEY POINTS • Monogenic disorders of lipoprotein metabolism affect structural proteins in the lipoproteins or enzymes or receptors involved in their metabolism. They can cause major physiological disruptions (e.g. steatorrhea pr pancreatitis) or be associated with dramatically increased risks of atherosclerotic cardiovascular disease. Polygenic lipid disorders are much more common and the lipid phenotypes are usually less severe than those seen in the monogenic syndromes, but can overlap the latter.

DIAGNOSIS OF LIPID DISORDERS The diagnosis of a lipid disorder should be based on more than one measurement of serum lipids, because combined analytic and biological variations in serum lipids range from 10% to 20%. The technology for measuring LDL-­cholesterol levels directly has improved steadily over the years, but in most laboratories it remains a calculated value. To perform this calculation, the total and HDL-­cholesterol levels, as well as the triglyceride value, are measured. The LDL-­cholesterol concentration is then estimated using a formula first devised by Friedewald et al., which has subsequently undergone several modifications by later groups. The original formula was: LDL-­cholesterol = total cholesterol – [HDL-­cholesterol + triglyceride/5]. The triglyceride/5 factor represents an estimate of VLDL cholesterol. The validity of this formula for estimating LDL cholesterol has been confirmed by ultracentrifugal measurement of lipoprotein levels and remains reasonably accurate as long as the total triglyceride is less than 400 mg/dL. In order to obtain an accurate calculation,

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patients must fast for at least 12 hours to clear their blood of any chylomicrons; these lipoproteins distort the triglyceride ratio on which the Friedewald formula relies. If the triglyceride level is greater than 400 mg/dL, the LDL cholesterol must be determined by alternative methods, although a recent report suggested a modified calculation methodology may provide better LDL-­cholesterol estimation at triglyceride values above 400 mg/dL than does the Friedewald formula.108 Apo B100, which is present in both LDL and VLDL, can be measured directly to get an assessment of lower-­density lipoprotein particle numbers, though this entails additional expense. With increasing evidence for a greater atherogenicity of smaller, denser LDL particles, more sophisticated assessments of LDL number and composition are being introduced into clinical practice, such as nuclear magnetic resonance spectroscopy. The place of these more sophisticated and expensive assays in the routine diagnostic evaluation of most patients remains unsettled. It appears that most patients at high risk for CHD can still be identified using traditional lower-­ cost laboratory assays. Before embarking on a treatment plan in a patient with hyperlipidemia, one must exclude other medical conditions that cause lipids to rise as a secondary consequence. The most common clinical conditions that cause this to occur are obesity, diabetes, and hypothyroidism. The latter two are best screened using a serum glucose (or HbA1c) measurement and a thyrotropin-­stimulating hormone level, respectively. Many medications commonly cause a secondary hyperlipidemia, with antiretroviral therapy, a variety of psychotropic drugs, estrogens, and glucocorticoids heading the list. Increasingly, the diagnosis of severe hyperlipidemic syndromes due to rare mutations in key proteins of the lipid metabolism system can be made by genetic testing. Using buccal smears or circulating white blood cells present in a blood sample, DNA can be extracted and mutations easily detected. At the present time, these assays are typically used to guide family counseling or to provide interested patients insights into the cause of their disorder, but the results rarely affect clinical decision-­ making or therapeutic choices. While many of the genetic assays have moved from research tools to readily obtainable clinical tests, few are covered by medical insurance in the United States. It seems likely that the declining costs of these tests and the potential for complex genetics to offer better predictive power in assessing CV risk will shift this dynamic toward greater utility in the near future. KEY POINTS • Although a wide array of specialty assays for lipoprotein numbers and size are available clinically, the use of the standard lipid profile which measures the total cholesterol, HDL cholesterol, and triglyceride levels in the serum and calculates the LDL cholesterol from those numbers remains the standard for routine evaluation of a patient’s lipid status.

MANAGEMENT/TREATMENT Apart from the patient at risk for hypertriglyceridemic pancreatitis, for most hyperlipidemic patients the primary goal of treatment is to reduce the risk of atherosclerotic vascular morbidity and mortality. The approach to the treatment of hyperlipidemia that dominated clinical practice in the United States for the 25 years following the approval of the first statin in 1987 was initially delineated by the National Cholesterol Education Program (NCEP) Adult Treatment Panel I in 1988. All of the Adult Treatment Panel guidelines focused on LDL-­cholesterol as the major lipid risk promoting CHD, and therapy was intended to achieve specific target LDL cholesterol levels whose selections were based on an individual’s overall CV risk profile. For

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a given degree of LDL-­cholesterol elevation, the threshold for initiation of therapy decreased and the intensity of therapy increased with increasing global CHD risk. Dietary modification, complemented by exercise and weight reduction, was to be combined with appropriate pharmacologic therapy, when needed, to achieve the targets identified in the guidelines. In the fall of 2013, a joint American College of Cardiology/American Heart Association (ACC/AHA) expert panel issued the successor guidelines to the Adult Treatment Panel series.109 These guidelines sparked considerable controversy in the lipid field, with many dissenting voices raised over the wisdom of moving away from specific LDL targets as the goal of therapy. Others criticized the accuracy of the specific risk calculator instrument favored by these guidelines to determine if the 10-­year risk was greater than or equal to 7.5%, suggesting it led to overtreatment of many individuals whose LDL-­cholesterol values were less than 100 mg/dL.110 An update to the ACC/AHA guidelines was subsequently published.111 The 2018 ACC/AHA Guideline on the Management of Blood Cholesterol provides evidence-­based recommendations for how to safely and effectively lower LDL-­cholesterol levels and improve CV outcomes.111 The Guideline’s focus is 2-­fold: first, it provides recommendations for how to estimate a patient’s risk of developing atherosclerotic CV disease (ASCVD). Second, it suggests courses of treatment and management based on that estimated risk. The ACC/AHA continues to recommend the use of the pooled cohort equation (PCE), which takes into account cholesterol levels as well as age, smoking history, blood pressure, and diabetes status. The ACC/AHA delineates four levels of risk based on PCE results: low (under 5%), borderline (5%–7.49%), intermediate (7.5%–19.99%), and high (20% or higher). Both medication-­and lifestyle-­based treatment options exist for patients with elevated cholesterol levels. Importantly, the ACC/AHA emphasizes that PCE predictions alone should not determine whether a patient receives medications to reduce cholesterol levels. Factors including the response of a patient’s cholesterol levels to behavioral changes, as well as the financial burden of the medications, should be considered as patients work with their physicians to determine the best course of treatment. Among individuals who do not have ASCVD, the ACC/AHA highlights three populations for whom medications may be appropriate to reduce LDL-­cholesterol levels: individuals whose LDL-­cholesterol levels are greater than or equal to 190 mg/dL, patients with diabetes, and individuals between 40 and 75 years old whose LDL-­cholesterol levels are at or above 70 mg/dL and whose ASCVD risk, as calculated using the PCE, is a minimum of 7.5%. In the ladder group, calculation of a coronary artery calcium (CAC) score may help confirm the chosen course of treatment. The ACC/AHA 2018 guidelines reference studies published between 2015 and 2017 to suggest that medications may not be necessary for patients whose CAC score is equal to 0, as these patients are unlikely to develop ASCVD within 10 years. The guidelines also recommend that medications (statins) be considered for patients with CAC scores of 100 Agaston units and above, lowered from the 300 Agaston unit minimum recommended in the 2013 guidelines.109 Statins are recommended as the starting point of treatment for all three populations without ASCVD. Patients on “moderate” and “low-­intensity” treatment regimens are expected to experience LDL-­ cholesterol reductions of 30% to 49% and up to 30%, respectively. Patients for whom statin treatment is infeasible at the dosage corresponding to their risk level may consider cholesterol absorption inhibitors, such as ezetimibe, or bile acid sequestrants. For patients with ASCVD, the ACC/AHA suggests “high-­intensity” statin treatment, particularly for those under 75 years old. Individuals at “very high risk” may benefit from the addition of ezetimibe, known to reduce LDL-­cholesterol levels approximately 18% more than statins alone, an approach with documented outcomes benefit compared with

monotherapy with the same statin regimen.112 For “very high risk” patients whose LDL-­cholesterol levels remain at or above 70 mg/dL while taking statins and ezetimibe, the 2018 guidelines recommend that PCSK9 inhibitors be considered. Two recent clinical trials, FOURIER and ODYSSEY, found that the PCSK9 inhibitors evolocumab and alirocumab, respectively, can lower LDL-­cholesterol levels 43% to 64% more than statins alone and can improve outcomes in individuals with preexisting CV conditions.113,114 These data are reflected in ACC/ AHA’s 2018 guideline recommendations that support the use of “combination therapy” for aggressive lowering LDL-­cholesterol to reduce future ASCVD events. In addition, and in contrast with the 2013 guidelines, the new guidelines recommend the use of statins at “moderate intensity” in patients who are likely to live at least 3 to 5 years who have heart failure. This recommendation stems from the results of two smaller studies on the relationship between statin administration to individuals with heart failure and CV outcomes.115 While medications offer one useful approach to managing cholesterol levels and ASCVD outcomes, modifications to daily dietary and exercise habits are also often effective, either on their own or in conjunction with drug treatments. A metaanalysis reported in 2018 found an association between consumption of a diet rich in plant products and a fall in LDL-­cholesterol levels of approximately 17%.116 This decrease in LDL levels corresponded with a decline of approximately 13% in the likelihood of developing CHD within 10 years. The ACC/AHA recommends intake of a vegetable-­rich, sugar-­and saturated fat–poor diet (for example, the Dietary Approaches to Stop Hypertension diet), as well as frequent aerobic exercise (120–160 min weekly).109,117 Lifestyle-­based approaches to managing cholesterol levels are especially important in patients under 40 years, as fewer data are available regarding CV outcomes using drug therapies in this age group. The risk-­benefit calculus for utilizing preventive lipid therapies early in adulthood for individuals with average risk for CV disease is a hotly debated topic that is not informed by actual outcome data. Extrapolating benefit using genetic risk markers and short-­term studies in older individuals allows one to create a compelling argument for aggressive early preventive therapy, but the realities of medication and management costs, modest drug side effects, and poor compliance with long-­term preventive medications have limited the uptake of this approach in most medical practices. KEY POINTS • Optimum treatment of lipid disorders typically requires a combination of lifestyle approaches involving diet and activity improvements that may require adjunctive medications. Statins are the mainstay of drug treatment for those with LDL cholesterol elevations and can work well even in the patient who does not modify their lifestyle very much. Disorders of triglyceride-­rich lipoproteins frequently cannot be controlled by medications alone and require comprehensive metabolic improvements to achieve normal lipid levels.

Assessing the Need to Treat Patients With Hyperlipidemia As the magnitude of the benefit of treating hyperlipidemia is positively correlated with the overall degree of CHD risk, it is important to assess that risk before initiating treatment. Assessment should be comprehensive, extending beyond lipid levels to include consideration of blood pressure, smoking, diabetes, family history of premature CHD, age, sex, and presence of established CHD or other atherosclerotic disease. Although still in its early days, it is likely that the growing list of polygenic risk factors associated with atherosclerotic vascular disease will be incorporated into future risk-­stratification schema.103,118-­121

CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders Treatment recommendations follow directly from the degree of estimated CHD risk. Dietary modification is the sole mode of therapy for patients at the lower end of the CHD risk spectrum, while pharmacologic measures are reserved for patients at higher risk or for those who fail dietary intervention. The trend in preventive care over the past 20 years has been to push LDL levels ever lower, with no evidence to date that a value has been reached below which no further benefit is conferred. For the highest-­risk patients, the current LDL-­cholesterol target is to be under 70 mg/dL, although recent outcomes studies have demonstrated LDL-­cholesterol values in the 50 mg/dL range or lower are associated with improved outcomes over those of approximately 70 mg/dL.113,114,122 The concept that diabetes is a risk equivalent for CHD derives from the high risk of CHD in middle-­aged and older diabetic populations and was exemplified in the East-­West study of Finnish patients.123 In that study, patients with prior histories of myocardial infarction and no diabetes had a 7-­year incidence of new CHD events of 18.8%, whereas diabetic patients without prior myocardial infarctions had an incidence of 20.2%. The hazard ratio for death from CHD comparing these two groups did not differ. A follow-­up report by the same investigators, extending to 18 years of observation and with more CHD events recorded, reaffirmed this outcome and suggested that female diabetics were at particularly high risk.124 A finding of similar degrees of carotid intimal medial thickness in US patients with diabetes and no CHD compared with patients with CHD but no diabetes further strengthened the concept of diabetes as a CHD equivalent.125 Several other studies in different populations have supported this conclusion.126-­130 However, not all investigations of this topic have yielded similar conclusions, with some studies indicating lower rates of future CHD events in the diabetic population without CHD than are found in nondiabetics with CHD. Other studies have indicated that gender substantially influences this comparative outcome.131-­138 What all studies demonstrate, however, is that diabetes does confer a substantial increase in CHD risk, and that the combination of diabetes and CHD puts individuals at a very high risk of a future coronary event. Treating diabetes as a CHD risk equivalent in middle-­aged and older patients does, therefore, appear to be clinically appropriate. Other commonly assessed risk factors not included in most guideline calculations of risk include obesity, impaired fasting glucose, markers of inflammation (e.g., high sensitivity C-­reactive protein), homocysteine, endothelial dysfunction, and a thrombosis predilection. The role of these other risk factors in improving prognosis remains controversial, but clinicians may want to modulate the standard risk-­ factor treatment guideline based on the presence or absence of these nontraditional risks in their individual patients.

Dietary and Drug Treatment of Lipid Disorders

Diet/Lifestyle Therapies. With the advent of very potent lipid-­ lowering therapies, the use of dietary approaches to controlling lipid levels has become deemphasized in much of the developed world. While drug treatments (especially for elevated LDL-­cholesterol levels) are very effective, the role of diet in promoting hyperlipidemia should not be forgotten. The dietary approach to the treatment of elevated LDL-­cholesterol levels focuses on reductions in saturated fat, partially hydrogenated unsaturated fatty acids, and dietary cholesterol. Substituting foods that provide polyunsaturated and monounsaturated fats in place of saturated and trans unsaturated fat is particularly important, whereas the value of reducing the total fat intake is less clear.139 The 2013 clinical practice guidelines of the American Diabetes Association (ADA) recommend that saturated fat intake be restricted to less than 7% of calories, and that cholesterol intake not exceed 200 mg per day in patients with diabetes. These dietary recommendations

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mimic those proposed by the NCEP Adult Treatment Panel III guidelines for individuals without diabetes who have established coronary disease. For nondiabetics without coronary disease, the NCEP liberalizes saturated fat intake to less than 10% of calories and cholesterol to less than 300 mg/d. In both diets, it is recommended that trans fat intake be kept to a minimum. The adoption of a Mediterranean-­style diet, with substitution of monounsaturated fats for saturated fat, has demonstrated benefits on LDL cholesterol reduction, insulin sensitivity, and endothelial reactivity.11,140,141 The magnitude of the LDL reduction achieved with dietary interventions is quite variable and depends on the preintervention diet, as well as metabolic and genetic factors that influence diet-­dependent lipid responses. LDL reductions of 5% to 20% are typical for most patients adopting the reduced fat and cholesterol diets recommended by the ADA and NCEP, but reductions of over 50% can occur in selected individuals.142 For patients with VLDL elevations, which include a large portion of the diabetic population, carbohydrate and alcohol consumption may be more important dietary factors to address than the intake of cholesterol or saturated fat. The current United States Department of Agriculture–recommended daily allowance of carbohydrate is 130 g/d, but studies of long-­term carbohydrate-­restricted diets are limited. Recently, investigations comparing low-­carbohydrate and low-­fat diets have demonstrated that low-­carbohydrate diets can produce as good or even modestly better weight reduction than can low-­fat diets in 6-­to 12-­month timeframes.143-­145 The lower-­carbohydrate, higher-­fat diets typically produce greater reductions in VLDL (serum triglycerides) and greater increases in HDL-­cholesterol than do the fat-­restricted, higher-­carbohydrate diets. On the low-­carbohydrate, higher-­fat diets, the LDL-­cholesterol level can vary significantly, and if a substantial amount of saturated fat is used in these diets, the rise in LDL could be substantial.146 Therefore, the use of low-­carbohydrate diets should be accompanied by the use of monounsaturated and polyunsaturated fats as the major sources of fat in the diet. Weight loss (if obese), aerobic exercise, and smoking cessation can increase HDL levels and contribute to the dietary lowering of lower-­ density lipoproteins and CHD risk. They also reduce CHD risk by decreasing blood pressure. In the Diabetes Prevention Program (DPP), subjects in the lifestyle intervention group reduced their fat intake to 28% of calories after 1 year (down from 34%), and most were able to maintain the goal of 150 minutes per week of moderate physical activity.147,148 On this program, in which a body weight loss of 7% was targeted, subjects experienced less progression to diabetes and reductions in multiple CV risk factors, including dyslipidemia and hypertension.149,150 Although the DPP was conducted in an academic environment with substantial investigative resources devoted to ensuring compliance with the program, more recent efforts modeled on DPP suggest that the results can be translated to community-­based outreach programs.151,152 The clinical trials data that demonstrate improvements in CAD outcomes as a result of dietary interventions alone have generally employed diets that restrict fat and/or cholesterol intake much more dramatically than the ADA and NCEP diets mentioned earlier. In the St. Thomas’ Atherosclerosis Regression Study (STARS), cholesterol intake in men was reduced to 100 to 120 mg/d, and excess weight was addressed by prescribing a physical activity program. After 3 years, the group treated with a low-­cholesterol diet had a slower rate of progression of coronary disease and a higher rate of regression as determined by angiography.153 The Lifestyle Heart Trial employed an even more stringent diet, reducing cholesterol intake to less than 10 mg/d, combined with an exercise and behavioral modification program. This intervention led to regression of coronary lesions as measured by angiography and a reduction in symptoms of ischemia.154 This study was conducted in a very small number of highly motivated volunteers; it

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would be challenging to replicate the same interventions in more typical clinical care settings. Nevertheless, a longer-­term follow-­up of a small number of participants in this study demonstrated that further regression of coronary atherosclerosis could be obtained.155 A study with potentially greater applicability than either STARS or the Lifestyle Heart Trial is the Lyon Diet Heart Study.156-­158 Conducted using a Mediterranean diet rich in alpha-­linolenic acid, with participants who had experienced a prior myocardial infarction, this trial demonstrated improvements in its primary endpoint (myocardial infarction and death) as well as several CV and secondary endpoints. These results have been validated in the Spanish PREDIMED study of 7447 individuals with either type 2 diabetes mellitus or at least three major CV risk factors who were randomized to a Mediterranean or control diet.159 Participants in the Mediterranean diet groups experienced a 30% relative reduction in CV events, which was driven by a decrease in stroke rates. The Lyon Diet Heart Study and PREDIMED results, combined with other investigations of the impact of similar diets on lipids and lipoproteins, provide substantial support for the utility of Mediterranean-­style diets in the prevention and treatment of CHD.

Outcome Studies of Pharmacologic Therapy for Hyperlipidemia The addition of drug therapy to a diet and exercise program can greatly enhance lipid-­lowering results and dramatically effect CHD outcomes. Numerous investigations over the past 30 years have demonstrated that the use of one class of drugs, the HMG CoA reductase inhibitors, or statins, leads to significant reductions in nonfatal and fatal cardiac events (i.e., myocardial infarction, revascularization, cardiac death) in appropriately selected populations. Reductions in all-­cause mortality have also been demonstrated in lipid-­lowering drug trials, particularly in higher-­risk populations. With intensive drug therapy, the rate of plaque progression falls, and modest plaque regression can be demonstrated in major coronary vessels and systemic arteries. There is also evidence that lipid-­lowering medication (statins) can reduce the risk of stroke in persons with atherosclerotic carotid disease. These benefits have been shown to accrue to men and women, diabetics and nondiabetics, and middle-­aged and older individuals. A summary of the major outcomes studies using statins is provided in the coming paragraphs, followed by a brief review of the data available for outcomes using drugs that are not HMG CoA reductase inhibitors. KEY POINTS • The value of lipid-lowering in improving cardiovascular outcomes has been one of medicine’s most intensely investigated topics. Statins and other more recently approved LDL cholesterol lowering drugs, when given to patients with clinically documented ASCVD and elevated LDL cholesterol levels, have proven benefits. For those with lower risks for future ASCVD events the benefit of preventive lipid therapy is not as great in the shortterm but may still be valuable over longer timeframes.

Coronary Heart Disease Outcomes with 3-­ H ydroxy­3-­Methylglutaryl Coenzyme A Reductase Inhibitors (Statins). The current era of lipid-­lowering therapy began in 1984 with the publication of the Lipid Research Clinics Coronary Prevention Trial. A randomized, double-­blind study employing cholestyramine compared with placebo, this trial demonstrated that an 8% reduction in total cholesterol yielded a 19% reduction in CHD death or myocardial infarction.160,161 With the advent of statin therapy in 1987, much more effective LDL-­cholesterol–lowering agents became available. During the 1990s, a series of landmark CV prevention trials were

completed using these agents. In 1994, the Scandinavian Simvastatin Survival Study (4S) established for the first time that a lipid-­lowering agent could prolong overall survival in individuals with preexisting CHD.162 Randomizing 4444 patients with a prior myocardial infarction or angina to simvastatin or placebo, the drug treatment cohort of 4S had baseline LDL-­cholesterol levels fall from approximately 190 mg/dL to 120 mg/dL. With a trial endpoint of death for 10% of the original study enrollees (444 study subjects), it took a little over 5 years to complete the study. The result was that 8% of the simvastatin patients died versus 12% of the placebo-­treated individuals. This 30% reduction in mortality established statin therapy as an essential intervention in CHD patients with significant LDL elevations. In addition to the mortality benefit, 4S also produced substantial reductions in major coronary events, overall CHD death (19% vs. 28%, a relative risk reduction of 42%), and cerebrovascular events (2.7% vs. 4.3%). Unlike several of the pre–statin era cholesterol intervention studies, there were no increases in other causes of mortality to counterbalance the benefits on the CV system. When 4S was examined in greater detail, and after longer follow-­up to determine if the therapy had been confined to any subset of patients, these analyses concluded that the benefit was widespread.163-­165 Individuals at the highest risk appeared to benefit the most, particularly those with diabetes.166 Though only 202 of the 4444 patients in 4S were diagnosed with diabetes, these individuals experienced relative risk reductions in mortality (43%) and major CHD events (55%) that exceeded those in the nondiabetic population. An equally impressive outcome was noted in individuals who, in addition to their elevated LDL-­cholesterol levels, had lipid phenotypes characteristic of the metabolic syndrome (low HDL-­ cholesterol levels and high triglycerides). Study subjects with this lipid triad had the highest event rates among those receiving placebo (35.9%) and the greatest reduction in relative risk with treatment (0.48; 95% CI: 0.33–0.69).167 Subsequent studies have further refined our understanding of the benefits of statins in patients with and without preexisting CHD. The West of Scotland Coronary Prevention Group study (WESCOPS) enrolled hyperlipidemic men who had not experienced a prior myocardial infarct but who had high levels of other CHD risk factors. Pravastatin reduced LDL-­cholesterol levels from approximately 190 mg/dL to 140 mg/dL and, compared with the placebo-­treated group, lowered CHD death and myocardial infarction by 30%.168 An improvement of similar magnitude in a younger population of lower-­risk patients was observed in the Air Force/Texas Coronary Atherosclerosis Prevention Study when lovastatin was given to men and women with starting LDL-­cholesterol levels of 150 mg/dL.169 When compared with the placebo-­treated patients, those receiving lovastatin reduced their LDL on average to 115 mg/dL and decreased their incidence of major coronary events by 30% to 40%. In the Cholesterol and Recurrent Events Trial (CARE), pravastatin was given to patients with myocardial infarctions whose LDL-­cholesterol levels were substantially below (mean of 139 mg/dL) those of the 4S and WESCOPS patients. With a primary endpoint of a fatal coronary event or a nonfatal myocardial infarction, the pravastatin-­treated group had a 24% relative risk reduction compared with placebo-­treated patients. Stroke was reduced 31%.170 A post hoc analysis of CARE by the investigators who performed it led to the conclusion that reduction of LDL-­cholesterol in the range from 174 mg/dL to 125 mg/dL lowered the coronary event rate, but that further reductions below 125 mg/dL did not appear to confer additional benefit.171 The value of LDL reductions to levels substantially below 125 mg/dL has been a topic of considerable controversy ever since CARE was published, but several subsequent studies have indicated that further reductions of LDL do confer benefit. For example, the Long-­Term Intervention with Pravastatin in Ischaemic

CHAPTER 25  Lipoprotein Metabolism and the Treatment of Lipid Disorders Disease study group enrolled over 9000 patients with recent evidence of CHD and demonstrated benefits in overall mortality and CHD death using pravastatin.172 When stratified by starting LDL-­cholesterol, the magnitude of the benefit appeared to be maintained even in those with starting levels below 116 mg/dL.173-­175 The Treating to New Targets trial enrolled 10,000 patients with stable CHD and randomized treatment to high-­dose (80 mg/d) or low-­dose (10 mg/d) atorvastatin.176 Pretreatment LDL-­cholesterol levels were between 130 mg/dL and 250 mg/dL, and posttreatment LDL-­cholesterol values were 77 mg/dL and 101 mg/dL, respectively, in the high-­and low-­dose groups. The primary endpoint of the study, which was major CV events, declined 22% more in the high-­dose group than the low-­dose cohort, as did rates of myocardial infarction and stroke. However, overall mortality was not lower in the high-­dose group, owing to an increase in non-­CV deaths. Cancer deaths accounted for most of these events, a finding not replicated in other studies using the same drug. The Heart Protection Study (HPS), like several of its predecessor trials, attempted to examine the question of LDL-­cholesterol lowering in patients with moderate hyperlipidemia. HPS was a much bigger trial, randomizing 20,536 patients to placebo or simvastatin (up to 40 mg/d). The inclusion criteria required evidence of prior CV disease and permitted a broad range of LDL-­cholesterol levels, with a third of the patients having values less than 116 mg/dL. After an average follow-­up of 5.5 years, all-­cause mortality was reduced in the statin-­ treated cohort, as was CHD death and major CV events.177 Notably, over 5000 diabetics were included in the study, and they also achieved a substantial relative risk reduction in myocardial infarction and stroke.178,179 Particularly striking was the finding that the benefits of LDL-­cholesterol lowering were produced in all three of the tertiles into which patients’ baseline LDL-­cholesterol were stratified. Even those with starting LDL-­cholesterol levels approximating the current NCEP target goal of 100 mg/dL (for those with CHD) had a benefit when their value was reduced substantially below that number. Both men and women, as well as older and younger patients, derived similar benefits from the statin treatment. As with HPS, the Collaborative Atorvastatin Diabetes Study demonstrated a significant benefit in diabetic patients with lower LDL-­cholesterol levels (102 cm Men Women >88 cm Triglycerides >150 mg/dL HDL Cholesterol Men Women Blood pressure Fasting glucose

>40 mg/dL >50 mg/dL >130/80 mm Hg >110 mg/dL*

*American Diabetes Association subsequently lowered fasting glucose level to 100 mg/dL. HDL, High-­density lipoprotein. Data from Grundy SM, Brewer HB, Cleeman JI, et al. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association Conference on scientific issues related to definition. Circulation. 2004;109:433–438.

insulin resistance, hyperinsulinemia, and modest increases in plasma triglycerides, blood pressure, and glucose that by themselves may not be considered significant even in young normal-­weight individuals (phase 1).

The clustering of the major components of metabolic syndrome, such as obesity, type 2 diabetes, hypertension, and dyslipidemia, has long been recognized25; however, its delineation as a distinct entity took place only after its linkage to insulin resistance, hyperinsulinemia, and cardiovascular disease (CVD) became more apparent.21,26,27 Insulin resistance has been defined as a state (of a cell, tissue, system, or body) in which greater than normal amounts of insulin are required to elicit a normal biological response.28 In humans, it is currently diagnosed on the basis of increased plasma insulin concentrations, either fasting or during a glucose tolerance test, or by a decreased rate of glucose infusion required to maintain euglycemia during a hyperinsulinemic-­ euglycemic clamp.8 The much greater prevalence of insulin resistance in patients with type 2 than type 1 diabetes was appreciated 70 years ago by Himsworth and colleagues, based on their substantially higher insulin requirement29,30 and diminished response to exogenous insulin.31 Shortly after the development of the insulin immunoassay by Yalow and Berson in 1960,32 this suspicion was confirmed,33,34 and other disorders associated with insulin resistance and hyperinsulinemia were identified, including coronary heart disease and several of its risk factors,21,35-­40 as well as obesity itself (see Fig. 26.1). In general, most adults with insulin resistance and hyperinsulinemia are obese (BMI >29 kg/m2) or overweight (BMI 25–29 kg/m2). However, a significant

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PART 3  Obesity and Nutrition 50

Prevalence (%)

40

Men Women

30 20 10 0 20–29

30–39

40–49

50–59

60–69

70

Age (yrs) Figure 26.2  Prevalence of the metabolic syndrome in the United States according to age, based on National Health and Nutrition Examination Survey data and Adult Treatment Panel III criteria (see Table 26.1). Since these data were obtained, the criteria for abnormal glucose levels in patients with metabolic syndrome diminished from 110 mg to 100 mg. In addition, the prevalence of obesity and type 2 diabetes has increased in all of these age ranges and the general population (by as much as 50%). (Adapted from Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA. 2002;287:356–359.)

percentage are normal-­weight by BMI but show increases in visceral fat (central obesity),4,8 increased ectopic lipid deposition in liver and skeletal muscle,7,41 and/or enlarged fat cells.5,42 The presence of central obesity has been shown to correlate strongly with an individual’s predisposition to most of the diseases indicated in Fig. 26.1, including coronary heart disease,43,44 and this is the reason waist circumference is one of the principal diagnostic criteria for metabolic syndrome, as discussed later. Interestingly, hyperinsulinemia and insulin resistance are also present in normal-­weight offspring of individuals with type 2 diabetes,7,45-­47 hypertension,48 and hypertriglyceridemia8,49,50 and in individuals at increased risk for coronary heart disease,51-­54 suggesting that they are early markers or pathogenetic factors for these disorders. Studies such as these, plus the presence of a high rate of ischemic heart disease in patients with type 2 diabetes at the time of diagnosis (20%– 50%),55-­57 and to a somewhat lesser extent individuals with impaired glucose tolerance,58 have led to the suggestion that treatment of metabolic syndrome at an early stage may be needed for preventing or at least delaying coronary heart disease.8,22,39,59,60 The presence of insulin resistance in otherwise normal offspring of patients with type 2 diabetes, hypertriglyceridemia, and hypertension has led to the notion that it is a causal factor for metabolic syndrome or an early pathogenetic event.21 According to this widely held view, insulin resistance affects a number of organs (e.g., muscle, liver, adipose tissue, blood vessels), and hyperinsulinemia due to increased insulin secretion by the pancreatic β cell and decreased insulin degradation by the liver is a compensatory phenomenon.14,21,35 The observation that therapies that increase insulin sensitivity and lower plasma insulin levels (e.g., lifestyle modification such as diet and exercise)61-­63 or treatment with metformin61 and, to an even greater extent, the thiazolidinediones (TZDs)64-­65 prevent or delay the onset of diabetes in individuals with glucose intolerance is compatible with this notion, as is the efficacy of these therapies in some individuals with other disorders associated with metabolic syndrome, such as nonalcoholic fatty liver disease66,67 and polycystic ovarian syndrome (PCOS).68-­70 Left unexplained by this hypothesis is

the molecular mechanism by which insulin resistance develops initially and how it leads to hyperinsulinemia. Also, the possibility that hyperinsulinemia is the more primary of the two events or occurs simultaneously with the insulin resistance has not been ruled out.70 Hypothetically, metabolic syndrome could be related to genetic abnormalities in the insulin signaling cascade. In keeping with this possibility, mutations in IRS1 and IRS2, which encode the initial targets of the insulin receptor tyrosine kinase, have been shown to lead to insulin resistance and diabetes in transgenic mice.71,72 Evidence that these or other genetic defects in the insulin signaling cascade are common in humans with metabolic syndrome or type 2 diabetes and account for observed signaling defects73 is still lacking, however. KEY POINTS  • Metabolic syndrome is becoming increasingly common. As of 15 years ago, upward of 50 million individuals older than 20 years of age are affected in the United States, and the diagnosis is becoming increasingly common in children and adolescents.

The Lipid Theory Insulin resistance and hyperinsulinemia in humans and experimental animals have been linked to obesity and dysregulation of cellular lipid metabolism in a wide variety of circumstances.73-­76 Early studies focused on fatty acid release from adipose tissue and assumed that insulin resistance occurs in skeletal muscle when plasma free fatty acid (FFA) levels are increased as a consequence of central obesity. More recently, it has become apparent that insulin resistance is associated with alterations in lipid metabolism in tissues other than skeletal muscle, and that a number of newly discovered hormones and intracellular regulatory mechanisms affect its appearance. In addition, it has been demonstrated that metabolic syndrome occurs in individuals who lack adipose tissue, as well as in those with excess adiposity, and that in both groups it is associated with triglyceride deposition in ectopic sites such as muscle, liver, and visceral fat. In this section, we will attempt to review the current status of this increasingly complex but intriguing area. We will discuss several distinct but often interrelated mechanisms that have been put forth to explain the link between altered lipid metabolism and components of metabolic syndrome.

Excess Free Fatty Acids. Over 50 years ago, Philip Randle and colleagues proposed the existence of a glucose–fatty acid cycle.77 More specifically, they demonstrated that increased fatty acid levels diminish insulin-­stimulated glucose utilization in a perfused rat heart preparation. They showed that this effect occurs within minutes, and that it is associated with enhanced mitochondrial fat oxidation that leads to both inhibition of glucose oxidation at the pyruvate dehydrogenase step and an increase in the cytosolic concentration of citrate. Randle and associates also demonstrated that the increase in citrate inhibited glycolysis at phosphofructokinase, and that the secondary increase in glucose 6-­phosphate inhibited hexokinase and secondarily diminished insulin-­stimulated glucose uptake78 (Fig. 26.3). It was suggested that a similar mechanism in skeletal muscle may account for the insulin resistance observed in humans with obesity or type 2 diabetes, in both of whom plasma FFA levels were known to be elevated.79 Over the next 25 years, most investigators were unable to reproduce these findings in skeletal muscle, however,75,80-­82 except in special circumstances.83 For this reason, the acute contribution of elevated plasma FFA levels to the insulin resistance observed in most humans with obesity, type 2 diabetes, and other metabolic syndrome–associated disorders remained unclear.

CHAPTER 26  Metabolic Syndrome ↑ Citrate

Glucose

−

−

−

HK

PFK

PDH

↑ G-6-P → → Pyruvate

↑

Acetyl CoA CoA ↑

NADH NAD

GLUT 4 Plasma glucose Plasma fatty acid Figure 26.3  Inhibition of glucose uptake and oxidation by fatty acids as described in heart muscle by Randle, Garland, Hales, and Newsholme (1964, 1965). (See text for details.) (Adapted from Shulman GI: Cellular mechanisms of insulin resistance. J Clin Invest. 2000;106:171–176.)

431

unclear whether plasma FFAs are elevated in individuals with metabolic syndrome in its very early stages. Only modest increases in plasma FFAs, if any, have been observed in normal-­weight insulin-­ resistant individuals who are at risk for developing diabetes because of family history.45,100

Altered Fatty Acid Metabolism: Malonyl Coenzyme A, Mitochondrial Dysfunction, and Adenosine Monophosphate– Activated Protein Kinase. A second abnormality in lipid metabolism that could lead to insulin resistance is a disturbance in fatty acid metabolism in which the oxidation of cytosolic long-­chain fatty acyl CoA (FACoA) by mitochondria is impaired, and its esterification and metabolism by other nonmitochondrial processes is enhanced.76,101 This could occur if either the intrinsic ability of mitochondria to oxidize fatty acid is decreased or the activity of carnitine palmitoyltransferase 1, the enzyme that regulates the transfer of cytosolic long-­chain fatty acyl CoA into mitochondria, is diminished.

Malonyl CoA. Altered fatty acid partitioning between the mitoThis changed in 1991 when Boden and colleagues84 definitively demonstrated that raising plasma FFAs (by infusing a lipid emulsion with heparin to activate lipoprotein lipase activity) in humans during a euglycemic-­ hyperinsulinemic clamp inhibits insulin-­ stimulated peripheral glucose uptake. Importantly, they found that this effect required 4 to 6 hours rather than a few minutes to become evident, and that it was not accompanied by an increase in citrate. Subsequent investigations by Shulman’s laboratory in which 31P magnetic resonance spectroscopy (MRS) was used to noninvasively measure intramyocellular glucose-­6-­phosphate found that its concentration was reduced under the same conditions, suggesting that fatty acids principally inhibit insulin-­stimulated glucose transport or phosphorylation activity85 and not the phosphofructokinase reaction, as suggested by Randle. Later, studies by the same group, in which 13C MRS was used to assess intramyocellular glucose concentrations, revealed that insulin-­stimulated glucose transport and not phosphorylation was the step inhibited by high plasma fatty acids, and that this defect was associated with lipid-­induced defects in insulin-­stimulated PI3 kinase activity.86 Other studies have demonstrated that insulin resistance in human muscle, caused by infusing lipids to increase plasma FFAs, is associated with impaired insulin signaling,73 increases in the concentrations of muscle triglyceride, long-­chain fatty acyl CoA87 and diacylglycerol, and increases in protein kinase C (PKC) activity and the translocation of various PKCs from the cytosol to a membrane fraction.76,88-­90 Another key finding was a decrease in IKBα abundance, suggesting activation of NF-­κB and proinflammatory events.90 As discussed later, similar abnormalities have been found in rodent liver following a sustained exposure to fatty acids,91-­94 in rodent muscle in a wide variety of states associated with insulin resistance,76,88,89 and in liver and muscle of massively obese insulin-­resistant humans with type 2 diabetes.95,96 Thus, the intracellular changes produced by an excess of fatty acids are associated with insulin resistance in many tissues. A still unanswered question is whether an increase in plasma FFAs is an early pathogenetic event in metabolic syndrome. Elevated concentrations of plasma FFAs attributable to increased adipose tissue mass and the relative insensitivity of large fat cells and visceral fat to insulin35,87 are present in individuals with obesity and type 2 diabetes, and they appear to contribute to insulin resistance when these disorders are established.74,87,97,98 On the other hand, even some severely obese individuals remain insulin-­sensitive (see later section entitled “Bariatric Surgery Patients: Insulin-­Resistant and -­Sensitive”), suggesting that other factors are involved.99 It is also

chondria and cytoplasm as a cause of insulin resistance was suggested by studies from the Ruderman laboratory in denervated rat muscle, in which enhanced diacylglycerol (DAG) synthesis and PKC activation were observed,102 and later by similar findings in muscle of obese, insulin-­resistant KKAy mice.103 In these and other instances, insulin resistance in rodent muscle correlated with an increase in the concentration of malonyl CoA,76 an allosteric inhibitor of carnitine palmitoyltransferase. As shown in Fig. 26.4, an increase in malonyl CoA by decreasing the oxidation of cytosolic FACoA would increase its availability for the formation of DAG, triglycerides, ceramide, and possibly other factors linked to insulin resistance. In keeping with such a mechanism, McGarry concurrently demonstrated that treatment with etomoxir, a pharmacologic CPT1 inhibitor, both increases triglyceride accumulation and causes insulin resistance in rat skeletal muscle.101,104 A number of other findings also support this notion: (1) mice lacking functional acetyl CoA carboxylase 2 (ACC2, the principal isoform that generates the malonyl CoA that regulates CPT1 in skeletal muscle) are more insulin-­sensitive than control rats105,106; (2) the administration of an ACC inhibitor diminishes obesity and insulin resistance in fat-­fed rats107; (3) antisense oligonucleotides directed at hepatic ACC1 and ACC2 diminish both hepatic steatosis and insulin resistance in mice fed a high-­fat diet108; and (4) low rates of fatty acid oxidation have been reported in preobese humans,109,110 Zucker diabetic rats,111 and interleukin-­6 (IL-­6) knockout mice112 prior to the onset of diabetes and obesity. Malonyl CoA was not assayed in muscle in any of these studies; however, in two of the rodent models113,114 and in adipose tissue of obese insulin-­resistant humans,115,116 a decrease in the activity of AMPK was found, suggesting that its concentration was elevated (see later section entitled “AMP-­Activated Protein Kinase”).

Mitochondrial Dysfunction. Altered fatty acid partitioning in muscle and other tissues could also occur if fatty acid oxidation is depressed as a consequence of mitochondrial dysfunction. Decreases in mitochondrial function, and in some instances number and size, have been found in muscle of individuals with type 2 diabetes associated with obesity,117 in lean insulin-­resistant older adults,118 and in lean insulin-­resistant offspring of diabetic parents.7 In the latter, the reduction in mitochondrial function was associated with a similar decrease in mitochondrial content.119 Likewise, decreases in the mRNA for PGC1α, a transcriptional coactivator that enhances genes for mitochondrial biogenesis and function, have been observed in

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BOX 26.1  AMP-­Activated Protein Kinase AMP-­activated protein kinase (AMPK) is a heterotrimer containing α, β, and γ subunits, each of which has at least two isoforms. The α subunit contains the catalytic site; the β subunit contains a glycogen-­binding domain; and the γ subunit contains two AMP-­binding sites. All three subunits are necessary for full activity.298,299 In general, AMPK is found in the cytosol of a cell; however, the A2 isoform of the enzyme is also present in the nucleus.299 Decreases in the energy state of a cell that lead to increased binding of AMP and ADP in place of ATP on the γ subunit activate AMPK by a number of mechanisms, including allosteric activation and especially covalent modification due to phosphorylation of its catalytic subunit on Thr-­172 as a result of the actions of the AMPK kinase LKB1. In addition, factors that increase cellular Ca2+ and CaMKKb also activate AMPK.300,301 When activated, AMPK enhances a number of processes that increase ATP generation, including fatty acid oxidation and glucose transport (in skeletal muscle), and it decreases others that consume ATP, but are not acutely necessary for survival, including to varying extents fatty acid triglyceride and protein synthesis. In addition, AMPK can alter the expression of a wide variety of genes, including several that alter mitochondrial function (e.g., PGC1a, UCP3) and lipid synthesis (SREBPIC).

Figure 26.4  AMP-­ activated protein kinase (AMPK) concurrently activates (+) or inhibits (–) multiple aspects of fatty acid and glucose metabolism. By phosphorylating and inhibiting ACC and activating MCD, AMPK diminishes the concentration of malonyl CoA. This relieves the inhibition of CPT1 by malonyl CoA and results in both increased fatty acid oxidation and a decrease in the availability of cytosolic FACoA for TG, DAG, and ceramide synthesis, and possibly lipid peroxidation and protein acylation. Conversely, AMPK activation independently decreases the expression of glycerophosphate acyltransferase GPAT, fatty acid synthase (not shown), and SPT, decreasing the synthesis of glycerolipids, fatty acids (de novo), and ceramide (de novo), respectively. A decrease in AMPK produces the opposite effects. The basis for the ability of AMPK to inhibit oxidant stress (ROS generation) inflammation, and ER stress (not shown) in some settings is not completely understood. Whether AMPK activation enhances or inhibits a process or an enzyme in this scheme is denoted by plus signs and minus signs, respectively. Not shown in the diagram is that AMPK achieves these changes in some instances by phosphorylating the indicated enzyme and/or in other instances by regulating its expression at the level of the gene. For instance, AMPK can enhance mitochondrial biogenesis and function by enhancing the expression of the transcriptional coactivator PGC1α. ACC, Acetyl CoA carboxylase; CPT1, carnitine palmitoyltransferase 1; DAG, diacylglycerol; FACoA, cytosolic long-­chain fatty acyl CoA; GPAT, glycerophosphate acyltransferase; MCD, malonyl CoA decarboxylase; ROS, reactive oxygen species; SPT, serine palmitoyl transferase. (Adapted from Ruderman N, Prentki M. AMP kinase and malonyl-­CoA: targets for therapy of the metabolic syndrome. Nat Rev Drug Discov. 2004;3:340–351.)

some120 but not all119 studies of nondiabetic first-­degree relatives of patients with type 2 diabetes, as well as in individuals with type 2 diabetes and impaired glucose tolerance.121 Whether these mitochondrial changes are hereditary or secondary to metabolic events (e.g., lipotoxic changes due to abnormalities in intracellular lipid metabolism) or abnormalities in AMPK regulation (see the following section) remains to be determined. However, given the likely role for increased intracellular fatty acid metabolites in mediating insulin resistance, impaired mitochondrial function leading to decreased fatty acid oxidation will almost certainly exacerbate the problem. Also to be determined is whether the changes observed in the offspring of diabetic parents reflect a difference in muscle fiber type, because the ratio of mitochondrial-­rich type 1 fibers to glycolytic type 2 fibers may be decreased in these individuals.7,122

AMP-­Activated Protein Kinase. AMPK (Box 26.1) is a fuel-­sensing enzyme that appears to play a key role in regulating both cellular metabolism and mitochondrial function. In addition, an increasing body of evidence has suggested that its dysregulation could be a cause of metabolic syndrome (animal studies) as well as a target for its prevention and therapy (human and animal studies).3,75 Furthermore, it has been shown definitively in rodents that exercise in vivo (treadmill running) activates AMPK not only in muscle but also in liver, adipose tissue,123 and aortic endothelium.124 As shown in Fig. 26.4, when AMPK is activated (e.g., during exercise or in some tissues by caloric deprivation), it phosphorylates and inhibits acetyl CoA carboxylase, the enzyme that catalyzes the synthesis of malonyl CoA and (by a still undetermined mechanism) activates malonyl CoA decarboxylase, the enzyme that catalyzes malonyl CoA degradation.123 In addition, in many tissues and cells, activation of AMPK concurrently inhibits the use of cytosolic FACoA for the synthesis of various factors that could lead to insulin resistance, including DAG and ceramides, and it diminishes the generation of lipid peroxides, ER stress, and activation of NF-­κB caused by elevated concentrations of specific fatty acids and other factors.75,125,126,127 Furthermore, it can do so in multiple tissues and by cells such as macrophages, which could contribute to inflammation in various tissues.128 Thus, AMPK could protect cells and tissues against lipotoxicity by multiple mechanisms. For instance, in addition to modulating the above events acutely by phosphorylating specific proteins, AMPK chronically regulates the effects of transcriptional regulators (e.g., SREBP1C) that govern the synthesis of acetyl CoA carboxylase and other key enzymes that regulate fatty acid oxidation (see Fig. 26.4). Of specific relevance to this chapter is that decreases in AMPK activity are associated with insulin resistance in muscle (Table 26.2) and liver in many situations, and failure to activate AMPK in the fat cell during lipolysis is associated with increases in oxidative stress and inflammation.126 In addition, hormones and other factors that decrease AMPK in peripheral tissues, such as glucocorticoids129 and resistin,130 cause insulin resistance, whereas hormones (e.g., adiponectin and leptin) and pharmacologic agents (e.g., metformin, TZDs, α-­lipoic acid, salsalate, AICAR) that activate AMPK diminish it in experimental animals, as does exercise75 (Table 26.3). Finally, as already noted, closely linked to mitochondrial theories of insulin resistance is PGC1α (PPARγ-­coactivator 1α), a transcriptional coactivator of mitochondrial biogenesis whose expression is increased when AMPK is activated (e.g., by exercise or AICAR).131-­134

CHAPTER 26  Metabolic Syndrome

433

TABLE 26.2  Abnormalities Associated With Insulin Resistance in Tissues of Humans

and Experimental Animals Model

TG

DAG

Malonyl CoA

PKC Activity

Activated IKK-­NF-­kB

AMPK Activity

fa/fa rat Glucose-­infused rat Fat-­fed rat Fat-­infused humans Obese insulin-­resistant humans

(+) (+) (+) (+) (+)

(+) (+) (+) (+) ND

(+) (+) (+/–) ND (+/–)

(+) (+) (+) (+) (+)

ND ND (+) (+) ND

(–) (–) ND ND (+/–)

AMPK, AMP-­activated protein kinase; DAG, diacylglycerol; IKK, inhibitor of NF-­κB kinase; NF-­kB, nuclear factor kappa B; PKC, protein kinase C; TG, triglyceride; (+), increased; (–), decreased; ND, not determined. Data are for skeletal muscle. They are taken from Ruderman N, Prentki M. AMP kinase and malonyl-­CoA: targets for therapy of the metabolic syndrome. Nat Rev Drug Discov. 2004;3:340–351 and are from the laboratories of the authors and those of Turinsky, Kraegen, Caro, Boden, and Shoelson. Many of these changes have also been demonstrated in liver in insulin-­resistant obese humans, fat-­fed and glucose-­infused rats, and fa/ fa rats. Whether AMPK activity is depressed and the concentration of malonyl CoA elevated in skeletal muscle of obese insulin-­resistant humans is controversial; however, it has been clearly observed in adipose tissue (see the section entitled “AMPK and the Metabolic Syndrome in Humans”). Studies, primarily in vitro, suggest that similar events occur in the liver, pancreatic β cell, and cultured vascular endothelium.

TABLE 26.3  Effect of Therapies That Activate AMP-­Activated Protein Kinase in Humans (H)

and/or Rodents on Various Manifestations of the Metabolic Syndrome Factor

Insulin Resistance

Pancreatic β Cell Dysfunction

Endothelial Cell Dysfunction

Coronary Heart Disease Risk

NAFLD/NASH

Exercise (H) Calorie/weight reduction (H) Adiponectin AICAR Leptin Metformin (H) TZDs Polyphenols

(–) (–) (–) (–) (–) (–) (–) (–)

ND (–) (–) (–) (–) (–) (–) ND

(–) (–) (–) (–) (–) (–) (–) (–)

(–) (–) (–) ND ND (–) (–) (–)

(–) (–) (–) ND (–) (–) (–) (–)

AICAR, 5-­Aminoimidazole-­4-­carboxamide riboside; AMPK, AMP-­activated protein kinase; NAFLD/NASH, nonalcoholic fatty liver disease/nonalcoholic steatotic hepatitis; TZDs, thiazolidinediones; (–), decreased; ND, not determined. Where studied, these factors also alter ectopic lipid deposition in keeping with their effects on AMPK and malonyl CoA. Inactivity, caloric excess (glucose), and deficiencies of leptin or adiponectin, where studied, have been shown to have opposite effects. Studies with AICAR and experimental polyphenols (resveratrol, SI-­17834) have only been carried out in rodents. Polyphenols stimulate AMP-­activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-­deficient mice. Resveratrol improves health and survival of mice on a high-­calorie diet.

Inflammation and Oxidative and Endoplasmic Reticulum Stress In recent years, considerable attention has been given to the theories in which the combination of oxidative and ER stress and NF-­κB activation play a central role in the pathogenesis of metabolic syndrome. As noted by numerous investigators, all of these factors have been linked to insulin resistance in humans and experimental animals with a wide variety of disorders.9,10,135-­140 Because of this, it has been suggested that an abnormality of the innate immune system could be a proximal event in the pathogenesis of metabolic syndrome.10 Although this possibility cannot be disproven, and it is unquestioned that proinflammatory events and oxidative and ER stress are often an integral component of metabolic syndrome, a number of observations including the following suggest that dysregulation of lipid metabolism that can lead to oxidative and ER stress and inflammation is likely to be a more primary factor: (1) the close correlation of metabolic syndrome in its early stages with weight gain, central obesity, ectopic lipid deposition, and elevated plasma FFA levels; (2) the presence of metabolic syndrome and ectopic lipid deposition in humans and experimental animals deficient in peripheral adipose tissue (lipodystrophy) and the reversal of these abnormalities in rodents by the implantation of fat141-­143 and in humans and/or

rodents by the administration of leptin,144,145 adiponectin,146 and in some instances TZDs, all of which activate AMPK75; (3) the observation that elevating plasma FFA levels in humans and rodents by itself acutely (hours) leads to insulin resistance and proinflammatory cha nges87,90,147; and (4) the very early occurrence of alterations in cellular lipid metabolism and mitochondrial function in normal-­weight normoglycemic, young offspring of diabetic parents.7 To our knowledge, evidence of a proinflammatory state has not been reported in the latter group. These considerations aside, the possibility that proinflammatory changes leading to alterations in lipid metabolism are the cause of metabolic syndrome has not been ruled out; indeed, studies in rodents suggest that TNFα-­induced inflammation can lead to a decrease in AMPK activity in skeletal muscle.148 In addition, it has recently been demonstrated that treatment with the antiinflammatory drug salsalate improves glycemic status modestly (less than 0.24% reduction in hemoglobin A1c) and reduces systemic inflammation in obese adults at risk for developing type 2 diabetes.149 This agent has also been shown to activate AMPK, however.150 Because therapies that target lipid metabolism, oxidative stress, and in some instances inflammatory events have all been shown to diminish insulin resistance in specific situations,94,136-­138 what is clear is that these factors are almost certainly interrelated.

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PART 3  Obesity and Nutrition ? P Ceramide Caspase1

NLRP3 Inflammasomes

AMPK ↓AMPK

TG

FFA

P ROS

ULK1

IFNγ Autophagy

IL-1β Macrophage

↓Autophagy

Dysfunctional mitochondria

T cells Quality control of mitochondria (mitophagy)

Adipocyte

↑ROS

↑NLRP3 Inflammasome

A

B

↑IL-1β

Figure 26.5  Hypothetical mechanisms for the activation of inflammasomes in adipose tissue of obese insulin-­resistant subjects. A, It is proposed that increased free fatty acid release from lipid droplets in the adipocyte lead to changes in adjacent macrophages (ceramide, caspase 1 activation) that activate the inflammasome, which generates interleukin (IL)-­1B and other inflammatory cytokines. B, Effects of AMP-­activated protein kinase (AMPK) on events in the macrophage. The identity of the factor(s) that decrease AMPK activity in cells in which inflammasomes are found is uncertain, although oxidative stress and inflammatory cytokines such as IL-­1B and TNFα could be factors. Not shown here is that activation of caspase 1 in the inflammasome complex cleaves and presumably inactivates SIRT1. ULK1, a regulator of the autophage lysosome is phosphorylated and activated by AMPK. (Adapted from Choi AM, Nakahira K. Dampening insulin signaling by an NLRP3 “meta-­flammasome.” Nat Immunol. 2001;12:379–380.)

Fat cells (+) FFA, IL1-B, TNFα, ROS (–) Adiponectin Inflammatory cells

TG, ceramide DAG-PKC IKKB-NFκB JNK1 ROS ER stress

Cellular dysfunction Insulin resistance

Adipose tissue Plasma Liver, Endothelium Figure 26.6  Hypothetical mechanism by which dysregulation of adipose tissue in an obese insulin-­ resistant individual leads to insulin resistance and dysfunction in liver, aorta, and possibly other tissues. Adipose tissue of such patients releases increased amounts of free fatty acids, reactive oxygen species, inflammatory cytokines, and decreased amounts of adiponectin, all of which could contribute to insulin resistance and dysfunction in other tissues. AMP-­activated protein kinase activity is decreased in adipose tissue of such individuals and in liver, aorta, and in some studies, muscle; however, it is uncertain whether it causes or is a result of the above changes.

Specific Tissues

Adipose Tissue. A number of lines of investigation have linked abnormalities in adipose tissue to the pathogenesis of metabolic syndrome. They also indicate that this likely reflects abnormalities both in adipocytes and in macrophages and other inflammatory cells in the stroma.128,151 Thus, as already noted, elevated plasma FFA levels, attributable to an increase in their release from adipocytes in grossly or centrally obese individuals, correlate with the presence of insulin resistance in most patients.87,152 In addition, when the function of the adipocyte as a store for lipid is impaired,141,142 as it is in many insulin-­resistant obese individuals,99 it has been proposed that the excess fatty acid release could be a key event leading to the generation of the inflammatory cytokines

IL-­1, IL-­8, TNFα, and others by stromal macrophages (Fig. 26.5). As shown in Fig. 26.6, an increase in the release of these cytokines, as well as of reactive oxygen species and FFA (from the adipocyte), are thought to be largely responsible for the cellular dysfunction and decreased AMPK observed in liver and other tissues of obese individuals with metabolic syndrome. As reviewed recently by Steinberg and Schertzer,128 an overwhelming body of evidence indicates that AMPK is at the crossroad of metabolically driven macrophage inflammation, presumably by controlling its mitochondrial metabolism. In keeping with this conclusion, they point to multiple studies showing that decreasing AMPK activity by various means increases inflammation in the macrophage, whereas AMPK activation decreases it.128

CHAPTER 26  Metabolic Syndrome

Adiponectin. A deficiency of the adipokine adiponectin, also referred to as ACRP30, has been most closely linked to insulin resistance in humans. It is produced exclusively or at least predominantly in adipose tissue and is present in the circulation in trimeric, hexameric, and high–molecular-­weight (HMW) forms. The biological relevance of the three oligomers153,154 and still other forms of adiponectin is not completely understood. These considerations aside, there is abundant evidence that low immunoassayable adiponectin levels in plasma (accounted for mainly by the HMW form) are present in obese insulin-­ resistant individuals and in individuals at risk for type 2 diabetes146,155 and coronary heart disease,156 even in the absence of overt obesity. In addition, polymorphisms of the adiponectin gene have been associated with metabolic syndrome in some populations and a predisposition to type 2 diabetes in others.146 Adiponectin, like exercise, activates AMPK and stimulates AMPK-­ mediated events such as glucose transport and fatty acid oxidation in muscle157,158 and inhibition of glucose production in liver (in rodents).159 In addition, adiponectin has antiinflammatory actions.160 Whether the insulin-­ sensitizing effect of adiponectin is AMPK-­ mediated has not been proven definitively; however, treatment with TZDs increases plasma adiponectin, and the insulin-­sensitizing effect of these agents and their ability to activate AMPK are both markedly attenuated in adiponectin knockout mice.161,162 Furthermore, decreased levels of adiponectin are one of the hallmarks of bariatric surgery patients who are insulin-­resistant,163 as is decreased AMPK activity in their adipose tissue.115,116

Leptin. Since its discovery by Friedman and colleagues,164 interest in leptin has for the most part focused on its role as an appetite suppressant. However, it has also been recognized for some time that leptin increases oxidative metabolism and fatty acid oxidation in peripheral tissues, owing both to a direct action165 and to an effect on the hypothalamus that appears to be mediated by the sympathetic nervous system.166 As first reported by Minokoshi and Kahn and their coworkers, both the direct and centrally-­mediated effects of leptin on peripheral tissues are associated with AMPK activation,167 whereas its action on hypothalamic nuclei is associated with a decrease in AMPK activity.168 When leptin is lacking or its receptor is not functioning in peripheral tissues, lipid accumulates and cellular damage may result. Unger and associates169,170 have reported that, in the Zucker diabetic fatty (ZDF) rat, which lacks the leptin receptor, such ectopic lipid accumulation occurs in liver, muscle, and the pancreatic β cell, and that it antedates the presence of diabetes and pancreatic β cell apoptosis. Furthermore, it has been found that AMPK is deficient in tissues of these rats,114 and that treatment with AICAR114,171 and exercise, which also activates AMPK in these animals,171 prevents ectopic lipid accumulation, pancreatic β cell damage, and the development of diabetes. More recent studies have found that the antidiabetic effects of leptin may be attributed to its inhibition of the hypothalamic-­pituitary-­adrenal axis, leading to reduction in lipolysis and hepatic gluconeogenesis.172 Vascular Endothelial Cells and Atherogenesis. An impressive case has been made that atherogenesis is essentially an inflammatory response to a variety of risk factors, and that the consequences of this response include acute coronary artery and cerebrovascular syndromes.173 An early site at which this inflammatory response appears to occur is the endothelial cell173; indeed, increases in NF-­κB expression have been observed in endothelium at sites predisposed to atherosclerotic plaque formation175 and in vitro in endothelial cells exposed to elevated concentrations of glucose176 and fatty acids.177 Likewise, impaired endothelium-­dependent relaxation and increases in circulating adhesion molecules (VCAM1, ICAM, selectins), markers

435

of cellular dysfunction and incipient ASCVD, have been observed in humans with type 2 diabetes and metabolic syndrome135,178 and in normal individuals in whom plasma FFA levels are increased by a lipid infusion.179 Conversely, endothelial cell dysfunction is diminished in humans by factors that diminish the proinflammatory state, including exercise and caloric restriction180,181 and by treatment with TZDs.178,182 As already noted, all of these interventions have been reported to activate AMPK in rodents. Studies with endothelial cells in culture also support such a protective role for AMPK. Thus, increases in oxidative stress and NF-­κB–mediated gene expression observed in cultured human umbilical vein endothelial cells (HUVECs) incubated with palmitate are inhibited by AICAR and other AMPK activators.125,183,184 Also, AICAR and (where studied) expression of a constitutively active AMPK have been shown to inhibit apoptosis, mitochondrial dysfunction, DAG synthesis, and the development of insulin resistance (diminished Akt activation) in HUVECs incubated in a high-­glucose medium.185 Finally, it has been demonstrated that the administration of atorvastatin, a cholesterol-­lowering agent, by gavage activates AMPK and eNOS in the rat aorta,186 and it has a similar effect when incubated with HUVECs. Whether this accounts for the antiatherogenic effect of statins attributed to its antiinflammatory action is an intriguing possibility.187 Finally, it has long been appreciated that increases in macrophages are a hallmark of arteries undergoing atherosclerotic changes. As recently reviewed, an increasing body of evidence strongly suggests that treatments that activate AMPK combat atherosclerosis by diminishing proinflammatory events in macrophages, as they do in adipose tissue and presumably other sites.128

Liver. Changes similar to those in muscle and the endothelial cell occur in the liver in insulin-­resistant states. Thus, as in muscle, an association between hepatic lipid deposition and insulin resistance has been clearly demonstrated in humans.188,189 Also, in rats infused with a lipid emulsion to increase plasma FFA levels during a euglycemic-­ hyperinsulinemic clamp91,147 or with short-­ term fat feeding,93 the development of hepatic insulin resistance is associated with increases in DAG content, PKC activation, and a decrease in IKBα abundance— changes almost identical to those observed in human muscle.90 Similar alterations in PKC have been noted in the livers of massively obese, insulin-­resistant humans95,190 and of fat-­fed rats with hepatic steatosis.93 Also, knockdown of PKC epsilon expression by antisense oligonucleotides protects rats from fat-­induced hepatic insulin resistance.191 As discussed later, bariatric surgery in such rodents is followed by increases in the activity of AMPK, SIRT1, and LKB1 in liver, as well as an increase in insulin sensitivity.192

Pancreatic β Cell. Insulin resistance in muscle and liver does not

initially result in hyperglycemia, because it is accompanied by hyperinsulinemia. As noted previously, it is unclear whether the hyperinsulinemia is compensatory or results from the same factors that cause insulin resistance, in which event it may occur at the same time or even precede it.70,193 In this context, it is noteworthy that increases in plasma FFA have been shown acutely to increase insulin secretion in certain settings, whereas chronic increases in the concentration of saturated fatty acids and glucose cause dysfunction and damage to the β cell and ultimately result in apoptosis.75,101,194 Work from a number of laboratories169,170,195,196 has both delineated the events that lead to these phenomena in the β cell and revealed their similarity to the changes observed in endothelium and other cells when exposed to high concentrations of FFAs or glucose. As already noted, in the ZDF rat, the leptin-­receptor–deficient rodent characterized by Unger,169 the activity of AMPK in multiple tissues is depressed, and treatment with troglitazone or AICAR or caloric deprivation prevents or at least

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PART 3  Obesity and Nutrition

markedly attenuates the development of β cell damage and dysfunction and hyperglycemia.114,197 Likewise, it has been demonstrated that AMPK activation prevents the apoptosis and mitochondrial dysfunction observed in pancreatic β cells when incubated with saturated fatty acids at a high glucose concentration.195 Finally, although theories linking triglyceride accumulation to β cell dysfunction are attractive and have led to interesting hypotheses,22,182,198,199 to our knowledge there have been no definitive studies showing that triglyceride accumulates in human islets in patients with type 2 diabetes.

Molecular Mechanisms of Insulin Resistance and Cellular Dysfunction According to the Lipid Theory (Muscle and Liver). Based on studies reviewed in the preceding sections, a model can be proposed in which insulin resistance and cellular dysfunction in liver and muscle are due to an increase in intracellular fatty acid– derived molecules and metabolites such as fatty acyl CoA DAG89,91,200 and ceramides, possibly secondary to dysregulation of AMPK (Fig. 26.7; see Table 26.2). According to this scheme, such changes activate a serine-­threonine kinase cascade that includes conventional and/ or novel protein kinase C isoforms,89,91,201,202 IKKB,203,204 and Jun-­ activated kinase, one or more of which phosphorylate serine residues on IRS-­1 in muscle, and likely other tissues.87 Similar changes in IRS1 in response to hyperglycemia may also result from activation of the mTOR/p70s6k signaling mechanism.205 Serine phosphorylation of IRS-­1 in turn impairs its ability to associate with P13-­kinase, leading to diminished activation by insulin of Akt and PKC zeta, glucose transport, glycogen synthesis, and other downstream events. Studies demonstrating that transgenic mice with a muscle-­specific alteration in IRS-­1 Ser→Ala are protected from fat-­induced insulin resistance in skeletal muscle strongly support this hypothesis.206 Similar changes appear to occur in liver, except that the inhibitory actions of insulin on gluconeogenesis and glycogenolysis are impaired,87,93,191 and IRS1 and IRS2 may be differentially affected.207 Also possibly involved in this chain of events are increases in oxidative and ER stress, ceramide synthesis, NF-­κB activation, and NF-­κB–mediated gene expression that could explain, at least in part, the proinflammatory state associated with metabolic syndrome.73,75,136 Interestingly, the hallmark of this insulin-­resistant state is an increase in intracellular triglyceride in liver and muscle that can be quantified noninvasively with magnetic resonance imaging.208,209 Triglyceride accumulation in muscle and liver is generally regarded as a marker of lipid-­induced insulin resistance and cellular dysfunction, rather than a cause.210 On the other hand, by providing an additional source of intracellular FFAs, it could play a more pathogenetic role.

The Hypothalamus, Food Intake, and Insulin Resistance. Leptin, which activates AMPK in peripheral tissues by a direct action, diminishes AMPK activity in the hypothalamus. This in turn leads to decreased food intake and activation of the sympathetic nervous system and, secondary to this, further activation of AMPK in peripheral tissues.205 Conversely, glucocorticoids increase AMPK in the hypothalamus, leading to an increase in food intake, and they decrease AMPK and cause insulin resistance in peripheral tissues (see discussion of Cushing syndrome in the section entitled “AMPK and Metabolic Syndrome in Humans” later in the chapter and in Christ-­Crain and associates129). Various antipsychotic drugs have also been found to activate AMPK in the hypothalamus, and, like glucocorticoids, they increase food intake and cause insulin resistance.211 Why some agents that decrease or increase AMPK activity in the hypothalamus have opposite effects on peripheral tissues is not known. On the other hand, the possibility that a drug that activates or inhibits AMPK activity in peripheral tissues has the opposite effect in the hypothalamus or

elsewhere in the central nervous system needs to be considered in evaluating its clinical efficacy and side effects. KEY POINTS  • An increasing body of evidence suggests that a likely pathogenetic mechanism in most patients is an abnormality of cellular lipid metabolism that causes lipotoxic changes, including: insulin resistance, oxidant and ER stress, inflammation, and mitochondrial dysfunction in one or more tissues. Proposed causes of these abnormalities in cell lipid metabolism are elevated plasma FFA levels, dysregulation of AMPK, SIRT1, or malonyl CoA, and primary mitochondrial defects.

AMPK and SIRT1 Sirtuins are a group of histone 1 protein deacetylases that are regulated by changes in the cellular redox state (NAD+/NADH+ ratio) and increases in nicotinamide phosphoribosyltransferase (NAMPT), the rate-­limiting enzyme for NAD synthesis. The seven sirtuins, and in particular SIRT1, the most studied member of the family, were evaluated initially because of their role in combatting aging.212 As reviewed elsewhere,213 SIRT1 responds to overfeeding, starvation, changes in energy expenditure, and exercise, as well as to adiponectin, much as does AMPK, although with somewhat different timing.13,54,213 Interestingly, it has been shown that SIRT1 can activate AMPK by deacetylating its activator LKB1, which promotes its translocation from the nucleus to the cytosol, where LKB1 is activated, and it in turn phosphorylates and activates AMPK (which is already substantially located in the cytosol).127,214,215 Conversely, AMPK can activate SIRT1 by increasing the NAD+/NADH ratio or the expression/activity of NAMPT.216 These findings suggest the existence of an AMPK/SIRT1 cycle that links the cell’s energy and redox states.217 In keeping with this notion, AMPK and SIRT1 (and probably other sirtuins) act on common transcriptional activators and coactivators, including members of the FOXO family and PGC1. In addition, both AMPK and SIRT1 activators have been shown to decrease atherosclerosis and prevent diabetes in experimental animals.75,217,218 The latter could be related, at least in part, to the fact that changes in both AMPK and SIRT1 appear to exert effects on inflammation and fuel metabolism in M1 macrophages and T helper cells, both of which are more numerous in inflamed adipose tissue.148 How AMPK and SIRT1 may interact with each other and with NF-­κB and oxidative and ER stress in these cells to combat metabolic syndrome is illustrated in Fig. 26.8.

AMPK and Metabolic Syndrome in Humans Although existing data in rodents with decreased AMPK activity and the beneficial effects of exercise and various pharmacologic agents that activate AMPK have linked it to metabolic syndrome in humans,219 until recently, direct AMPK measurements in muscle of insulin-­resistant humans were not strongly supportive, or yielded mixed results.212 Since then, decreased AMPK activity has been clearly demonstrated in adipose tissue of two distinct groups of insulin-­resistant individuals.

Cushing Syndrome. Korbonits and coworkers220 observed decreased AMPK activity in the visceral fat of patients with Cushing syndrome, most of whom had elevated plasma cortisol levels due to a functioning adrenal adenoma. Such individuals are classically characterized by insulin resistance, visceral adiposity, hypertension, type 2 diabetes, and a predisposition to ASCVD and other diseases. Similar abnormalities also have been observed in patients treated with high doses of glucocorticoids for extended periods, although whether the incidence of ASCVD is increased is somewhat controversial.221 Also of note, in contrast to most individuals with metabolic syndrome, patients with Cushing syndrome show little, if any, evidence of inflammation, and

CHAPTER 26  Metabolic Syndrome

Muscle Fatty acid

pS ↓β-oxidation

↑DAG

GLUT4

pS pS

n

PH ↓AKT2

↓T ra n

sl o

ca tio

as e

pS pY

↑PKC-θ Ser/Thr kinase

↑LCCoA

PIP3 PIP3 PIP3

pY pY

Glucose

SH ↓P 2 13 -k in

pY pY

PTBP PIP2 H

Insulin receptor

↓Glucose ↓G6p

P ↓GSK

↓UDP-glucose

IRS-1

↓GS activity

↓Mitochondrial density

↓Glycogen synthesis

A Liver

↑DAG

↑LCCoA

↓β-oxidation? ↑de novo lipid synthesis

B

pY pY

IRS-1

PH ↓AKT2

se

PIP3 PIP3 PIP3

GLUT4

↑Gluconeogenesis

na

↑PKC-θ Ser/Thr kinase

pY pY

Glucose

SH ↓P 2 13 -k i

pY pY

PTBP PIP2 H

Insulin receptor

Fatty acid

P ↓GSK3

P ↓FOXO

↓Glycogen synthesis

↑FOXO

NUCLEUS

↑PEPCK ↑G6Pase

Figure 26.7  Proposed mechanisms for fatty acid induced insulin resistance in skeletal muscle (A) and liver (B). A, Muscle. Increases is intramyocellular LCCoA and DAG, due to increased fatty acid delivery and/or decreased mitochondrial fatty acid oxidation, trigger a serine/threonine kinase (Ser/Thr) cascade initiated by nPKCs and possibly involving IKKB and/or JNK-­1. This ultimately induces serine/threonine phosphorylation of critical IRS-­1 sites in muscle, which inhibit IRS-­1 tyrosine phosphorylation and activation of PI 3-­kinase. This in turn results in reduced insulin-­stimulated glucose transport and glycogen synthesis. B, Liver. Increases in intracellular DAG, due to enhanced lipogenesis and/or decreased mitochondrial fatty acid oxidation, activate PKCs (ε and/or possibly δ), which bind to and inactivate the insulin receptor kinase. This results in reduced insulin-­stimulated IRS-­1 and IRS-­2 tyrosine phosphorylation, and decreased insulin activation of PI3-­kinase and AKT2. This in turn leads to diminished GSK3 and FOXO phosphorylation, which decrease insulin-­stimulated liver glycogen synthesis and hepatic gluconeogenesis, respectively. DAG, Diacylglycerol; FOXO, forkhead box protein O; GLUT, glucose transporter; G6P, glucose-­6-­phosphate; GSK3, glycogen synthase kinase 3; IRS, insulin receptor substrate; IKKB, IκB kinase β; JNK-­1, Jun-­activated kinase 1; LCCoA, long-­chain acylcoenzyme A; PEPCK, phosphoenolpyruvate carboxykinase; PI3-­kinase, phosphatidylinositol-­3-­ kinase; PTB, phosphotyrosine-­binding domain; PH, pleckstrin homology domain; SH2, src homology domain. (Adapted from Savage DB, Petersen KF, Shulman GI. Disordered lipid metabolism and the pathogenesis of insulin resistance. Physiol Rev. 2007;87:507–520.)

437

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PART 3  Obesity and Nutrition Inflammation (NF-κB)

AMPK Oxidative stress

ER stress

in AMPK and SIRT1 and LKB1 activity were observed in liver 9 weeks after bariatric surgery.192 Possibly other factors contribute to differences between severely obese individuals who are insulin-­sensitive and insulin-­resistant, including differences in their microbiome, collagen VI deposition, and lipid droplet proteins,213 all of which have yet to be intensively examined. With respect to the microbiome, it has been shown that germ-­free mice are characterized by a substantial increase in AMPK activity,223 suggesting that bacteria and possibly the inflammation they cause diminish AMPK activity in mammalian organisms.

SIRT 1,3

DIAGNOSIS Figure 26.8  Proposed interrelations of AMP-­ activated protein kinase (AMPK) and sirtuins 1 and 3 (SIRTs) with oxidative and ER stress and inflammation. AMPK and SIRT1 both activate each other and diminish oxidative and endoplasmic reticulum (ER) stress and low-­ grade inflammation in various settings. Conversely, oxidative and ER stress and inflammation, which activate each other, appear to diminish AMPK and SIRT1. In principle, any of these factors could be targeted to combat insulin resistance and the development of metabolic syndrome–associated disorders; however, to date, the most success has been observed with therapies that target AMPK. (Adapted from Ruderman NB, Carling D, Prentki M, et al. AMPK, insulin resistance and the metabolic syndrome. J Clin Invest. 2013;123:2764–2772,)

to our knowledge ER stress in adipose tissue has not been studied. In what respects the lack of these abnormalities in patients with Cushing syndrome distinguishes them from most other individuals with metabolic syndrome in whom they are present has yet to be determined.

Bariatric Surgery Patients: Insulin-­Resistant and -­Sensitive. Perhaps even more compelling evidence of a link between decreased AMPK in adipose tissue and metabolic syndrome–associated disorders has been provided by studies in severely obese patients undergoing bariatric surgery. Gauthier and colleagues116 and Xu and associates115 observed that AMPK activity is significantly diminished (30%–50%), and that chronic oxidative stress (protein carbonylation) increased in adipose tissue of the 75% of such individuals who were insulin-­resistant compared with equally obese subjects who were insulin-­ sensitive (homeostasis model assessment insulin resistance 6.7 vs. 2.3). They also observed that AMPK activity and mitochondrial gene expression were lower in visceral fat than in subcutaneous fat in both groups, and that the expression of inflammatory genes was lower in both fat depots in the patients with insulin resistance.115 In another study of bariatric surgery patients in which AMPK was not measured, infiltration of adipose tissue with macrophages and decreases in plasma adiponectin (an AMPK activator) were the strongest correlates of insulin resistance.163 These studies in adipose tissue have interesting implications, because bariatric surgery reverses type 2 diabetes and other disorders associated with metabolic syndrome, including dyslipidemia, hypertension, and PCOS.24,151 It also diminishes long-­term mortality from coronary heart disease (30%–50% at 20 years),222 and the prevalence of solid tumors (by 70% 5 years postsurgery).151 Interestingly, the beneficial effect of the surgery on coronary heart disease was most prominent in patients in the two highest quintiles of plasma insulin concentration preoperatively.222 As noted earlier, such individuals have been found to be insulin-­resistant, with decreased AMPK activity and increased oxidative stress, inflammation, and presumably ER stress and decreased mitochondrial function in their adipose tissue.213 Whether the decreases in AMPK and, when it occurs, SIRT1 in adipose tissue of these patients increases rapidly post–bariatric surgery is not known. Interestingly, in rats made obese by fat-­feeding, substantial increases

No single definitive diagnostic test for metabolic syndrome is yet available. As discussed earlier, historically it has been diagnosed based on the presence of general or abdominal obesity, dyslipidemia, hypertension, and impaired fasting glucose or glucose intolerance in various combinations. In addition, the presence of premature coronary heart disease, type 2 diabetes in its early stages, and other disorders associated with insulin resistance have sometimes been considered diagnostic criteria. Several organizations have published standards for diagnosis, including the National Cholesterol Education Program, ATP III, and the World Health Organization (WHO).224 They differ principally in that the WHO criteria place more emphasis on measures of insulin resistance, the presence of microalbuminuria, and the use of the glucose tolerance test, whereas the ATP III emphasizes abdominal obesity and risk factors for CVD such as dyslipidemia and hypertension. A more recent set of criteria proposed by the International Diabetes Federation is similar to that of the ATP III but with somewhat lower cutoff points for blood pressure and fasting glucose.17 In part because of its relative simplicity, for clinical purposes the ATP guidelines (see Table 26.1) appear to be in widest use. On the other hand, they probably underestimate the prevalence of insulin resistance in the general population.225 Also of note is that modified criteria have been developed for different ethnic groups, because metabolic syndrome is not as closely associated with obesity (based on BMI) in some groups (e.g., South Asians) as it is in Caucasians.17,226

CORONARY HEART DISEASE AND TYPE 2 DIABETES The notion that metabolic syndrome, or its surrogate markers hyperinsulinemia and insulin resistance, antedate and contribute to the pathogenesis of coronary heart disease, diabetes, and at least some cases of hypertension was proposed many years ago.21,35 Coronary heart disease in the setting of metabolic syndrome can to a great extent be attributed to dyslipidemia (increased dense low-­density lipoprotein [LDL] cholesterol, diminished HDL cholesterol, and hypertriglyceridemia),227 as well as to elevations in blood pressure and blood glucose and the presence of a procoagulant, proinflammatory state.22,224 In addition, some studies suggest that hyperinsulinemia and insulin resistance, as well as hyperglycemia, may be independent risk factors.51 Whether elevated FFA levels or a dysregulation of intracellular fatty acid metabolism contribute to atherosclerosis by directly altering the function of endothelium (see the section entitled “Vascular Endothelial Cells and Atherogenesis”) or other cells in the vascular wall remains to be determined. Relevant to this discussion, low levels of adiponectin are associated with an increased risk for coronary heart disease in humans,156 whereas, as noted earlier, overexpression of adiponectin or its globular subunit diminishes the severity of atherosclerosis in ApoE–/– mice.228,229 More definitive evidence that metabolic syndrome per se predisposes to coronary heart disease and cerebrovascular disease has been

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CHAPTER 26  Metabolic Syndrome ↑ Triglyceride

19.2% CHD prevalance (%)

18

↓ HDL ↑

13.9% 12 8.7%

↑ De novo lipogenesis

Glycogen 7.5%

↑ NAFLD

6 Glucose

0 + Metabolic syndrome – – + + Diabetes – + – % of population studied 54.2 28.7 2.3 14.8 Figure 26.9  Age-­adjusted prevalence of CHD in the US population over 50 years of age, categorized by the presence of metabolic syndrome (Adult Treatment Panel III criteria) and diabetes. Data are from the National Health and Nutrition Examination Survey study. The complete absence of an increase in CHD incidence in the diabetic patients without metabolic syndrome should be viewed with caution because of the small size of this group. CHD, Coronary heart disease. (Data from Alexander CM, Landsman PB, Teutsch SM, et al. NCEP-­ defined metabolic syndrome, diabetes, and prevalence of coronary heart disease among NHANES III participants age 50 years and older. Diabetes. 2003;52:1210–1214.)

reported. Thus, a 2-­fold to 4-­fold increase in subsequent cardiovascular events has been described in men and women with metabolic syndrome (modified WHO criteria) even in the absence of type 2 diabetes or impaired glucose tolerance.230-­232 Qualitatively, similar results have been obtained when metabolic syndrome is defined by ATP III criteria23,73,234 (Fig. 26.9). In a compilation of multiple studies, the presence of metabolic syndrome had a greater impact on the risk for developing diabetes (5-­fold) than ASCVD (2-­fold).22,182,199 In addition, where studied, the rate of cardiovascular events was higher in patients who had diabetes and metabolic syndrome than in individuals with only metabolic syndrome.22,235 KEY POINTS  • In both adolescents and adults, metabolic syndrome substantially increases the risk for type 2 diabetes, coronary heart disease, and other associated disorders.

LINKAGE OF METABOLIC SYNDROME TO OTHER DISORDERS From a practical point of view, the ATP III and WHO guidelines focus on the relationship of metabolic syndrome to obesity and the risk for developing type 2 diabetes and ASCVD. However, insulin resistance and hyperinsulinemia are also associated with other disorders in individuals who, for genetic or other reasons (e.g., drug therapy), are more susceptible to them. A few of these disorders will be briefly discussed.

Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis It was estimated over 10 years ago that nearly 20 million individuals in the United States have the diagnosis of nonalcoholic fatty liver disease (NAFLD), and approximately 10% of these individuals develop nonalcoholic steatohepatitis (NASH), a disorder characterized by mitochondrial dysfunction, increases in oxidative stress and cell cytokines, and a predisposition to cirrhosis (approximately 20% of patients with NASH), and less commonly hepatocellular carcinoma.67,236-­239

Glycogen

Glycogen

Insulin Sensitive Insulin resistant Figure 26.10  Schematic of whole-­ body energy distribution after high-­ carbohydrate mixed meals in young insulin-­ sensitive and insulin-­resistant individuals. (Adapted from Petersen KF, Dufour S, Savage DB et al. The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome. Proc Natl Acad Sci USA. 2007;104:12587–12594.)

NAFLD is also seen with increasing frequency in children and adolescents in parallel with the increasing prevalence of obesity in these populations.240 Presumably, mildly abnormal liver function tests in the presence of obesity or type 2 diabetes or other manifestations of insulin resistance would help to identify individuals with NAFLD at an early point in time. Recent studies in young, lean, insulin-­resistant individuals have demonstrated that insulin resistance in skeletal muscle, as reflected by decreased glycogen synthesis, can promote atherogenic dyslipidemia by changing the fate of ingested carbohydrate away from skeletal muscle glycogen synthesis into hepatic de novo lipogenesis, resulting in an increase in plasma triglyceride concentrations and a reduction in plasma HDL concentrations (Fig. 26.10). Furthermore, insulin resistance in these subjects was independent of changes in the plasma concentrations of TNFα, IL-­6, HMW adiponectin, resistin, retinol-­binding protein 4, or intraabdominal obesity, suggesting that these factors do not play a primary role in causing insulin resistance in the early stages of metabolic syndrome.241 Further evidence in support of this hypothesis comes from a recent study that found that a single bout of exercise was sufficient to promote increased muscle glycogen synthesis and decreased de novo lipogenesis and hepatic triglyceride synthesis following carbohydrate ingestion in young, lean individuals with muscle insulin resistance.242

Polycystic Ovarian Syndrome Another common disorder that becomes more prevalent in the presence of metabolic syndrome is PCOS.68,69 PCOS is characterized by genetically determined increases in ovarian androgen production and disordered gonadotropin secretion that may be exaggerated by hyperinsulinemia or insulin resistance. Perhaps because of its association with metabolic syndrome and often, although not always, with obesity, PCOS is a leading risk factor for glucose intolerance and type 2 diabetes in adolescent and premenopausal women.243 It may also increase their risk for premature CVD.244 PCOS is associated with increases in such inflammatory factors as plasminogen activator inhibitor, C-­reactive protein, and TNFα.68 Like NAFLD/NASH, type 2 diabetes, and other disorders associated with metabolic syndrome, PCOS often responds to treatments that activate AMPK and/

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TABLE 26.4  Recommended Treatments for Metabolic Syndrome in Adults, Children,

and Adolescents

ADULTS

CHILDREN AND ADOLESCENTS

Treatment

Nondiabetic

IGT

Diabetic

Nondiabetic

Diet and exercise Metformin TZDs

+ ND ND

+ + +*

+ + +*

+ ND ND

+, Currently recommended treatment; ND, as yet no definitive evidence for or against use. *See text for discussion of controversy concerning TZD use. For diabetic children and adolescents, diet and exercise are the recommended therapy. Where possible, however, metformin and insulin are often used. Data from Alberti G, Zimmet P, Shaw J, et al. Type 2 diabetes in the young: the evolving epidemic: the International Diabetes Federation Consensus Workshop. Diabetes Care. 2004;27:1798–1811.

or lower the concentration of malonyl CoA, such as diet and exercise, metformin, and TZDs68 (see later section entitled “Treatment of Metabolic Syndrome”).

Certain Cancers The reason for the link between metabolic syndrome, obesity, type 2 diabetes, and cancers of the colon, breast, liver, and other sites is less clear. It has generally been held that insulin-­like growth factor 1 (IGF-­1) could be one factor, because insulin both stimulates the synthesis of IGF-­1 by liver and inhibits the synthesis of its binding protein IGFBP-­1.245 Another possibility relates to the fact that an upstream kinase that activates AMPK is LKB1,246,247 a tumor suppressor that is deficient in individuals with Peutz–Jeghers syndrome and places them at increased risk for developing carcinomas of the colon, stomach, and pancreas and adenocarcinomas of the lung. The possibility that LKB1 and AMPK link metabolic syndrome to these cancers and are targets for their prevention or therapy warrants consideration.248-­250 Of note, agents that activate AMPK, such as metformin, have been shown to diminish the growth of cancer cells.251

Alzheimer Disease Alzheimer disease and other disorders associated with dementia are more common in individuals with metabolic syndrome and type 2 diabetes. As reviewed elsewhere,252 Alzheimer disease appears to be associated with insulin resistance as well as inflammation and mitochondrial dysfunction in the brain. Also of note, physical activity, which has been shown to activate AMPK in many tissues in rodents,123 has demonstrated efficacy for preventing and treating Alzheimer disease in both humans and experimental animals.3,252 Likewise, benefits from treatment with TZDs and metformin have been reported in some studies.253

Cushing Syndrome and Related Disorders As already noted, patients with primary Cushing syndrome or Cushing syndrome due to therapy with glucocorticoids typically demonstrate central obesity, insulin resistance, hyperinsulinemia, and a predisposition to diabetes and hypertension.254 In addition, like other patients with this clustering of events, they may be at increased risk for ASCVD.255,256 The value of treatments aimed at AMPK and malonyl CoA in individuals with Cushing syndrome, when its primary cause cannot be corrected, has to our knowledge not been evaluated. It is noteworthy, however, that glucocorticoids diminish AMPK in peripheral tissues in rodents and, as discussed earlier, AMPK activity is decreased in visceral fat of patients with Cushing syndrome.129 The possibility that an increase in cellular 11-­β-­dehydrogenase activity resulting in local increases in cortisol could be a cause of metabolic syndrome has been discussed elsewhere.257

Lipodystrophy Patients with primary lipodystrophy and lipodystrophy secondary to drug therapy (e.g., protease inhibitors in HIV patients) appear to be subject to the same lipotoxicity observed in individuals with metabolic syndrome for other reasons. Two factors appear to contribute to an insulin-­resistant state in these patients: (1) decreased peripheral fat cells cause more plasma FFA and lipoprotein triglyceride to be shunted to ectopic sites, and (2) plasma leptin and adiponectin levels are very low. The importance of the latter is suggested by successes in treating some patients with leptin144,145 or with TZDs.258 Interestingly, in the latter study, the beneficial effect of rosiglitazone was accompanied by an increase in adiponectin but not in subcutaneous fat mass.

Hyperalimentation As suggested by Unger,259 hyperalimentation can cause lipotoxic damage and, by inference, metabolic syndrome in some individuals. He hypothesized that at one extreme are normal individuals who eat an excess number of calories for an extended period and become hyperinsulinemic and insulin-­resistant when the ability of their adipose tissue to deposit the excess lipid in their diet is exceeded.260 At the other extreme are hyperalimented patients with an extensive loss of subcutaneous adipose tissue due to third-­degree burns. Such burn patients are historically very insulin-­resistant, which can be attributed at least in part to increased plasma concentrations of inflammatory cytokines, such as TNFα and IL-­6. It remains to be determined if the burns contribute to their insulin resistance and whether the latter responds to pharmacologic agents that enhance insulin sensitivity.

Additional Disorders. Metabolic syndrome has also been associated with an increased prevalence of such disorders as gout, sleep apnea, gallstones, and chronic kidney disease.199 Like other metabolic syndrome–related disorders, they are associated with obesity, type 2 diabetes, and a predisposition to ASCVD. KEY POINTS  • The prevalence of metabolic syndrome is markedly increased in individuals who lack adipose tissue, as well as in individuals who are obese. In both populations, a common occurrence is ectopic lipid deposition in muscle, liver, and often visceral fat.

TREATMENT OF METABOLIC SYNDROME The demonstration that metabolic syndrome increases the risk for developing other disorders in both otherwise normal individuals with type 2 diabetes strongly suggests that it warrants treatment (Table

CHAPTER 26  Metabolic Syndrome 26.4). Less clear are which therapies should be used in a given circumstance and when they should be started.

Lifestyle Modification (Weight Loss and Physical Activity) The treatment of metabolic syndrome with the aim of preventing CVD has been reviewed by a clinical conference jointly sponsored by the American Heart Association, the National Heart, Lung and Blood Institute, and the American Diabetes Association (AHA/NHLBI/ ADA). The consensus was that lifestyle modifications consisting of diet for the treatment of obesity and overweight and physical activity were the first line of therapy.224,261,262 The efficacy of this approach for preventing disease has not been assessed specifically in patients with metabolic syndrome diagnosed by ATP III or WHO criteria; however, exercise has been shown to reverse the defects in insulin-­stimulated glucose transport and muscle glycogen synthesis in young, lean, insulin-­resistant offspring of parents with type 2 diabetes,46 and in several prospective studies61-­63 the combination of diet and exercise has proven quite effective in delaying or preventing the onset of diabetes in patients with impaired glucose tolerance (most of whom probably had metabolic syndrome). Also, numerous epidemiologic studies have shown a 30% to 50% decrease in the risk for developing coronary heart disease, as well as type 2 diabetes, with the maintenance of a physically active compared with a sedentary lifestyle.262 It must be emphasized, however, that the incidence of diabetes in the treated patients with impaired glucose tolerance in these studies was still higher than in the general population,61,63 suggesting that, to be maximally effective, lifestyle changes may have to be introduced even earlier. Whether lifestyle changes have a similar effect on coronary heart disease is less certain. However, in follow-­up studies of patients in the US and Finnish diabetes prevention program who were insulin-­resistant, diet and exercise diminished nontraditional risk factors for coronary heart disease, such as C-­reactive protein and fibrinogen, as well as some of the more classic risk factors associated with metabolic syndrome.263,264

Drug Therapy When recommended therapeutic goals are not achieved with diet and exercise, pharmacologic therapy is necessary. The recommendations of the AHA/NHLBI/ADA at different stages of metabolic syndrome, based on Framingham risk scores for developing ASCVD, have been presented elsewhere.22,235 Suffice it to say, they vary with an individual’s risk for developing coronary heart disease and cerebrovascular disease over given time intervals. Thus, in an individual already at risk for these disorders because of diabetes, the use of statins and other agents to diminish plasma cholesterol has a lower goal (LDL cholesterol 95th percentile).286 Furthermore, because the incidence of obesity until recently has been increasing in the years since these data were obtained, these are likely underestimates of its prevalence at the present time. Where studied, metabolic syndrome in children and adolescents has been associated with insulin resistance, central adiposity, dyslipidemia, elevations in blood pressure, and increases in intramyocellular and intrahepatic lipids, much as in adults, although with different absolute measurements.19,20 In addition, as in adults, the prevalence of NAFLD240 and PCOS68 are increased, as are plasma levels of the proinflammatory markers C-­reactive protein and IL-­6, and immunoassayable adiponectin in plasma is diminished.20 Also, the prevalence of CVD in these individuals later in life may be increased.287-­289 Current treatment recommendations for children with type 2 diabetes, most of whom have metabolic syndrome, have been discussed critically by an International Diabetes Federation Consensus Workshop.18

LOW BIRTH WEIGHT INFANTS (EPIGENETIC CHANGES) It has been known for some time that a low birth weight, independent of gestational age, predicts the development of type 2 diabetes, coronary heart disease, central obesity, and other aspects of metabolic syndrome in middle age in some individuals.290,291 Recent reports suggest that the greatest risk is in infants with low birth weight who gain weight rapidly in childhood.292 It has been suggested that an altered intrauterine environment (e.g., poor nutrition) could be responsible for the low birth weight in these children; however, the precise nature of these alterations is not known. Because of the long period between the malnutrition and the development of disorders, many years later it was suggested that epigenetic changes may be involved. This question was subsequently investigated in individuals prenatally exposed to food deprivation during the Dutch famine at the end of World War II (1944–1945). Six decades later, it was found that those individuals who were malnourished during the first trimester of pregnancy showed less DNA methylation of the maternally imprinted insulin-­like growth factor 2 (IGF2) gene compared with their unexposed same-­sex sibilings.293 Interestingly, in middle age, these individuals were predisposed to obesity, altered lipid profiles, CVD, and accelerated cognitive aging.294

THE THRIFTY GENOTYPE AND THE INCREASING INCIDENCE OF METABOLIC SYNDROME The recent increase in the prevalence of metabolic syndrome and type 2 diabetes has been attributed to the epidemic of obesity that began in the second half of the twentieth century and continues unabated. As reviewed elsewhere,295 this is almost certainly a reflection of environmental factors, most notably the increased availability of food (and possibly the higher carbohydrate content of our diet over the past 50 years) and the decrease in physical activity that occurred in many industrialized societies during this time period. In 1962, Neel296 raised the question of why diabetes (type 2), which has adverse effects on health, persisted in humans during the course of evolution. He suggested that it could be related to the existence of a “thrifty gene” that predisposes individuals to obesity, and secondarily diabetes, but had survival value for our hunter-­gatherer ancestors. More specifically, he proposed that in the feast-­famine environment of early humans “individuals exceptionally efficient in the uptake and utilization of food and its storage as fat” would have had a selective advantage. Neel further suggested that, in our modern environment of calorie surplus, such a gene (or genes) that protected us from death when food sources were scarce may make us prone to obesity and diabetes (and metabolic syndrome). Over the years, many candidate thrifty genes have been proposed, including UCP2, UCP3, and those encoding B3-­adrenergic receptors. Genes governing the AMPK/malonyl CoA fuel-­ sensing network, which has been linked to the regulation of both food intake105,168,297 and energy expenditure,106,111 have also been suggested. This is an attractive notion, because a decreased ability to oxidize fatty acids appears to be characteristic of preobese humans.76,110 For the same reason, genes that regulate mitochondrial biogenesis also need to be considered.7 Decreased mitochondrial content/activity, be it acquired or inherited, may promote obesity by two mechanisms. First, by decreasing daily energy expenditure, even by a small amount, it could lead to progressive weight gain. For instance, a 50-­calorie/day reduction in energy expenditure in a typical adult, if not accompanied by a comparable decrease in food intake, would lead to a weight gain of approximately 5 lb/year. Second, a decrease in mitochondrial activity in skeletal muscle, by predisposing an individual to altered cellular

CHAPTER 26  Metabolic Syndrome lipid metabolism (as reflected by an increase in intramyocellular triglyceride), like alterations in the AMPK/malonyl CoA network,75 can lead to insulin resistance and hyperinsulinemia.118 The latter, in turn, could promote obesity by increasing lipogenesis in liver and adipose tissue and by inhibiting lipolysis until a new, more obese, steady state is achieved.

ACKNOWLEDGMENTS This work was supported in part by USPHS grants DK19514, DK49147, P01-­HL-­68758, R01 DK-­40936, R01 DK-­49230, R24 DK-­085638, P30 DK-­45735, U24 DK-­059635, a grant from the Novo Nordisk Foundation for Basic Metabolic Research, and a grant from the Juvenile Diabetes Research Foundation. The authors thank Christina Nielsen-­ Campbell and Laura DelloStritto for their assistance in preparing this chapter. • For your free Expert Consult eBook with bibliographic citations as well as the ability to take notes, highlight important content, search the full text, and more, visit http://www.ExpertConsult.Inkling.com.

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Young Committee (Council on Cardiovascular Disease in the Young) and the Diabetes Committee (Council on Nutrition, Physical Activity, and Metabolism). Circulation. 2003;107:1448–1453. 290. Barker DJ, Hales CN, Fall CH, et al. Type 2 (noninsulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia. 1993;36:62–67. 291. Phillips DI. Birth weight and the future development of diabetes. A review of the evidence. Diabetes Care. 1998;21:B150–B155. 292. Bhargava SK, Sachdev HS, Fall CH, et al. Relation of serial changes in childhood body-mass index to impaired glucose tolerance in young adulthood. N Engl J Med. 2004;350:865–875. 293. Heijmans BT, Tobi EW, Stein AD, et al. Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci U S A. 2008;105(44):17046–17049. 294. Schulz LC. The Dutch Hunger Winter and the developmental origins of health and disease. Proc Natl Acad Sci U S A. 2010;107:16757–16758. 295. Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature. 2001;414:782–787. 296. Neel JV. Diabetes mellitus: a “thrifty” genotype rendered detrimental by “progress”. Am J Hum Genet. 1962;14:353–362. 297. Hu Z, Cha SH, Chohnan S, et al. Hypothalamic malonyl-CoA as a mediator of feeding behavior. Proc Natl Acad Sci U S A. 2003;100:1262–12629. 298. Kemp BE, Stapleton D, Campbell DJ, et al. AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans. 2003;31:62–168. 299. Hardie DG, Scott JW, Pan DA, et al. Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett. 2003;546:113–120. 300. Sanders MJ, Grondin PO, Hegarty BD, et al. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J. 2007;403:139–148. 301. Towler MC, Hardie DG. AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res. 2007;100:328–341.

27 Bariatric Procedures and Operations Todd Andrew Kellogg, Barham K. Abu Dayyeh, and Michael R. Rickels

OUTLINE Introduction/Overview, 450 Commonly Performed Bariatric Operations, 450 Vertical Sleeve Gastrectomy, 451 Roux-­en-­Y Gastric Bypass, 451 Biliopancreatic Diversion With Duodenal Switch, 453 Single Anastomosis Duodenal Ileostomy Bypass With Sleeve, 453 Laparoscopic Adjustable Gastric Banding, 453 Endoscopic Bariatric and Metabolic Therapies, 453 Evolving Evidence Profile for the Use of Endoscopic Bariatric and Metabolic Therapies in Clinical Practice, 455 Space-­Occupying Devices, 455 Aspiration Therapy, 456 Small Bowel Intervention, 456 Eligibility for Bariatric Operations and Procedures, 458 Assessing Outcomes after Bariatric Interventions, 458 The Physiology of Weight Loss After Bariatric Operations and Procedures, 459 Weight Loss Mechanisms, 459 Caloric Restriction, 459 Gastrointestinal Anatomy Changes, 459 Gastrointestinal and Adipocyte Hormonal Changes, 459 Central Nervous System Control, 459 Vagal Signaling, 460 Energy Expenditure, 460

Physiologic Effects of Bariatric Surgery on Cardiometabolic Health and Mortality, 460 Diabetes Remission Following Bariatric Surgery, 460 The Role of Insulin Sensitivity and Insulin Secretion in Diabetes Remission After Gastric Bypass, 460 The Hindgut and the Foregut Hypotheses, 461 Branched-­Chain Amino Acids and Aromatic Amino Acids, 461 Bile Acids, 461 Gut Microbiota, 461 Cardiovascular Disease and Mortality, 461 Impact of Bariatric Surgery on Other Obesity-­Related Comorbid Conditions, 462 Polycystic Ovarian Syndrome and Fertility, 462 Pulmonary Disease, Including Obstructive Sleep Apnea, 463 Gastroesophageal Reflux Disease, 463 Pseudotumor Cerebri, 463 Cancer, 463 Complications of Bariatric Operations and Procedures, 463 Early Complications, 463 Late and Chronic Complications, 464 Weight Regain, 464 Dumping Syndrome, 464 Alimentary Hypoglycemia With Neuroglycopenia, 464 Summary, 467

 

INTRODUCTION/OVERVIEW

COMMONLY PERFORMED BARIATRIC OPERATIONS

Healthy food choices with portion control, as well as consistent exercise, are the mainstays of a healthy lifestyle and maintenance of a healthy weight. An imbalance of these factors can contribute to obesity with its associated diseases. Surgically-­induced weight loss is the most potent form of weight loss intervention, particularly in individuals with class 2 and class 3 obesity, and can treat many obesity-­associated diseases. The potential health benefits of surgically-­induced weight loss include improved survival, improvement and remission of type 2 diabetes, and a reduced incidence of diabetes, cardiovascular disease, and cancers in women. The epidemic of medically severe obesity coupled with the development and widespread utilization of less invasive laparoscopic techniques has led to a consistent increase in the annual number of bariatric operations performed, as well as the development of endoscopic bariatric interventions and together provide a multidisciplinary approach with escalating obesity interventions (Fig. 27.1). We will begin by discussing the various bariatric operations and procedures, followed by discussion of the metabolic and physiologic consequences.

With the development of endoscopic procedures for weight loss, it has become necessary to differentiate between bariatric surgical operations and bariatric endoscopic procedures. We will begin by discussing the anatomy and general mechanisms of the operations most commonly performed for weight management (Fig. 27.2). The mechanisms behind bariatric operations have components of restriction, malabsorption, and neurohormonal changes. All operations have a varying combination of these three components as their mechanism, resulting in a unique anatomy and physiology that can affect caloric intake, nutrient handling, and nutrient sensing. These changes likely influence each operation’s unique outcome, as discussed later in this chapter. Although much has been learned regarding these mechanisms, much remains to be understood. All these operations are performed laparoscopically, which benefits the patient because there are fewer wound complications, less pain, and earlier return to activities compared with open operations.1,2 Outcomes after bariatric surgery are categorized into the effect on weight and the effect on weight-­related comorbid disease, particularly metabolic disease. Potential adverse effects to be considered include nutritional complications.

450

CHAPTER 27  Bariatric Procedures and Operations

451

Efficacy

Surgical procedures Endoscopic procedures Medications Lifestyle

Risk Figure 27.1  Escalating interventions in the treatment of obesity.

Vertical Sleeve Gastrectomy

Roux-­en-­Y Gastric Bypass

Sleeve gastrectomy (SG) is currently the most commonly performed bariatric procedure in the United States and worldwide, comprising approximately 65% of all bariatric operations in the United States. Its rapid adoption can be attributed to it being a less technically demanding operation that has very good weight loss, as well as improvement of obesity-­related comorbid diseases. Although the SG is a promising operation for treating obesity, the development of gastroesophageal reflux disease (GERD) postoperatively and long-­term weight regain are growing concerns3 and thus, as with any bariatric operation, careful patient selection is of paramount importance. The SG is a restrictive operation with a neurohormonal component.4 The restrictive component occurs by way of an approximately 75% to 80% reduction of the gastric volume via resection of the gastric greater curvature including the gastric fundus (see Fig. 27.2B). Its neurohormonal effects are not completely understood but are thought to be the result of the reduction of ghrelin-­producing oxyntic gland endocrine P/D1 cells5-­7 located in the gastric fundus and parietal cells of the gastric greater curvature. A prospective, double-­ blind study that compared Roux-­en-­Y gastric bypass (RYGB) and laparoscopic SG demonstrated significant postprandial suppression of ghrelin after SG and no change after RYGB. Appetite reduction and excess weight loss (EWL) were greater after SG, suggesting an association with the suppression of ghrelin levels. In the same study, peptide YY (PYY) levels were increased similarly after either operation.8 An increase in gastric emptying with upregulation of glucagon-­ like peptide 1 (GLP-­1) has also been proposed as a mechanism for the effects of SG. However, the evidence is mixed, which may be secondary to variations in surgical technique. SG is a very safe operation, with a 30-­day mortality rate of 0.2% to 0.5%.9,10

Although the SG is currently the most commonly performed bariatric operation in the United States, in many ways the RYGB is still considered the gold standard bariatric operation. It has a solid track record of efficacy, durability, and safety over more than 20 years of follow-­up, which the SG lacks. Although its effects are mostly due to restriction and neurohormonal changes, it also has a small malabsorptive component. The RYGB creates a small approximately 15-­to 30-­mL gastric pouch that is constructed by dividing the upper aspect of the stomach, and this small pouch is then attached to a 75-­cm to 150-­cm jejunal Roux limb (see Fig. 27.2C). The so-­called “biliopancreatic limb” is typically 40 cm to 100 cm in length and carries bile and digestive enzymes from the duodenum and from the remaining portion of the stomach (gastric remnant). The result of this anatomic configuration is that the ingested food and digestive juices are separated for the length of the Roux limb, after which they enter a long common channel of small bowel (the remainder of the jejunum and the entire ileum), where mixing of food with bile and digestive enzymes occurs. This anatomy results in the ingested food bypassing the majority of the stomach, the entire duodenum, and the first portion of the jejunum. In general, the relatively small length of bypassed intestine (roughly 30%) is not enough to cause protein-­calorie malabsorption. However, it does impact the absorption of some micro-­and macronutrients, and specifically calcium, iron, vitamin B12, and vitamin D. The mechanism of action leading to weight loss and comorbidity improvement is multifactorial. Like the SG, the RYGB is mainly a restrictive procedure with additional neurohormonal effects. However, unlike the SG, bypass of the duodenum and proximal jejunum as well as rapid delivery of chime to the distal small bowel are thought to be important mechanisms resulting in alteration in of the entero-­endocrine axis. The changes observed in gut peptides caused by rapid delivery of nutrients to the jejunum (glucose-­dependent insulinotropic polypeptide and GLP-­1) after bypass of the duodenum may be an important long-­term mechanism for durable weight loss and comorbidity remittance.11

Esophagus Proximal pouch of stomach

“Short intestinal Roux limb

Bypassed portion of stomach

Pylorus

Duodenum

A

Gastric “Sleeve”

Pylorus

B

Excised stomach

C

Sleeve stomach

Duodenal anastomosis

Resected stomach

Biliopancreatic limb

Common channel

D

E

Figure 27.2  A, Adjustable gastric band. A silicone band is looped around the proximal stomach to create a 15-­to 20-­mL pouch with an adjustable outlet. The stomach is wrapped around the band anteriorly to prevent the band from slipping out of position. The band consists of a rigid outer ring and an inner inflatable balloon reservoir connected by tubing to a subcutaneous port that can be accessed through the skin to adjust the tightness. B, Sleeve gastrectomy. A narrow gastric sleeve is created by stapling the stomach vertically. The fundus and greater curve of the stomach are surgically removed. C, Roux-­en-­Y gastric bypass (RYGB). A small gastric pouch (15–30 mL) is created by division of the upper stomach connected to a 100-­to 150-­cm limb of jejunum called the Roux limb. The small gastric pouch results in restriction of food intake. D, Biliopancreatic diversion with duodenal switch (BPD-­DS). To avoid acid reflux, marginal ulcers, and dumping syndrome by maintaining the pylorus, the operation was modified with the pouch based on the lesser curve of the stomach and an anastomosis at the first portion of the duodenum. E, Single anastomosis duodenal ileal bypass with sleeve (SADI-­S). A variation of the BPD-­DS in which a single anastomosis is constructed 250–300 cm proximal to the ileocecal valve.

CHAPTER 27  Bariatric Procedures and Operations Gastric bypass is a very safe and extremely effective bariatric operation. The associated 30-­day mortality is 0.2%, incidence of unplanned intensive care unit admissions is 1.3%, and incidence of 30-­day reoperations is 2.5%.12 This low perioperative risk is accompanied by a nearly 30% to 35% total body weight loss (TBWL). The weight loss outcomes are sometimes reported as “excess body weight loss,” meaning the patient has lost an amount of weight above “ideal” body weight, which for RYGB would be approximately 70% excess body weight loss. In addition to the highly significant weight loss, there is a high rate of improvement or remission of obesity-­related comorbidities,13 which makes the RYGB a good choice for treatment of obesity and its complications.

Biliopancreatic Diversion with Duodenal Switch Biliopancreatic diversion with duodenal switch (BPD-­DS) is the most powerful bariatric procedure in terms of weight loss and the treatment of diabetes and other obesity-­related comorbidities. It is a technically challenging operation and has a higher incidence of complications compared with RYGB and SG. Primary BPD-­DS represents less than 1% of all bariatric operations in the Unites States [https://asmbs.org/ resources/estimate-­of-­bariatric-­surgery-­numbers]. However, BPD-­DS is beginning to gain popularity as a revision operation, especially in the setting of weight regain after SG.14 The effects of the BPD-­DS can be attributed to components of restriction, malabsorption, and neurohormonal changes and, compared with the SG and RYGB, is the only bariatric operation that employs true protein-­ calorie malabsorption as a mechanism for weight loss (see Fig. 27.1D). The BPD-­DS combines a modest SG with an extensive bypass of the small bowel while preserving the pylorus. Preservation of the pylorus theoretically decreases the risk of developing dumping syndrome and reactive hypoglycemia.15,16 The BPD-­DS anatomy is a combination of restriction (vertical SG) and malabsorption (extensive intestinal bypass). The malabsorptive component is profound, and its contribution is what creates the most amount of weight loss. The intestinal bypass has a metabolic effect thought to be due to early exposure of nutrients to the ileum leading to increased levels of PYY and GLP-­1.17 The length of the intervening bowel between the alimentary limb-­biliopancreatic limb anastomosis and the cecum, termed the “common channel,” is the main determinant of the degree of malabsorption. As the length of the common channel becomes shorter, the malabsorptive power of the operation increases, and with it the potential weight loss. However, the risks of the adverse effects associated with aggressive malabsorption also increase and can lead to severe macro-­and micronutrient deficiencies, including sometimes fatal protein-­calorie malnutrition. The BPD-­DS is the most effective bariatric operation currently performed, producing the most weight loss and the best rates of diabetes remission.18-­20 However, due mainly to its impact on nutrition, it is associated with higher morbidity compared with other procedures, with a reported 8% to 11% overall complication risk.21 BPD-­DS may be best reserved for patients with body mass index (BMI) greater than 50 kg/m2 or as a revision procedure for patients with inadequate weight loss after SG. Because of the potential for malnutrition after BPD-­DS, this operation should be avoided in patients who are unlikely to be compliant with postoperative dietary guidelines and nutrition supplements. In our experience, those with a heavy burden of mental health issues, lack of stability in their personal lives, and a poor social support system, as well as those who exhibit dubious compliance preoperatively, are not considered candidates in our program.

Single Anastomosis Duodenal Ileostomy Bypass With Sleeve The single anastomosis duodenal ileostomy bypass with sleeve (SADI-­S) is a modification of the classic BPD-­DS and was initially described by Torres over 10 years ago.22 The SG portion of the operation is the

453

same. However, rather than a Roux-­en-­Y configuration of the bypassed small bowel, the SADI-­S has a loop configuration with one anastomosis between the proximal duodenum and the ileum constructed 250 to 300 cm proximal to the ileocecal valve, which eliminates the biliopancreatic diversion portion (see Fig. 27.2E). This operation has recently been endorsed by the American Society for Metabolic and Bariatric Surgery (ASMBS) as effective and safe. It can be offered as a primary bariatric operation or for revision of SG in the setting of weight regain. Early studies have suggested that weight loss and comorbidity outcomes are similar to those of the BPD-­DS. One retrospective study demonstrated 85.7% EWL at 4 years postoperatively, with 97.6% patients being able to maintain hemoglobin A1c (HbA1c) less than 6% (with or without diabetic medications).23 Other retrospective studies have shown more weight loss in BPD-­DS patients compared with SADI-­S patients.24 The postulated advantages over the traditional BPD-­DS include a lower risk of internal hernias and lower risk of malnutrition and diarrhea.25

Laparoscopic Adjustable Gastric Banding The adjustable gastric band (AGB) is a Silastic device that is comprised of a band with an inner balloon attached to tubing that leads to a subcutaneous port (see Fig. 27.2A). Its relatively simple concept, straightforward surgical procedure, low mortality, and reversibility drove its initial popularity to the point where it was the most popular weight loss operation in 2007. However, it has lost favor due to poor long-­term weight loss and unacceptable long-­term complication and reoperation rates, as well as the emergence of the SG as the restrictive operation of choice. The AGB currently comprises approximately 1% of all bariatric operations. KEY POINTS  • The vertical sleeve gastrectomy (SG), a restrictive operation with a neurohormonal component, is currently the most commonly performed bariatric operation in the United States. • The Roux-­en-­Y gastric bypass provides restriction as its main mechanism, coupled with a neurohormonal component. The limited malabsorptive component is not designed to provide protein-­calorie malabsorption as a weight loss mechanism. • The biliopancreatic diversion with duodenal switch and single anastomosis duodenal ileostomy with sleeve gastrectomy are primarily malabsorptive operations and can result in profound protein-­calorie malabsorption. The SG portion of these operations also provides restriction, and there is an associated robust neurohormonal component. • The adjustable gastric band is a purely restrictive operation that currently comprises less than 2% of the bariatric operations being performed.

Endoscopic Bariatric and Metabolic Therapies The burden of obesity is compounded by an insufficient treatment arsenal. Behavioral changes are thwarted by low durability and patient compliance.26,27 Pharmacologic therapies are associated with significant cost and potential adverse events, require lifelong compliance to maintain response, and, given their targeted nature of redundant evolutionary pathways defending body weight, are often inflicted with weight recidivism and attenuation of response.28,29 Bariatric surgery, while associated with significant and sustained weight loss, is limited by its invasiveness, expense, long-­term risks, and patient acceptance.30-­32 These barriers have prevented bariatric surgeries from reaching more than 2% of eligible patients, with the vast majority of patients with mild to moderate obesity not qualifying for surgery.32,33 Furthermore, obesity equally inflicts younger patients, who will likely outlive the durability of bariatric surgery and require additional revision surgery, which significantly compounds the short-­and long-­term risks and cost.34,35 This landscape has provided an opportunity for

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PART 3  Obesity and Nutrition

Gastric EBMT Weight loss dependent pathways

Small bowel EBMT Weight loss dependent and independent pathways

Improved satiation Improved satiety Alteration in gastric accomodation Alteration in gastric emptying Improved insulin resistance

Improved satiety Regeneration of gut mucosal barrier Gut microbiome Gut neurohormonal signaling Improved insulin resistance Bile acid signaling

Improved insulin sensitivity

Improved gut dysbiosis + Improved leaky gut + Decreased metabolic endotoxemia

Alteration in bile acid signaling

Figure 27.3  Proposed pathways for effectiveness of endoscopic bariatric and metabolic therapies (EBMT).

minimally-­invasive and anatomy-­preserving endoscopic bariatric and metabolic therapies (EBMTs) to address the gaps in obesity care and increase access to effective therapies capable of producing significant weight loss and improvement in excess adiposity–related comorbidities.36 Recognizing the noncurable, chronic, relapsing nature of obesity, an evolving management strategy pivoting on an intensive weight loss intervention utilizing EBMT for those with moderate forms of the disease is needed. The goal of such a strategy is to produce enough upfront weight loss that the patients will engage in lifelong weight stabilization and reintensification efforts, similar to any other chronic disease, where interventions are layered and sequenced for effective disease management.37 EBMTs are generally considered in patients who do not qualify for or who wish to pursue bariatric surgery, especially in those with class 1–2 obesity (BMI 30–40 kg/m2) who have previously failed to lose weight using lifestyle methods. Furthermore, EBMTs can be utilized as a bridge to definitive bariatric surgery in patient with very high BMIs and prohibitive surgical risk.38-­40 Other indications include: 1. Prior to organ transplantation, allowing pretransplant candidates to meet transplant eligibility.41-­43 2. Weight reduction prior to orthopedic procedures and for infertility treatment.44 3. For management of obesity-­related comorbidities such as nonalcoholic steatohepatitis and type 2 diabetes.45-­48 EBMT approaches are thought to target peripheral and central gastrointestinal pathways implicated in the regulation of energy intake and can be divided based on their anatomic manipulation into gastric or small intestinal interventions (Fig. 27.3).49 1.  Gastric interventions include space-­ occupying devices that most commonly take the form of temporarily placed prostheses (intragastric balloons [IGBs]). The TransPyloric Shuttle (BAROnova Inc,

Goleta, CA) intermittently seals the pyloric channel and delays gastric emptying in the fed state to induce early satiation and prolonged satiety. The Full Sense Device (BFKW, Grand Rapids, MI) is a modified, fully covered gastroesophageal stent with a cylindrical esophageal component and a gastric disc connected by struts that is thought to induce weight loss by exerting constant gentle, pressure on the gastric cardia, thereby triggering afferent vagal signaling to the central nervous system and a sensation of fullness. Additional approaches that target the stomach include gastric remodeling techniques that reduce the gastric reservoir by endoscopically creating a tubular sleeve along the greater curvature of the stomach through transoral suturing (Overstitch, Apollo Endosurgery, Austin, TX) or plication (POSE, USGI Medical, San Clemente, CA) and aspiration therapy that allows patients with obesity to dispose of a portion of their ingested meal via a specially designed percutaneous gastrostomy tube, known as the A-­Tube (Aspire Bariatrics, King of Prussia, PA). 2. Small bowel interventions: it has been hypothesized that the proximal small intestine plays a role in the pathogenesis of metabolic disease, resulting in insulin resistance and chronic inflammation. The pathways that have been proposed include alteration of neurohormonal signaling, changes in the gut microbiome and mucosal barrier function, and activation of innate immune reactions caused by an excess of free fatty acids, bacterial lipopolysaccharides, chemokines, and cytokines.50 The approaches that have been designed to address these pathways include the exclusion of the proximal small intestines by impermeable polymer duodenojejunal bypass liners (EndoBarrier, GI Dynamics, Lexington, MA; Metamodix, Minneapolis, MN) and ablative duodenal resurfacing techniques that regenerate the proximal small intestinal mucosal barrier by thermal or nonthermal ablation methods (Fractyl Laboratories, Cambridge, MA). Some of these therapies have shown promise in the

CHAPTER 27  Bariatric Procedures and Operations management of obesity and metabolic disease. Another theory is that primary and secondary bile acid concentrations and composition in the intestinal, portal, and systemic circulation play roles in insulin secretion and sensitivity, metabolic rate, liver lipogenesis and inflammation, and liver fibrosis progression through FXR/ fibroblast growth factor (FGF)15 and FGF19/TGR5 signaling.51 In hopes of favorably altering bile acid profiles, endoscopic treatments have been developed that deploy self-­assembling magnets (GI Windows, Boston, MA) to create a dual-­path enteral bypass between the proximal jejunum or duodenum and ileum in order to partially divert bile to the terminal ileum.

EVOLVING EVIDENCE PROFILE FOR THE USE OF ENDOSCOPIC BARIATRIC AND METABOLIC THERAPIES IN CLINICAL PRACTICE Space-­Occupying Devices Space-­occupying devices can be divided into balloon and nonballoon devices. At present time of this publication, there are four US Food and Drug Administration (FDA)-­approved space-­occupying devices (ReShape Duo, Orbera, Obalon, and the Transpyloric Shuttle). The ReShape Duo balloon is no longer being marketed. Two additional IGBs (Spatz3 and Elipse) have completed pivotal US regulatory trials and are awaiting approval in the United States. 1. Orbera is the most-­studied IGB for obesity. The device is a single fluid-­filled spherical balloon system that requires endoscopy to confirm placement. Once placed in the stomach, the balloon is filled with 450 to 700 mL of saline with optional methylene blue to detect spontaneous deflation. At 6 months, the balloon requires endoscopic removal. Balloon intolerance was reported to be 7.5%.52 A prior metaanalysis of 55 studies that included over 6500 Orbera IGB placements demonstrated a pooled estimate of 13.2% TBWL at 6 months and 11.3% at 12 months.53 In a pivotal US multicenter randomized controlled clinical trial, investigators compared subjects (n = 125) who received 6 months of balloon therapy plus lifestyle intervention with those who received lifestyle intervention alone (n = 130). At time of removal (6 months), the %TBWL in the Orbera group (n = 116) was 10.2 ± 6.6% versus 3.3 ± 5% in the control group (n = 99). At 12 months, patients in the balloon arm maintained more than 70% of their weight lost at 6 months.52 In a metaanalysis of over 65 studies, accommodative symptoms after placement, such as abdominal pain and nausea, were frequent side effects, occurring in 29% to 34% of patients, but these symptoms mostly dissipated after a few days of therapy. Serious side effects such as migration and gastric perforation were 1.4% and 0.1%, with 50% of the perforations occurring in those with prior gastric surgery, which is a contraindication for placement in the United States.53,54 2. The Obalon balloon is a gas-­filled balloon system packaged within a gelatin capsule. The capsule contains a self-­sealing valve that is connected to a thin catheter extending from the stomach to the mouth when the capsule is ingested. Capsule entry into the stomach is verified by fluoroscopy or a percutaneous locating device that does not use radiation; upon entry into the stomach, the gelatin dissolves, freeing the balloon. The catheter is then used to inflate the balloon with a nitrogen gas mixture. The inflated balloon detaches from the catheter and remains in place until it is removed endoscopically after 12 to 26 weeks. Up to three capsules can be swallowed during the same session, or preferably sequentially throughout therapy as weight loss plateaus. In a US pivotal multicenter randomized sham-­controlled clinical trial, investigators

455

compared 198 patients receiving up to three consecutive balloon capsules plus lifestyle intervention to 189 patients receiving sham capsules in addition to lifestyle intervention. %TBWL in the IGB balloon arm was 6.6 ± 5.1% at 6 months from the first swallowed capsule compared with 3.4 ± 5.0% in the control group. The safety profile of the Obalon IGB system is favorable. In the pivotal trial, no unanticipated adverse device events occurred, and only one serious adverse event occurred, which was a gastric ulcer in the setting of nonsteroidal antiinflammatory medication use. Transient mild to moderate nausea and abdominal pain were common and self-­ limited, with the vast majority of patients completing therapy.55 3. Spatz is an adjustable fluid-­filled IGB placed endoscopically. The balloon has an extractable thin, flexible tube that allows for endoscopic volume adjustment while the balloon is in place. The balloon volume may be decreased to improve tolerability or increased to aid in increased weight loss. Prior generations of the Spatz adjustable balloon resulted in up to 20% TBWL at 12 months but had device-­ related complications.56 The new-­ generation device overcomes these design flaws and is approved for 12-­month placement outside of the United States. A US multicenter pivotal trial randomized 288 participants (187 IGB and 101 open-­label control). At 32 weeks the IGB cohort achieved 14.9 ± 7.2% TBWL compared with 3.6 ± 5.8% in the control group, for a difference of 11.3%([97.5% lower confidence bound 9.1%). The serious adverse events rate was 5.3%, and these events were mostly related to persistent accommodative gastrointestinal symptoms.57 The device is currently awaiting regulatory approval. 4. The Elipse balloon is the first procedureless IGB that does not require an endoscopy to place or to remove. It is administered by swallowing a capsule with an attached microcatheter through which the balloon is filled once the balloon capsule is confirmed to be within the stomach on an abdominal x-­ray. The balloon subsequently self-­deflates after degradation of its valve mechanism, permitting release of balloon contents and the entire device and contents are expected to pass through the gastrointestinal tract and be excreted spontaneously in the stool approximately 4 months after administration. In a systematic review and metaanalysis including 2013 unique patients with mild to moderate obesity, the pooled early removal rate was 2.3% (95% confidence interval [CI]: 1.1%–3.5%; I2 31%) and pooled %TBWL after completion of treatment (4–6 months) was 12.8% (95% CI: 11.6%–13.9%; I2 83%) and at 12 months was 10.9% (95% CI: 5.0%–16.9%, I2 98%). Three patients in this study developed small bowel obstruction with balloon exit, and one patient had gastric perforation requiring surgery.58 A pivotal US regulatory multicenter randomized sham-­ controlled trial has concluded, and the balloon is awaiting regulatory approval. 5. The TransPyloric Shuttle consists of a large spherical bulb attached to a smaller cylindrical bulb with a flexible tether. The device is delivered and removed through an overtube with endoscopic guidance. The large bulb remains in the stomach, while the smaller bulb can traverse to the duodenum during peristalsis. It creates an intermittent gastric outlet obstruction to delay the gastric emptying of ingested meals and induce satiety. The FDA approved this device in April 2019 for use in patients with a BMI of 30 to 40 kg/m2 for 12 months. A US pivotal multicenter randomized double-­blinded sham-­controlled trial (ENDobesity II) involving 270 patients (181 TransPyloric Shuttle and 89 sham) led to this approval. At 12 months, the %TBWL was 9.5% for the TransPyloric Shuttle group, compared with 2.8% in the sham group. The incidence of serious adverse events in the TransPyloric Shuttle group was 2.8%, in which one patient experienced esophageal rupture that occurred

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PART 3  Obesity and Nutrition

A

ESG

B

POSE1

C

POSE2

Figure 27.4  Gastric contour following different endoscopic gastric remodeling techniques. A, Endoscopic sleeve gastroplasty. B, Primary obesity surgery endoluminal 1. C, Primary obesity surgery endoluminal 2.

during an unsuccessful delivery attempt. The early removal rate due to adverse events was14.8%.(https://www.accessdata.fda.gov/ cdrh_docs/pdf18/P180024B.pdf)

Gastric Remodeling Techniques

Endoscopic Sleeve Gastroplasty. Endoscopic sleeve gastroplasty (ESG) is an incisionless gastric volume reduction technique that creates a restrictive endoscopic sleeve. This endoscopic sleeve is achieved using a commercially available full-­thickness suturing device (OverStitch; Apollo EndoSurgery, Austin, TX, USA) by placing a series of triangular full-­thickness sutures through the gastric wall along the greater curvature of the stomach from the prepyloric antrum to the gastroesophageal junction, leaving a small pouch in the fundus (Fig. 27.4A).59 In a systematic review and metaanalysis of eight studies involving 1776 patients with a follow-­up of up to 2 years, the body weight loss at 6, 12, and 18 to 24 months was 15.1%, 16.5%, and 17.2%, respectively. The reported rate of serious adverse events was 2.2%.60 Although most studies evaluated the impact of ESG on body weight, two evaluated its association with important metabolic parameters.61,62 Sharaiha and colleagues observed a significant improvement in HbA1c, systolic blood pressure, triglycerides, and alanine aminotransferase 12 months post-­ESG.61 In another study, all patients with hypertension and 76.5% of patients with early type 2 diabetes mellitus achieved remission of these entities.62 Limited longer-­term studies have reported outcomes beyond 1 year.63 However, beyond sustained weight loss, the postprocedural anatomy has not been routinely assessed beyond the immediate postprocedural phase. It is likely that considerable variability may be present, as the techniques, suture patterns, and follow-­up protocols continue to evolve. Primary Obesity Surgery Endoluminal. In its first iteration, primary obesity surgery endoluminal (POSE) used a peroral incisionless operating platform (USGI Medical, San Clemente, CA) to place transmural tissue anchor plications that reduce accommodation of the gastric fundus. Three additional plications are placed in the distal gastric body to delay gastric emptying (see Fig. 27.4B). POSE has been associated with both improved satiety, indicating a gut neurohormonal response.64 A pivotal US multicenter randomized blinded clinical trial that compared 221 patients receiving POSE in addition to a low-­intensity lifestyle intervention for 12 months with 111 patients receiving lifestyle intervention alone failed to meet its primary efficacy endpoints, with a reported weight loss at 12 months for the POSE group of 5 ± 7% compared with 1.4% ± 5.6% in the control group.65 An alternative plication method, POSE2.0, where plications are placed in the gastric body between the gastroesophageal junction and

level of the incisura, with none placed in the fundus, thereby generating a small food reservoir in the fundus with a tubular configuration of the remainder of the stomach, is being investigated, with encouraging early results (see Fig. 27.4C).66

Aspiration Therapy Aspiration therapy (AT) is another endoscopic treatment approach involving partial disposal of an ingested meal to reduce overall caloric intake through a modified percutaneous endoscopic gastrostomy (PEG) tube. This device is FDA-­approved for use in the United States in patients with a BMI between 35 and 55 kg/m2, a higher BMI range than for IGBs, for up to 5 years. It requires endoscopic placement and removal, unlike standard PEG tubes. The AT process uses a specifically designed percutaneous gastrostomy tube (A-­Tube, Aspire Bariatrics, King of Prussia, PA) made of silicone. The tube is inserted in a similar fashion as standard PEG tubes. After 2 weeks, the A-­tube is shortened to the level of the skin, and the skin port is attached. The skin port allows attachment to the aspiration device, which fits in a small zippered bag. Once the device is connected to the skin port, a water reservoir flushes tap water into the stomach to facilitate disposal of food. Patients are instructed to perform aspiration approximately 20 minutes after ingesting a meal, removing and discarding approximately one third of the meal. The process takes approximately 10 to 15 minutes to complete. The device has a built-­in counter that locks the device to avoid overuse. In a US pivotal multicenter randomized controlled trial, 111 subjects underwent AT with lifestyle intervention for 12 months compared with 60 subjects who underwent lifestyle intervention alone. At 12 months, %TBWL for those who completed AT (n = 82) was 14.2% ± 9.8% compared with 4.9% ± 7% in the control group (n = 31). Because aspiration removes around 30% of ingested calories, it is estimated that AT accounts for 80% of the reported weight loss. AT requires patients to chew food thoroughly and eat slower, potentially leading to new eating behaviors. As far as safety, the adverse events included stoma granulation tissue (40.5%), stoma infection (14.4%), peritonitis (0.9%), and gastric ulcer (0.9%). Careful review of disordered eating behavior questionnaires found no evidence of worsening eating behaviors with therapy.67

Small Bowel Intervention

Duodenal-­Jejunal Bypass Liner. The Endobarrier (GI Dynamics, Lexington, MA) is a 65-­ cm-­ long Teflon-­ coated duodenal jejunal bypass sleeve. It is anchored at the duodenal bulb by an anchoring crown and terminates at the proximal jejunum, allowing ingested nutrients to bypass the duodenum to the jejunum, thereby altering proximal duodenal interaction with ingested nutrients and subsequent

.

l

457

CHAPTER 27  Bariatric Procedures and Operations

Insulin-sensitive

Insulin-resistant

Altered microbiome

Mucus

LPS/ inflammation

Mucus Goblet cell

Healthy microbiome LPS/ inflammation Glucose Goblet cell Enteroendocrine cell

Enterocyte cell Gut hormones

Glucose

Enterocyte cell

Enteroendocrine cell Vagal neuron Spinal neuron

Proliferation zone

Enteric neuron Proliferation zone Paneth cell

Stem cell

Stem cell

Paneth cell Capillaries Nerves Figure 27.5  Intestinal adaptation to excess caloric intake. These hypothesized pathophysiologic adaptations have been targeted by endoscopic technologies capable of resurfacing the mucosal layer of the duodenum in an attempt to reset the abnormal metabolic signaling and improve insulin sensitivity.

gut hormonal and enteric nervous system response. This device is placed and removed endoscopically and remains in the small intestine for 12 months. A US pivotal multicenter randomized sham-­controlled trial (the ENDO trial) was terminated early after enrollment of 325 of 500 patients because of a 3.5% incidence of hepatic abscess. All were managed conservatively, but this was outside the predefined adverse event rate. An analysis of published trials of this device identified two studies that included a sham control arm with a total of 77 volunteers who received treatment for 12–24 weeks. The analysis was specifically aimed at assessing the effects of this approach on T2DM outcomes; for these two trials there was not a significant improvement in HbA1c between intervention and control studies. A redesigned clinical trial of the same device with a modified protocol designed to reduce the likelihood of adverse events has been approved by the FDA to begin enrolling patients. Furthermore, a newer atraumatic bypass liner system is being developed.

Duodenal Mucosal Resurfacing. Intestinal adaptation to excess caloric intake and states of metabolic disease has been hypothesized and is illustrated in Fig. 27.5.50,68,69 These pathophysiologic adaptations have been targeted by endoscopic technologies capable

of resurfacing the mucosal layer of the duodenum in an attempt to reset abnormal metabolic signaling and improve insulin resistance. Duodenal mucosal resurfacing (DMR; Fractyl, Lexington, MA), or the Revita procedure, is an endoscopic procedure that involves circumferential hydrothermal ablation of the duodenal mucosa. This procedure is primarily aimed at glycemic control. Though the precise mechanism is not yet defined, it is hypothesized that DMR induces remodeling of the diseased duodenum with an overgrowth of enteroendocrine cells and dysregulated secretion of gastrointestinal hormones, which have been observed in patients with diabetes. The first human study of 39 patients with type 2 diabetes demonstrated a 1.2% reduction of HbA1c and a 2.5-­kg weight reduction at 6 months. A longer segment ablation resulted in more potent glycemic control. Three patients developed duodenal stenosis, which was successfully treated with endoscopic dilation.70 In a double-­blind, randomized controlled trial (56 DMR, 52 sham) of patients with poor glycemic control despite greater than or equal to one stable oral antidiabetic medications with 24 weeks’ follow-­up, the median HbA1c decreased by 10.4 mmol/mol in the DMR group versus 7.1 mmol/mol in sham group (P = 0.147). Because of the heterogeneity between the two cohorts participating in the trial (European vs. Brazilian) the

458

PART 3  Obesity and Nutrition

groups were analyzed separately, as prespecified in the statistical analysis plan. In the European modified intention to treat cohort (39 DMR, 37 sham) the median HbA1c change was –6.6 mmol/mol versus –3.3 mmol/mol post-sham (P = 0.03). The Brazilian cohort showed a statistically nonsignificant trend towards DMR benefit in HbA1c. Patients with high baseline fasting plasma glucose, greater than or equal to 10 mmol/L, had significantly greater reductions in HbA1c post-­DMR versus sham (P = 0.002). One jejunal perforation was observed in this study requiring surgical repair.47 A US multicenter randomized sham-­controlled trial of hydrothermal ablation is underway. A newer ablation technique, utilizing electroporation in hopes of reducing serious adverse events and to enable safe re-treatment of the duodenum, is currently in early human trials.

Incisionless Magnetic Anastomosis System. The Incisionless Magnetic Anastomosis System (GI Windows, West Bridgewater, MA) consists of two self-­assembling magnets that are deployed, one each in the proximal jejunum by enteroscopy and in the ileum by colonoscopy, with ileoscopy creating jejunoileal anastomosis, which results in partial jejunoileal bypass. The coupled magnets then pass spontaneously after a week. The first human proof-­of-­concept study involving 10 patients demonstrated a %TBWL of 14.6% and a significant reduction of HbA1c of 1.9% at 12 months utilizing laparoscopic assistance and monitoring. All anastomoses were patent at a 12-­month follow-­up.71 Special Considerations and Future Directions. The magnitude and duration of weight loss varies by EBMT device and technique. Therefore, EBMTs should be administered with the support of a comprehensive team of primary care providers, gastroenterologists, nutritionists, dieticians, and behavioral therapy professionals. Moving forward, personalized EBMTs targeting clinical phenotype and responders’ profiles will be imperative. The sequential and tandem use of EBMTs in conjunction with antiobesity pharmacotherapy and comprehensive lifestyle interventions is an ongoing area of research, which may yield a robust and durable weight loss response, achieving a lasting effect on obesity and its comorbidities. In a proof-­of-­concept propensity score-­matched study of 52 patients, those receiving endoscopic sleeve gastroplasty with liraglutide achieved 25% TBWL at 12 months (7 months after the initiation of liraglutide) compared with 21% in the ESG-­only group.72 KEY POINTS  • Endoscopic bariatric and metabolic therapies (EBMT) are minimally invasive peroral management options that could augment the treatment spectrum for obesity and metabolic disease. • EBMTs are generally considered in patients who do not qualify for or wish to pursue bariatric surgery, especially in those with class 1–2 obesity who have previously failed to lose weight using lifestyle methods. • EBMTs include gastric space–occupying devices, gastric remodeling techniques, and small bowel interventions targeting metabolic pathways. • EBMTs should be administered with the support of a comprehensive multidisciplinary team. The sequential and tandem use of EBMTs in conjunction with antiobesity pharmacotherapy and comprehensive lifestyle interventions is an ongoing area of research.

Eligibility for Bariatric Operations and Procedures According to the current National Heart, Lung, and Blood Institute guidelines, candidates for bariatric surgery in the United States include patients with a BMI greater than 40 kg/m2 or with a BMI of 35 to 39.9 kg/m2 and one or more obesity-­associated comorbidity

(type 2 diabetes, cardiovascular disease, sleep apnea, dyslipidemia, hypertension, GERD, or osteoarthritis).72a These indications, which were established more than 25 years ago, may expand as more societies adopt this safe and durable weight loss option. In 2018, the ASMBS published a statement on the role of metabolic surgery in class 1 obesity. The issued statement supported lowering the BMI cutoff of 35 kg/m2 to 30 kg/m2 in consideration of the high-­quality supporting data available.73 At the very least, prospective patients must undergo a thorough preoperative evaluation by a bariatric surgeon, a dietician, and a psychologist. An endocrinologist’s expertise is often necessary to assist in managing the metabolic complications of obesity. This multidisciplinary approach ensures consideration of the “whole patient,” thus leading to favorable and long-­lasting outcomes that are not solely dependent on behavioral changes induced by the operation. KEY POINTS  • To be eligible for bariatric surgery or endoscopic bariatric procedure, patients must have a body mass index (BMI) >40 kg/m2 or a BMI of 35–39.9 kg/m2 and one or more obesity-­associated comorbidity (type 2 diabetes, cardiovascular disease, sleep apnea, dyslipidemia, hypertension, gastroesophageal reflux disease, or osteoarthritis). • There is mounting evidence supporting the application of bariatric operations and procedures for class 1 obesity (BMI 30.0–34.9 kg/m2), particularly for patients in this weight class with type 2 diabetes mellitus.

Assessing Outcomes after Bariatric Interventions The two main outcomes important in determining the success or failure of bariatric operations and procedures are: 1) weight loss and 2) comorbidity resolution. Many previous bariatric surgery studies measured outcomes as percent EWL, which is defined as the percent change from excess to ideal body weight. However, %TBWL is currently preferable, because this standardization is more easily comparable to the medical literature. %TBWL has the lowest variation coefficient and best describes weight loss changes over time for patients in different weight categories.56,74 However, the data obtained from %TBWL may not accurately reflect the clinical benefit. For example, someone with greater excess weight will need to lose more weight to achieve the clinical benefits. For this reason, %EWL is sometimes preferred. Weight loss is greatest after BPD-­DS, and is generally in the range of %TBBL 30% to 40%, %EWL 70% to 90%, followed by RYGB (%TBL 28%–32%, %EWL 65%–80%) and SG (%TBL 25%–32%, %EBL 50%– 65%). Recent randomized studies comparing the RYGB with the SG have demonstrated equivalent weight loss at 5 years.75,76 However, it is generally believed that the RYGB results in superior weight loss compared with the SG over the long term. It should be remembered that weight loss outcomes depend on multiple factors, perhaps most importantly long-­term compliance to behavioral modifications, resulting in variable outcomes. Generally, the operations with the greatest weight loss also carry the greatest risk for surgical complications, including the development of nutritional deficiencies. Determining which operation is most appropriate for a specific patient depends on several factors, most importantly which diseases need treatment, in addition to the degree of obesity. An analysis of the risks and benefits of each operation for each patient should be undertaken that incorporates medical comorbidities, weight loss needed to obtain health goals, likelihood of patient compliance, and patient preference.

CHAPTER 27  Bariatric Procedures and Operations

THE PHYSIOLOGY OF WEIGHT LOSS AFTER BARIATRIC OPERATIONS AND PROCEDURES Weight Loss Mechanisms All commonly performed bariatric procedures involve a restrictive component that may or may not impact intestinal nutrient handling. Thus, in addition to caloric restriction, altered gastrointestinal anatomy may affect the entero-­neuro-­endocrine axis involving signaling from multiple gut hormones and metabolites that influence appetite and satiety through endocrine or neural pathways to a varying degree following different bariatric procedures (see Fig. 27.3).

Caloric Restriction Caloric restriction creates an energy deficit that leads to mobilization of stored energy and weight reduction. The rate and magnitude of weight reduction are related to the degree of energy deficit produced, with very low-­calorie diets ( T4 (∼8.0), rT3; T3S > of BBB, kidney (AM T4S; rT3S of distal nephron), liver (cholangiocytes), lung, ciliary body, placenta (syncytiotrophoblasts)

OATP1B1

SLCO1B1

OATP-­C, LST-­1, OAPT2, OATP6

Liver (BLM of hepatocytes)

T4S, T3S, rT3S > rT3 >T4 (∼3.0), T3 (∼2.7)

n.d.

OATP1B3

SLCO1B3

OATP8, LST-­2

Liver (BLM of hepatocytes, central vein>portal vein)

T3 (∼6.4) > T4

n.d.

OATP1C1

SLCO1C1

Brain (glial and neuronal (precursor) cells), BBB, choroid plexus, testis (Leydig cells), ciliary body, adipose tissue

T4 (∼0.09-­0.12) >T4S (∼3) > rT3 (∼0.13) > T3

n.d.

OATP2B1

SLCO2B1

OATP3A1 (two protein isoforms: V1 and V2)

SLCO3A1

OATP4A1

SLCO4A1

OATP4C1

SLCO4C1

OATP-­F, OATP-­RP5 (human), BSAT1 (rat), Oatp2 (mouse), Oatp14 OATP-­B, OATP-­RP2 (human), moat1 (rat), Oatp9 OATP-­D, OATP-­RP3 (human), Pgt2 (rat), MJAM (mouse), Oatp11 OATP-­E, OATP-­RP1 (human), oatpE (rat), Oatp12 OATP-­H

Ubiquitously (including [T4 (∼0.31-­0.77)] hepatocytes (BLM) syncytiotrophoblasts (BLM), enterocytes (AM, BLM), BBB (AM) Brain (white/grey mat- T4 ter, choroid plexus), testes, ciliary body, heart, ovary

Other Substrates (Km) Inhibitor Tauroursodeoxycholate (∼19 μM), DHEAS (∼7 μM), cholate (∼93 μM), taurocholate (∼60 μM), cholate (∼93 μM), BSP (∼20 μM), E3S (∼16-­59 μM), ouabain, N-­methyl quinine, PGE2, glycocholate BSP (∼0.3 μM), cholate (∼11 μM), taurocholate (∼10-­ 34 μM), glycocholate, E3S (various), DHEAS (∼22 μM), PGE2, E217βG (∼8.2 μM) Bilirubin (∼39 nM), BSP (∼0.4 μM), cholate (∼42 μM), taurocholate (∼6-­42 μM), glycocholate (∼43 μM), E3S (∼73 μM), methotrexate (∼25 μM), DHEAS, ouabain, digoxin, E217βG, PGE2 E3S, BSP, E217βG, taurocholate

BSP, rifampicin, flavanones, polymethoxyflavanones

n.d.

BSP (∼0.7 μM), E3S (∼6.3 μM), DHEAS

BSP, flavanones, polymethoxyflavanones

n.d.

E3S, PGE2 (∼0.22-­ 0.37 μM), vasopressin

Ubiquitous (except gastrointestinal tract and brain)

T3 (∼0.9)> rT3>T4

n.d.

Taurocholate (∼14.9 μM), PGE2

Kidney

T4, T3 (∼0.9)

T3: no T4: n.d.

Digoxin, ouabain, methotrexate

BSP, flavanones, polymethoxyflavanones

BSP

BSP

BSP

Continued

1114

PART 6  Thyroid

TABLE 66.1  Characteristics of Human Thyroid Hormone Transporters—cont’d. Tissue/Cells and Subcellular Localization

Substrate (Km Substrate in μM) Uptake Efflux

Transporter

Gene Name Aliases1

NTCP

SLC10A1

SLC17A4

SLC17A4

LAT1

SLC7A5

LAT2

SLC7A8

Kidney, placenta, brain, 3,3’-­T2 > T3; MIT spleen, prostate, testis, ovary

None

LAT3

SLC43A1

Placenta, liver, kidney, pancreas, skeletal muscle, heart

None

3,3’-­T2, MIT, (rT3)

LAT4

SLC43A2

None

3,3’-­T2, MIT, (rT3)

MCT8

SLC16A2

XPCT, MCT7

MCT10

SLC16A10

TAT1

Placenta, leukocytes, skeletal muscle, spleen, kidney and heart Developing and adult brain: vascular endothelial cells of BBB, choroid plexus, liver, kidney, thyroid, pituitary Kidney, skeletal muscle, placenta, heart, developing brain, hypothalamus, choroid plexus

1Ambiguous

NPT homolog

Liver (basolateral mem- T4S, T3S > T4, T3 brane of hepatocytes)

No

Gastrointestinal tract, liver, pancreas, kidney Brain, placenta, testis, leukocytes, fetal liver, bone marrow

T3, T4

n.d.

3,3’-­T2 > rT3 > T3 > T4; MIT (∼13)

3,3’-­T2

T3 (∼7.5 μM short T3, T4 isoform, ∼1 μM long isoform), T4 (∼3 μM) > rT3 > 3,3’-­T2 > 3,5-­T2 >3’,5’-­T2 T3 >3,5-­T2 >>T4, T3 rT3

Other Substrates (Km) Inhibitor Eprotirome, cholate, taurocholate (∼6-­34 μM), glycocholate (∼27 μM), chenodeoxycholate-­ 3-­sulfate, E3S (∼27-­ 60 μM), DHEAS, BSP (∼3.7 μM) p-­[glycyl-­2-­3H]p-­aminohippuric acid (PAH), (uric acid) Broad spectrum of (neutral) amino acids (excluding Gly, Ala, Ser) (∼12-­ 120 μM), L-­dopa (∼34 μM), pregabalin (∼288 μM), IMT (22.6 μM) Broad spectrum of (neutral) amino acids (including Gly, Ala, Ser) (∼35–200 μM) Neutral amino acids (Leu, Ile, Val, Phe > Met, Tyr > others) (∼8–30 μM) Neutral amino acids (Ile, Leu, Met, Phe >>> others) (∼100–200 μM) -­

Phe, Tyr, Trp (∼400–700 μM), L-­dopa (∼1.2 mM)

Myrcludex B, BSP, furosemide

n.d.

BCH, JPH203

BCH, NEM

BCH, NEM

BCH, NEM

Silychristin

Trp

protein names (similar names used for distinct proteins, tissue-­specific names) are indicated in bold. LST, Liver-­specific transporter, OATP, organ anion transporting polypeptide; OATP-­RP, organ anion transporting polypeptide–related protein; BSAT, brain-­specific anion transporter; E3S, estrone-­3-­sulfate; PGE2, prostaglandin E2; E217βG, 17β-­glucuronosyl estradiol; IMT, 3-­iodo-­α-­methyl-­L-­tyrosine; NEM, N-­ethylmaleimide; XPTC, X-­Linked PEST-­containing transporter; TAT, T-­type amino acid transporter; BLM, basolateral membrane; AM, apical membrane; BSP, sulfobromophthalein; BBB, blood-brain barrier; BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; DHEAS, dehydroepiandrosterone sulfate; MIT, monoiodotyrosine. If the transport of different iodothyronines has been studied in parallel, the substrates are listed in order of apparent substrate preference with a , (comma sign) denoting equal efficacy, and > denoting greater efficacy. If the transport of iodothyronines has not been studied in parallel, a ; (semicolon sign) is used, and substrates are not ranked in a specific order.

CHAPTER 66  Thyroid Hormone Transporters and Metabolism have been helpful to dissect their role.8 Table 66.2 provides the main features of Dio knockout (KO) mice models. Dio1 null mice exhibit raised T4 and rT3, with normal T3 and thyroid-­stimulating hormone (TSH) concentrations.9 The Dio2 null mice has high T4 and TSH but

DIT + I + hydroquinone T4-glucuronide

Ether link cleavage

UGT

I

I

HO SULT T4-sulfate

O I

CH2-CH-NH2 I

DIO1, DIO2

COOH

DIO1, DIO3

? -CH2-COOH TA4

T3 rT3 Fig. 66.1  Pathways of thyroid hormone metabolism. Deiodination by the three types of deiodinating enzymes (DIO1, DIO2, DIO3) catalyzes removal of iodine moieties. Sulfation and glucuronidation are catalyzed by sulfotransferases (SULTs) and UDP-­glucuronosyltransferases (UGTs), respectively. Ether link cleavage and side-­chain modification result in alternative products.

1115

normal T3 concentrations and shows growth retardation and defective auditory function.10,11 Murine Dio3 deficiency is associated with partial embryonic and neonatal lethality, and surviving mice show severe growth retardation, impaired fertility, and central hypothyroidism.12 The phenotypes of the murine Dio1 and Dio2 double KO mice are surprisingly mild, with serum T3 level, general health, and reproductive capacity apparently not affected, and brain function only somewhat impaired.13 However, the serum rT3 is elevated 6-­fold in the Dio1/Dio2 null mouse, while only 2-­fold in the Dio1 knock-­out mouse and not at all in the Dio2 KO mouse. A mouse model with complete DIO deficiency, the triple Dio1/Dio2/Dio3 KO, has survival and growth issues that occur mainly in the embryonic and neonatal period, and many of the mice are infertile. However, the healthy surviving Dio1/Dio2/ Dio3 mice have normal growth, can be bred, and have little gross neurological phenotype, and serum thyroid hormone concentrations are unremarkable.8,13 The systemic and localized control of thyroid hormone signaling is essential for development, growth, and normal adult life, requiring dynamic regulation to allow for constant adjustment to thyroid hormone signaling according to endogenous and environmental demands. However, the versatility of the system also can lead to the dysregulation of DIO expression, resulting in abnormal systemic or local thyroid hormone concentrations. A striking clinical condition that affects DIO activity is observed in critically ill patients with a substantial drop in circulating T3, at least in part attributed to changed DIO expression and activity in one or more affected tissues.14,15 Other

TABLE 66.2  Characteristics of Iodothyronine Deiodinases Characteristics

DIO1

DIO2

DIO3

Function

Plasma T3 production rT3 clearance T4 to T3, rT3 to T2 T4 to rT3, T3 to T2 rT3 >>T4=T3 ++++ ++++ +++ 1p32-­p33 Liver, kidney, thyroid, pituitary, white adipose tissue, brain

Local T3 production

T3 and T4 clearance rT3 production T4 to rT3 T3 to T2 T3 >T4 +/-­++ +++

Reaction catalyzed Substrate preference Inhibitor

PTU Aurochioglucose Iopanoic acid

Human chromosomal location Tissues

Activity in hypothyroidism Activity in hyperthyroidism Phenotypic features of knockout mice

↓ Liver, kidney ↑ thyroid ↑ Liver, kidney, thyroid Viable Normal growth Normal fertility ↑ Serum T4 and rT3 in adults Normal serum T3 and TSH in adults Enhanced fecal excretion endogenous iodothyroines

T4 to T3 rT3 to T2 T4 >rT3 + ++ ++++ 14q24.3 Pituitary, brain, brown adipose tissue, skeletal muscle, osteoblast, thyroid, heart ↑ all tissues ↓ most tissues ↑ thyroid Viable Mild growth delay in males Normal fertility ↑ Serum T4 and TSH Normal serum T3 Pituitary resistance to T4 Impaired thermogenesis Impaired hearing Mild impairment neurocognition Insulin resistance Enhanced susceptibility to diet induced obesity

14q32 Fetus, brain, skin, uterus, decidual tissue, placenta, pancreatic β cell ↓brain ↑ brain Increased perinatal mortality Marked growth retardation Impaired fertility Hyperthyroidism in the perinatal period Moderate hypothyroidism in adulthood Impaired responsiveness of the thyroid gland, pituitary gland and hypothalamus Impaired retinal development Impaired hearing

++++: highly sensitive; +++: moderate sensitive; ++: low sensitivity; + very low sensitivity; ↑-­ ↓ increased -­decreased levels. PTU, Propylthiouracil; TSH, thyroid-­stimulating hormone.

1116

PART 6  Thyroid

examples include patients with Graves disease16 and McCune–Albright syndrome,17 in which higher serum T3 concentrations correlated with higher thyroidal DIO1 and DIO2 activities. Changes in DIO activity are also reported in several neoplasias, with depletion or excess of thyroid hormone promoting modifications in tumoral growth and development.2 One of the most striking examples is a severe form of hypothyroidism due to high levels of DIO3 activity in vascular tumors such as hepatic hemangiomas.18 Depending on tumor size and the levels of DIO3 expression, the DIO3 activity may inactivate circulating thyroid hormones faster than the thyroid gland can secrete, resulting in a condition known as consumptive hypothyroidism. The knowledge that DIO activity can affect thyroid hormone concentrations dramatically, influencing patient treatment and potentially outcome, raises the possibility that patients can benefit from genome-­ wide association studies that identify single nucleotide polymorphisms (SNPs) in DIO genes associated with serum thyroid hormone concentrations. These studies show that common variations in DIO1, but not DIO2 or DIO3, have a moderate-­to-­strong relationships with systemic thyroid hormone concentrations.19,20 With regard to identifying SNPs in DIO3, their impact may be affected by genetic imprinting, with DIO3 being preferentially expressed from the paternal allele.21 There are several studies describing an association between DIO2 SNPs and clinical syndromes, including hypothyroidism, hypertension, type 2 diabetes, and mental disorders; however, these associations have not been universally reproduced in all population studies.2,19 This observation confirms the notion that serum thyroid hormone concentrations and related phenotypes are controlled by a multitude of factors, including genetic background, diet, the interplay between gene networks, and environmental factors. While there is increasing evidence that DIO2 polymorphisms may play a role in dysregulation of thyroid hormone metabolism, further research should be performed to clarify these associations. Several independent studies suggest that DIO1 polymorphisms are linked with abnormal thyroid hormone metabolism, and further investigation will determine if this should be taken in consideration for treatment of affected patients.19 The human DIO genes have been subject investigation for a long time; however, the identification of pathogenic genetic defects has remained elusive. Only recently, two unrelated families were identified to be heterozygous for pathogenic variants in DIO122 (see later section on Iodothyronine Deiodinase I Deficiency). All three DIOs have the amino acid selenocysteine (Sec) in their catalytic site, providing enhanced substrate affinity and a fast turnover rate for the deiodination reaction. In humans, more than 25 different Sec-­containing proteins, referred to a selenoproteins, have been identified. The Sec residue is encoded by a UGA triplet that in functions as a stop codon in nonselenoproteins. Accordingly, recoding of UGA, enabling incorporation of Sec during selenoprotein synthesis, depends on the presence of a SElenoCysteine Insertion Sequence (SECIS) element, a stem-­loop structure in the 3′ untranslated region of the mRNA. The SECIS element attracts a number of cofactors, including SECIS-­binding protein 2 (SECISBP2 or SBP2) and Sec-­tRNA, the selenocysteine tRNA encoded by TRU-­TCA1-­1. Genetic defects in this pathway, e.g., SECISBP2, SEPSECS, and TRU-­TCA1-­1, affects generation of most if not all selenoproteins and, consequently, results in a multisystem disorder. Homozygous and compound heterozygous mutations have been described in SEPSECS and are linked with a disorder classified as autosomal recessive pontocerebellar hypoplasia type2D (OMIM 613811), also known as progressive cerebellocerebral atrophy.27-­29 The effect of SEPSECS mutations on selenoprotein expression and thyroid hormone metabolism has not been studied in detail and suggests that, in contrast to SECISBP2 and TRU-­TCA1-­1 mutations, selenoprotein deficiency is not generalized; it is unclear if

thyroid hormone metabolism is affected, and therefore this will not be discussed any further. However, in patients with SECISBP2 and TRU-­ TCA1-­1 mutations, decreased levels of active DIOs mediate abnormal thyroid hormone metabolism, with elevated free T4 (FT4), low or low-­normal free T3, raised rT3, and normal or slightly high TSH concentrations.29 KEY POINTS  • Cellular thyroid hormone regulation is governed at multiple levels: (1) plasma membrane transporters facilitate influx and efflux of thyroid hormones, (2) intracellular metabolism of thyroid hormone is catalyzed by three deiodinating, selenocysteine-­containing enzymes, and (3) genomic actions of thyroid hormone are mediated through binding of 3,5,3’-­tri-­iodothyronine to its nuclear receptor.

DISORDERS OF THYROID HORMONE TRANSPORT MCT8 Deficiency

Clinical Phenotype. Disrupted thyroid hormone transport due to mutations in MCT8 (SLC16A2) underlies the severe clinical features of MCT8 deficiency. In 2004, mutations in MCT8 were linked to patients with an X-­linked form of intellectual and motor disability and abnormal thyroid function tests.30,31 Soon afterwards, it was realized that this disorder had been recognized decades before by Allan, Herndon, and Dudley, and ever since was documented as the Allan–Herndon–Dudley syndrome.32 Over 200 families have been reported, and the estimated prevalence is approximately 1 in 70,000 males.33,34 Most children are born after an uneventful pregnancy and delivery. The age of presenting symptoms is approximately 4 months, with developmental delay, hypotonia, poor weight gain, and feeding problems most commonly reported. At examination, most patients have a global hypotonia, with the inability to hold the head upright, as well as upper trunk slipping through. Dystonic posturing of limbs starts in the first year of life. Both dystonia and spasticity contribute to hypertonia and exaggerated deep tendon reflexes. As a consequence, scoliosis is present in the large majority of cases. Primitive reflexes (e.g., glabellar reflex) do not disappear over time. Electroencephalogram-­proven seizures are present in approximately a quarter of patients. Patients exhibit moderate-­to-­ severe intellectual disability with a pronounced delay in motor and language development (e.g., speech, as defined by the ability to speak one word, is present in less than 10% of cases). Body weight (corrected for age) shows progressive deterioration over time, with the majority of patients being severely underweight (Fig. 66.2A). Gastroesophageal reflux disease is commonly encountered. Blood pressure is frequently elevated. In the context of being immobilized, tachycardia is present in a large proportion of patients, with the frequent occurrence of premature atrial and ventricular contractions (Fig. 66.2B). Also, atrial fibrillation and nonsustained ventricular tachycardia have been reported in affected children. In addition, second-­degree and incomplete right bundle branch blocks have been reported in up to 12% of patients. The median survival is 35 years, with 30% of patients dying in childhood. The main causes of death are pulmonary infections, aspiration pneumonia, and sudden death. There is a strongly increased risk for death in patients not attaining full head control compared with patients who do attain head control (Fig. 66.2C) and for young children (1–3 years of age) who are underweight compared with patients who are of normal body weight. Biochemical evaluation reveals abnormal thyroid function tests. Although TSH concentrations are within the normal range, free and total T4 as well as rT3 concentrations are lower than normal in 90%

CHAPTER 66  Thyroid Hormone Transporters and Metabolism 2

Heart rate

0 Weight (z score)

1117

60

–2

90

100 bpm

120

140

100 1000 Episodes / 24h

10000

–4 Tachycardia

–6

PACs

–8

PVCs

–10 0

5

10 15 Age (years)

A

20

Overall survival (%)

10

Patients Events 95 23 4 23

100

50

HR: 3.46 (95% Cl 1.76-8.34, p=0.004)

0 0 Individuals at risk No head control Head control

1

B

60 20 40 Age at last follow-up (in years)

96

43

10

0

0

23

15

9

5

4

80

C Fig. 66.2  A, Natural course of bodyweight change in patients with MCT8 deficiency. Blue dots represent available historical body weight measurements (n = 300) in 86 untreated patients. B, Mean (standard error of the mean; black lines) occurrence of indicated features during 24-­hour cardiac monitoring. Blue dots represent the measurements of mean heart rate during 24 hours in 47 patients, or the frequency (per 24 hours) of indicated parameter in 45 patients (premature atrial contractions [PACs] and premature ventricular contractions [PVCs]) or 41 patients (tachycardia) with available data. C, Kaplan-­Meier estimates of MCT8-­specific survival in patients who attained head control (red line) by the age of 1.5 years versus those who did not (blue line).

of patients, while total T3 concentrations are above normal in 95% of patients. This results in highly abnormal T3:T4 and T3:rT3 ratios. T4 concentrations obtained from neonatal screening cards were low in the majority of patients, while TSH concentrations were under 15 mU/L in all tested patients. Serum concentrations of sex hormone–binding globulin were elevated in most patients. Serum alanine aminotransferase, aspartate aminotransferase, and glutamyl transferase concentrations were mildly elevated in a large proportion of patients. Magnetic resonance imaging (MRI) scans of the brain consistently show a global delay in myelination,33,35 which improves over age. In addition, diffuse atrophy is present with concomitant dilatation of the ventricles. Magnetic resonance spectroscopy shows an increased choline peak and a decreased N-­acetyl aspartate peak, which is compatible with aberrant myelination and general atrophy.

Molecular Genetics. The SLC16A2 gene is located on the X chromosome and encodes two possible translation start sites, resulting in a long MCT8 isoform of 613 amino acids or a short isoform of 539 amino acids. The relevance of the extended intracellular N-­terminal part of the long isoform is unclear. MCT8 consists of 12 TMDs that are organized in two symmetric bundles of six TMDs linked through

a large intracellular loop.1 Approximately half of the mutations are missense mutations, all of which are located in the TMDs. The remainder of the mutations include deletions and nonsense mutations resulting in truncated proteins. An overview of mutations is shown in Fig. 66.3. For certain mutations (e.g., large deletions, early premature stop codons) it can be inferred that no functional MCT8 is produced. In vitro thyroid hormone transport capacity assessment of missense mutations indicates that the majority render the transporter fully inactive, with some mutations showing residual transport activity.1,36 Although the definitive genotype–phenotype relationship remains to be elucidated, individuals that have MCT8 mutations with residual thyroid hormone transport appear to have a less severe phenotype, including less affected motor and cognitive abilities. Female carriers have FT4 concentrations between those seen in affected patients and healthy individuals,37 and the majority appear asymptomatic. However, the occurrence of minor symptoms in female carriers has never been systematically studied and thus remains speculative. MCT8 deficiency mainly occurs in males due to the X-­linked inheritance. In rare cases, traits of MCT8 deficiency can be present in females as a result of chromosomal translocations and unfavorable nonrandom X inactivation in female carriers.38

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PART 6  Thyroid

Del ex2-6 (2)* Del ex2-4 Del ex3-4 (2) Del ex3-5 (1) Del ex3

Del ex1 (5)* 5’

TLS1 TLS2

I

II

Exon 1 c.431-1G >C p.G41_S42dup p.E83X p.W89fs143X (1) p.W89fs156X p.Q90X p.Q94fs102X (1) p.E95X p.Q96X p.Q97X (1) p.Q97Rfs156X (2) p.P99fs103X p.S107fs111X p.E114X (1) p.E116Sfs156X (1) p.P122Afs144X p.E128X (1) p.R160fs p.Q167X p.E170Rfs194X (1) p.W183X p.I188N p.189 insl p.F189fs203X (1) p.H192R (1) p.H192P p.N193fs240X p.S194F p.S194fs250X p.G196V p.G196E p.Y199Sfs203X (2) p.N210fs240X (3)* p.R211fs235X (1) p.Q214fsX (1) c.652+1G >A(2)

Exon 2 p.W219X p.G221R (3)* p.A224T (5)* p.A224V (2)* p.A224E p.M227R (1) p.F230del (9)* p.V235M p.236insV p.T239P (1) p.R245X (6)* p.G251E (1) p.A252P p.A252Lfs268X (1) p.V254fs278X

III

Del ex6 IV

Exon 3 Exon 4 c.798-1G> C(3)* C1249-2A> G(1) p.R271H (4)* p.K418fs453X p.Y275X p.A432fs (1) p.G276R (4)* p.L433H (1) p.G282C (1) p.L434W p.C283Y p.C436fs473X p.P289L p.S441X (2) p.S290F (5) p.S441L (1) p.L291R p.R445C (2)* p.F298L (1) p.R445S (1) p.L304P p.R445L (1) p.L304_1539del p.S448fs454X p.N306fs312X p.S448X p.G307C (2) p.D453V (2) p.G312R c.1392+1G >A p.P321L (2 ) p.Q335X p.L340fs340X p.Y354C p.R355Pfs419X p.S367Qfs400X (1) p.R368X p.Q380X (1) p.R388X (5)* p.W398R p.G401R (2)* p.A405fs416X p.L407fs418X c.1239_1247dupTGTACACCT (1)

V Exon 5 c.1393-1G> C(1) p.L469P (1) p.L471P (1) p.C491Y (1) p.L492P (1) p.L494P p.G495A ( 3) p.D498N p.G499D (1) p.F501del (2) p.L512P p.Q520X p.Q520fs591X (1) p.G527S (1) p.G536R p.P537L p.P538del p.l539fs590X p.G541C

VI

3’-UTR

Exon 6 p.L543P (1) p.Y550fs566X p.A553D (1) p.F554del (1) p.A565Afs566X (2) p.G558D p.P561del p.H562fs590X (1) p.G564R (9)* p.G564E (3) p.L568P p.P609fs679X p.P609Ffs685X (2) p.P612fs679X

Fig. 66.3  Genetic pathogenic mutations in SLC16A2, the gene encoding MCT8.

Mechanisms of Disease. The mechanisms underlying the phenotypic features have been investigated in different animal models. Mct8 KO mice have elevated T3, decreased T4, and normal or slightly increased TSH concentrations compared with wild-­type mice, replicating human thyroid function tests. Different mechanisms may contribute to the abnormal endocrine profile. First, the increased T3:T4 ratio suggests that thyroid hormone metabolism is changed. Dio1 activity is increased in the liver and kidney of Mct8 KO mice, and additional inactivation of Dio1 in Mct8 KO (Mct8/ Dio1 double KO mice) normalizes the T3:T4 ratio, indicating the prominent role of Dio1 to the disturbed T3:T4 ratio.39 However, selective inactivation of hepatic Dio1 activity did not normalize thyroid hormone concentrations in Mct8 KO mice.40 Second, the thyroid gland may contribute to the abnormal serum thyroid hormones. Intrathyroidal T4 and T3 concentrations are increased in Mct8 KO mice, and the ability to secrete T4 is reduced.41,42 Those observations indicate a role of MCT8 in releasing thyroid hormones into the bloodstream, which is compatible with the basolateral expression of MCT8 in thyrocytes. The partial retention of T4 in the thyroid may lead to an enhanced conversion to T3 by thyroidal Dio1 activity. However, athyroid Pax8/Mct8 KO mice substituted with T4 had similar low T4 concentrations to Mct8 KO mice, indicating that extrathyroidal mechanisms are involved. Third, T4 accumulates in kidneys of Mct8 KO mice; however, it is unclear how the absence of MCT8 in the kidney contributes to the endocrine phenotype. In Mct8 KO mice, thyrotropin-­ releasing hormone (TRH) is upregulated in the hypothalamus,41 possibly reflecting the inability of locally produced T3 to exert its effects on the hypophysiotropic

TRH-­producing cells. The pituitary is relative insensitive towards thyroid hormone, although the precise mechanisms are unknown.41,42 The elevated T3 concentrations in Mct8 KO mice have adverse effects on energy metabolism, with increased energy expenditure, associated with a lower fat mass and enhanced glucose metabolism in muscle as result.43 In addition, Mct8/Oatp1c1 KO mice have episodes of tachycardia and bradycardia, possibly linked to defective input from the central nervous system.44 Although Mct8 KO mice mimic the endocrine phenotype seen in patients, no neurological phenotype was observed. The double Mct8/ Oatp1c1 KO mouse model did, however, result in cerebral hypothyroidism associated with abnormal brain development due to defective transport of T3 and T4 across the BBB as described in patients.45 Patient-­derived induced pluripotent stem cells differentiated towards endothelial cells of the BBB also indicate the importance of MCT8 for the translocation of both T4 and T3 across the BBB.46 In addition to the expression at the BBB, MCT8 is expressed in other cells of the brain (e.g., neurons, astrocytes, and tanycytes lining the third ventricle).47 Although the functional role in those cells remains to be elucidated, data indicate that MCT8 has a cell-­autonomous role in neurons.48 The clinical phenotype is a composite of tissues with different thyroid states. Depending on the expression of MCT8 and other thyroid hormone transporters, tissues either have insufficient thyroid hormone concentrations (e.g., brain) or are exposed to toxic T3 concentrations (e.g., liver and muscle). With MCT8 being expressed at the BBB and in neural cells, defective MCT8 precludes entry of thyroid hormone into the brain, resulting in abnormal cerebral thyroid hormone concentrations. Postmortem histological analysis of brain regions in patients with MCT8 deficiency are consistent with a hypothyroid state.49 Therefore,

CHAPTER 66  Thyroid Hormone Transporters and Metabolism given the critical role of thyroid hormone for many processes involved in brain development, absence of MCT8 will result in abnormal neurodevelopment. The strongly elevated serum T3 levels cause thyrotoxic signs and symptoms in tissues that are not dependent on MCT8; the clinical sequelae predispose to significant morbidity and mortality.

Clinical Management. Supportive care is recommended to address all common clinical features. The occurrence of contractures and scoliosis warrants referral to rehabilitation physicians early in life. Physiotherapists should acknowledge the dominance of the movement disorder in childhood compared with a stronger spastic component later in life. Empirical symptomatic treatment can be initiated on demand. Seizures may warrant antiepileptic drugs, and treatment with drugs to alleviate dystonia and drooling (e.g., anticholinergic drugs) can provide relief, but usually to a limited extent. The progressive decline in body weight due to the thyrotoxicosis and difficulties in swallowing, as well as the strong association with mortality, calls for close monitoring of nutritional status by dieticians, with percutaneous endoscopic gastrostomy feeding often required to meet daily calorie requirements. Given the high prevalence of cardiovascular risk factors, referral for cardiac evaluation is indicated. Levothyroxine replacement therapy has been offered empirically in many patients because of suspected central hypothyroidism (i.e., low serum FT4 concentrations with normal TSH concentrations). Typically, this approach is ineffective in normalizing FT4 concentrations, and rather aggravates the elevated serum T3 concentrations due to immediate conversion of T4 to T3.1 One case report has been described in which prenatal intraamniotic treatment with a high dosage of levothyroxine improved neurodevelopment.50 The combinatory therapy of propylthiouracil (PTU) (but not methimazol) with levothyroxine replacement can improve features of peripheral thyrotoxicosis.51,52 This strategy can have clinical benefits, although effects on neurodevelopment are not expected. In addition, PTU is associated with a risk of severe hepatotoxicity. Accordingly, PTU is not recommended as therapy for hyperthyroidism, and its use, particularly in children, is discouraged by current guidelines. The unfavorable safety profile of PTU is particularly relevant in the context of the frequent need to use other drugs (e.g., antiepileptic drugs) with hepatotoxic side effects in MCT8-­deficient patients. Therefore, risks and benefits counseling is appropriate if this therapy is considered, in particular the lifelong need for PTU. Effective therapy should aim to alleviate toxic effects of thyroid hormone in peripheral tissues while improving decreased thyroid hormone signaling in brain. Different possibilities have been explored as therapeutic options for MCT8 deficiency. Preclinical studies have investigated restoring MCT8 function through gene therapy and through application of chaperone molecules that have the capacity to rescue misfolded MCT8 mutants.53,54 The clinical utilization of those potential options needs to be further elucidated. Thyromimetic molecules that act as thyroid hormone receptor agonist, but whose cellular entry is not dependent on MCT8, have the potential to prevent or even reverse the neurological phenotype in patients with MCT8 deficiency. Also, such T3 analogs can negatively regulate TSH concentrations, thereby reducing endogenous thyroid hormone production and secretion. The T3 analogs Triac (triiodothyroacetic acid), DITPA (diiodothyropropionic acid), and sobetirome and the sobetirome prodrug Sob-­AM2 have been investigated in preclinical studies, with varying positive effects on different outcomes.55-­58 Clinical experience with DITPA application in four children with MCT8 deficiency has been reported.59 Serum T3 and sex hormone– binding globulin concentrations improved in all patients, although no consistent effect on body weight was observed. Triac treatment was

1119

evaluated in 46 (pediatric and adult) patients in an international trial.60 Triac treatment induced a strong and sustained reduction of the elevated T3 concentrations and, consequently, improved biochemical markers of thyroid hormone action in different tissues. Also, sustained improvements in clinically relevant outcomes including body weight for age, heart rate, and blood pressure were observed. Severe underweight and cardiovascular dysfunction are important clinical sequelae of chronic peripheral thyrotoxicosis, causing substantial morbidity and mortality in patients with MCT8 deficiency. Amelioration of the thyrotoxicosis with Triac treatment could benefit patients with MCT8 deficiency, irrespective of their age. Ongoing studies may reveal if Triac administration early in life can improve the neurocognitive phenotype. KEY POINTS  • Mutations in the X-­linked thyroid hormone transporter MCT8 cause profound neurodevelopmental delay due to cerebral hypothyroidism, as well as a wide range of severe clinical sequelae secondary to chronic peripheral thyrotoxicosis. 3,5,3’-­tri-­iodothyronine analog (e.g., Triac) therapy has the potential to ameliorate key clinical features of this rare disorder.

Organic Anion Transporting Polypeptide 1C1 Deficiency. One individual with a homozygous mutation in OATP1C1 has been reported.61 Cognitive and motor function were delayed in childhood, followed by the progressive loss of attained skills, resulting in the absence of verbal communication and spasticity, as well as difficulties in swallowing. Profound cold intolerance was noted, with normal serum thyroid function parameters. (18F)-­fluorodeoxyglucose–positron emission tomography scan showed a largely decreased glucose metabolism, and MRI examination of the brain showed progressive atrophy in the cerebral cortex, subcortical white matter, and cerebellum. The homozygous D252N mutation had decreased cellular T4 transport capacity due to impaired plasma membrane localization. Brain T4 concentrations were reduced in Oatp1c1 KO mice but did not result in neurological abnormalities, probably due to redundancy of other thyroid hormone transporters, including Mct8.62 Zebrafish oatp1c1 KOs showed abnormal locomotor changes, alterations in radial glial cell development, and shorter neuronal axons.63 Of note, oatp1c1 KO zebrafish demonstrated a large goiter, which was fully reversed in the presence of Triac, but not by T3 or T4. As OATP1C1 expression is different in human versus in rodents and fish, it is unclear to what extent findings in animal models can be extrapolated to humans.1 Moreover, it is unclear if the clinical phenotype can be fully attributed to dysregulation of thyroid hormone in the brain. Possibly, based on the OATP1C1 expression in astrocytes, impaired T4 accumulation in astrocytes results in less conversion to T3 by the DIO2 present in those cells. However, with OATP1C1 present in other cell types as well, defective OATP1C1 in such cells may also contribute to the clinical phenotype. The combination of levothyroxine and Triac resulted in improvement of alertness and also of swallowing.61 Future identification of more patients with OATP1C1 mutations will help to define the clinical features and to understand the mechanisms of disease underlying OATP1C1 deficiency.

DISORDERS OF THYROID HORMONE METABOLISM Iodothyronine Deiodinase I Deficiency Two unrelated families heterozygous for pathogenic variants in DIO1 mutations have been identified.22 In the first family, a 3-­year-­old girl with Down syndrome, from nonconsanguineous parents of Hispanic

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PART 6  Thyroid

origin, had slightly elevated serum TSH and was positive for thyroperoxidase antibodies, often associated with Down syndrome.23 The second proband, from nonconsanguineous Jewish parents, was also referred to the clinic with resistance to TSH. Genetic analysis identified in each family one rare heterozygous and one predicted pathogenic variant in DIO1: N94K and M201I, respectively. Multiple family members were found to be heterozygous for the DIO1 mutations, and all affected individuals showed no obvious abnormalities other than characteristic thyroid tests with elevated serum rT3 concentrations and rT3/T3 ratios compared with unaffected family members. One family was investigated in more detail and exhibited normal liver enzymes (alanine aminotransferase and aspartate aminotransferase), blood urea nitrogen, and creatinine concentrations. However, total cholesterol concentrations were high/normal to very high in the affected individuals, and need to be considered as a potential parameter for identifying patients. In vitro activity assays suggest that the mutations affect substrate affinity and enzyme velocity compared with the wild-­type enzyme. The underlying mechanism for the abnormal serum thyroid hormone levels in heterozygous DIO1-­patients is thought to be haploinsufficiency, similar to what is described in two mouse models. Dio1 deficiency, naturally occurring in the C3H/He mice strain, resulted in elevated serum rT3 level and rT3/T3 ratio.24,25 These results were confirmed in the genetically engineered heterozygous Dio1-­deficient mice,22 with the homozygous Dio1 KO exhibiting raised T4 and rT3 with normal T3 and TSH levels.9 Both Asn94 and Met201 are highly conserved amino acids within the DIO family over different species, suggesting an important function. In the absence of a protein structure for DIO1, the effect of these mutations can be assessed using a model generated from the highly homologous DIO3 structure (Fig. 66.4). The N94K and M201I mutations are fairly conservative, both in size and polarity, and their positions are not expected to result in major changes in the overall structure, but may affect specific local function of DIO1. Met201 is located near the substrate-­binding and catalytic activity core of the enzyme, with His174, part of the proposed proton transfer chain for reductive 5’-­deiodination, in close proximity. Asn94 is situated close to the hinge segment, between the TMD and the globular substrate-­ binding domain. KEY POINTS  • The human deiodinase genes have been subject to investigation for a long time; however, only pathogenic genetic defects in DIO1 have recently been described. Thyroid tests show elevated serum 3,5’,3’-­tri-­iodothyronine concentrations and 3,5’,3’-­tri-­iodothyronine/3,5,3’-­tri-­iodothyronine ratio, with a suggestion that total cholesterol concentrations are elevated.

SBP2 Deficiency

Clinical Phenotype. Compound heterozygous or homozygous SECISBP2 defects have been described in patients from diverse ethnic backgrounds, exhibiting similar clinical phenotypes29 (Table 66.3). Most cases were diagnosed in childhood with growth retardation (e.g., failure to thrive, short stature, delayed bone age) and developmental delay (e.g., delayed speech, intellectual and motor coordination deficits) as common features that brought probands to clinical attention. The biochemical phenotype in all affected cases suggests a defect in T4 to T3 conversion, with raised circulating FT4, normal to low free T3 (FT3), raised rT3 concentrations, and normal or slightly high TSH concentrations with no noticeable goiter.64 This pattern reflects deficiency of all three selenocysteine-­containing DIO enzymes, and,

consistent with this hypothesis, higher than normal amounts of exogenous T4 are required to suppress TSH levels, whereas the response to T3 is normal.65 Such abnormal thyroid function, together with low plasma selenium levels, reflecting decreased levels of SELENOP and GPX3, which constitute the major circulating selenoproteins, provides the biochemical signature that facilitates identification of putative cases of SBP2 deficiency due to SECISBP2 mutation. Fatigue and muscle weakness, at least partially due to progressive rigid spine muscular dystrophy affecting axial and proximal limb muscles, are recognized features in several patients and are very similar to the phenotype of selenoprotein N‐deficient myopathies.66 Circulating skeletal muscle–specific creatine kinase levels can be elevated, and selective fatty infiltration of the muscle groups (adductor, biceps femoralis) is visible on MRI scan even in childhood, prior to onset of clinical symptoms. Histological analysis shows type 1 oxidative fiber predominance and areas of sarcomere disorganization, termed minicores, in muscle from the SECISBP2-­ mutated adult proband.67 More marked abnormalities and symptoms in adult patients suggest that the myopathy is progressive and lead to reduced lung function (expiratory and inspiratory flow; total vital capacity), possibly requiring positive pressure ventilatory support. Mild bilateral high-­frequency sensorineural hearing loss is seen in some patients, with adults being most severely affected and sometimes requiring hearing aids, perhaps reflecting a progressive course. Increased whole-­body fat mass, with the excess adipose tissue being predominantly subcutaneous with relatively normal visceral fat, and high circulating adiponectin, possibly of peripubertal onset, have been noted in some cases. Paradoxically, these features are associated with low intrahepatic lipid, a favorable blood lipid profile, and preserved or enhanced tissue insulin sensitivity with possible propensity to spontaneous hypoglycemia.67 One adult SECISBP2-­mutated patient was investigated in great detail, and at presentation (age 35 years) exhibited additional features beside the phenotypes described already, including azoospermia (spermatogenic maturation arrest with preservation of early cell types, e.g., spermatogonia and spermatocytes, but lack of mature spermatids and spermatozoa), skin photosensitivity, severe Raynaud disease (digital vasospasm), rotatory vertigo, and reduced red blood cell and total lymphocyte counts, with impaired mononuclear cell cytokine secretion and T-­cell proliferation.67 Decreased circulating white blood cells and neutrophils were also described in a Japanese patient.68 An additional characteristic in patients is that many phenotypes (e.g., metabolic phenotype, hearing, muscle weakness, photosensitivity) worsen with advancing age.

Molecular Genetics. Selenium is an essential micronutrient that is incorporated as the amino acid Sec into at least 25 human selenoproteins. Most selenoproteins function as oxidoreductases, with the Sec residue involved in catalytic activity, and recognized functions include maintenance of redox potential, regulating redox sensitive biochemical pathways, protection of genetic material, proteins, and membranes from oxidative damage, metabolism of thyroid hormones, regulation of gene expression, and control of protein folding.69,70 Biosynthesis of selenoproteins requires an UGA codon within its mRNA to be recoded as the amino acid Sec, preventing its recognition as a premature stop signal. This process is achieved via unique Sec-­ insertion machinery comprising cis-­ acting SECIS elements located in the 3′ untranslated region of all selenoprotein mRNAs and the UGA codon, interacting with transacting factors (SECISBP2, Sec tRNA–specific eukaryotic elongation factor and Sec-­ tRNA[Ser]Sec) (Fig. 66.5).29,70

l

CHAPTER 66  Thyroid Hormone Transporters and Metabolism

SECISBP2, generating different protein isoforms.72 These events alter content of the dispensable N-­terminal region, but not the essential C-­terminal domain and are thought to play a role in regulation of SECISBP2-­dependent Sec incorporation and the hierarchy of selenoprotein expression in vivo. Homozygous or compound heterozygous mutations in SECISBP2 have been described in 13 individuals from 11 families (Table 66.3). Consistent with a recessive mode of inheritance, heterozygous individuals do not present with any of the described phenotypes. Most SECISBP2 mutations identified to date are premature stop codons, resulting in absence of full-­length SECISBP2 protein. However, for premature stops located in the N-­terminal part of the protein, initiation of translation from alternative, downstream ATG codons permits low-­level synthesis of shorter SECISBP2 isoforms.67,73,74 Some premature stop mutations are situated downstream of Met300 and may completely eliminate synthesis of functional protein. Conversely, stop mutations (e.g., R770X, Q782X), distal to the minimal functional domain might generate C-­terminally truncated proteins whose functions remain partially intact (Fig. 66.6). In one patient with defective mRNA splicing due to an intronic mutation IVS8ds+29G>A,64 expression of the correctly spliced transcript was only reduced by 50%, and it is possible that a similar mechanism operates with other splice site mutations, resulting in some preservation of normally spliced SECISBP2 mRNA. Three missense SECISBP2 mutations (R540Q, E679D, and C691R) are situated in the RNA-­binding domain of the minimal functional protein. The R540Q mutation fails to bind only a subset of SECIS-­ elements, and a mouse model revealed a tissue-­specific pattern of SECISBP2 protein expression, correlating with varying loss or preservation of synthesis of different selenoproteins.64,75,76 The C691R mutation is subject to enhanced proteasomal degradation,67 confirmed in a mouse model, which also suggests that the C691R mutant is unable to bind RNA and is nonfunctional.76 The E679D mutation is predicted to be deleterious (PolyPhen-­2 algorithm score of 0.998), possibly affecting its binding to RNA, but this has not been investigated.73 With only a small number of patients described, SECISBP2 mutations identified hitherto are uniformly distributed, with no particular mutation “hot spots” in the protein, although most result in a premature stop in the dispensable N-­terminal region (Fig. 66.6). All patients described to date have at least one allele that either directs synthesis of SECISBP2 at reduced levels or directs synthesis of a partially functional SECISBP2, due to synthesis of shorter N-­or C-­terminally truncated

From the early stages of selenoprotein research it was clear that dietary selenium intake affects systemic selenium status and selenoprotein expression. However, not all selenoproteins are affected in the same way, with synthesis of housekeeping selenoproteins (e.g., TrxR1, TrxR3, GPx4) being less affected by reduced circulating selenium levels than stress-­related selenoproteins (e.g., GPx1, GPx3, SEPW1). Such differential preservation of selenoprotein expression is attributed to the existence of a “hierarchy of selenoprotein synthesis,” the underlying molecular basis of which is unclear.71 SECISBP2 is an obligate and limiting factor for biosynthesis of selenoproteins and is a large (854 amino acids, 120 kDa) protein. The first 400 N-­terminal residues are dispensable for its function in vitro, whereas the C-­terminal region (amino acids 399–784) is both necessary and sufficient for binding to the SECIS element in the selenoprotein RNA and Sec incorporation. Alternative splicing events and multiple ATG start codons for initiation of translation have been described for

Sec

C-terminal His174

Met201 Asn94

N-terminal Fig. 66.4  Structural model of the deiodinase 1 with positioning of the mutated amino acids. The model represents the globular, active domain of deiodinase 1 (DIO1). The amino acid selenocysteine is shown in green, amino acids described to be involved in proton transfer during reductive 5-­deiodination are in yellow, those involved in substrate binding and catalytic activity in orange, and the two affected amino acids (Met201 and Asn94) are in red. The C-­terminal and N-­terminal ends of the protein are indicated, with the latter linking to the transmembrane domain. The model was generated using the phyre2-­server101 and is based on the crystal structure of DIO3 (protein data bank [PDB]: 4TR3 and 4TR426).

EEFSec Polypeptide

Sec SECISBP2

5’ mRNA

3’ AUG

1121

UGA

UAA

SECIS-element

Ribosomal complex Fig. 66.5  Mechanism of selenoprotein biosynthesis. The 3′ untranslated region of selenoprotein mRNAs contains a stem-­loop RNA structure (SElenoCysteine Insertion Sequence element) that interacts with a multiprotein complex which includes SECISBP2 and selenocysteine (Sec)-­specific elongation factor. This enables ribosomal recruitment of a specific transfer RNA (tRNA[Ser]Sec) to the recoded UGA codon, enabling Sec incorporation into the nascent polypeptide. Failure of this mechanism results in miscoding of the UGA as a stop codon, terminating protein synthesis.70

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PART 6  Thyroid

TABLE 66.3 Human SECISBP2 Mutations Family

Mutation

Protein Change

Alleles Affected

A

c.1619 G>A

R540Q

Homozygous

B

c.1312A>T c.IVS8ds+29 G>A

K438X fs431X

Compound heterozygous

C

c.382 C>T

R128X

Homozygous

D

c.358 C>T

R120X

Compound heterozygous

c.2308 C>T

R770X

c.668delT

F223fs255X

c.IVS7 -­155, T>A

fs295X+fs302X

c. 2017T>C

C691R

Compound

1-­5 intronic SNPs

fs65X + fs76X

Heterozygous

c.1529_1541dup CCAGCGCCCCACT c.235 C>T c.2344 C>T c.2045-­2048 delAACA c.589 C>T

M515fs563X

Compound heterozygous

Q79X Q782X K682fs683X R197X

c.2108 G> T or C

E679D

J

c.800_801insA

K267Kfs*2

Homozygous

K

c.283delT c.589 C>T

T95Ifs31* R197X

Compound heterozygous

E

F

G

H I

Compound heterozygous

Compound heterozygous Compound heterozygous

Suggested Mechanism Predicted to affect SECIS and ribosome binding Premature stop, no/ decreased full-­length protein Premature stop, no full-­ length protein Premature stop, no full-­ length protein Premature stop, no full-­ length protein Premature stop, no full-­ length protein Premature stop, no or decreased full-­length protein Predicted to affect SECIS and ribosome binding, increased degradation Premature stop, no full-­ length protein/splice variants affected Premature stop, no full-­ length protein Premature stop, no full-­ length protein Premature stop, no full-­ length protein/splice variants affected Predicted to affect SECIS and ribosome binding Premature stop, no full-­ length protein Premature stop, no full-­ length protein

Ethnicity Saudi Arabian Irish/Kenyan

Ghanaian Brazilian

British

British

Japanese

Turkish Argentinian

Turkish N/A

N/A, Not available; SNP, single nucleotide polymorphism; SECIS, SElenoCysteine Insertion Sequence.

forms of the protein (Table 66.3), suggesting that they are hypomorphic rather than completely null for loss of gene function. Nevertheless, because SECISBP2 is rate-­limiting for Sec incorporation, reduction in functional SECISBP2 protein compromises selenoprotein synthesis. The small number of patients described from different ethnic and geographical backgrounds, often with compound heterozygous mutations whose functional consequences have not been defined, together with limited knowledge of their phenotypes, makes it impossible to assess the effect of a specific SECISBP2 mutation or its correlation with the severity of phenotype.

Mechanisms of Disease. Disruption of SECISBP2 function prevents appropriate Sec incorporation into most if not all selenoproteins, resulting in a multisystem disorder. Some clinical phenotypes are attributable to deficiencies of particular selenoproteins, while others may have a complex, multifactorial basis, possibly due to unbalanced oxidoredox and/or protein folding pathways, reflected in increased cellular oxidative stress measured in patients’ cells, or unknown function of selenoproteins. SECISBP2 is essential for survival, as murine

Secisbp2 deletion is embryonic lethal.77 Accordingly, conditional and tissue-­specific murine Secisbp2 or specific selenoprotein KO models have provided informative insights into pathogenesis of the human disorder. Selenocysteine is present in the active center and required for enzymatic activity of all three iodothyronine DIOs, and, consonant with SECISBP2 deficiency, DIO2 activity was reduced in fibroblasts from one family.64 DIO2 converts T4 to T3, and its activity can change very rapidly, as its half-­life (approximately 20 minutes) is much shorter (15-­ fold) than that of DIO1 and DIO3. The combination of reduced DIO2 synthesis, together with its short half-­life, likely explains its low activity in patient cells. The thyroid phenotype of SECISBP2-­mutated patients is most closely recapitulated by a murine Dio1 and Dio2 double KO,13 suggesting that it is mediated by combined, partial deficiency of both DIOs. However, unlike human SECISBP2 patients, both double Dio1/ Dio2 and triple Dio1/Dio2/Dio3 KO mice have relative normal serum T3 levels, suggesting that a combination of partial deficiency of all three DIOs and improper fine-­tuning of specific DIO expression mediates the abnormal thyroid function test pattern seen in this disorder.

1123

CHAPTER 66  Thyroid Hormone Transporters and Metabolism Q79X R128X

R197X K267X FS295X

FS563X

FS683X

399 Met1

Met69 Met139

FS65X FS76X R120X

Met233

FS255X

779

Met300

FS302X

854

Minimal functional domain

FS431X

R540Q

E679D C691R

R770X

Q782X

Fig. 66.6  Schematic representation of the SECISBP2 protein with the location of mutations. The position of all mutations is superimposed, and details of mutations in patients are shown in Table 66.3. Neither homozygous or different, compound heterozygous, mutation combinations are predicted to completely eliminate synthesis of the minimal functional domain (amino acids 399–779, shaded) of SECISBP2. Internal methionine residues from which translation of shorter SECISBP2 protein isoforms can be initiated are shown.

The thyroid hormone biochemical phenotype seen in patients is recapitulated in an inducible mouse model of Secisbp2 deficiency, albeit associated with more robust increase in serum TSH levels.78 Growth retardation and developmental delay are the commonest symptoms in SECISBP2-­mutated patients, and the pathogenesis is probably multifactorial, with no clear link to deficiency of a single selenoprotein or pathway. Growth retardation is a recognized feature in Dio2 and Dio3 null mice,11,12 suggesting that the human phenotype is, at least in part, mediated by abnormal thyroid hormone metabolism. Consistent with this notion, treatment of SECISBP2-­deficient children with T3 alone (family C, F) or in conjunction with growth hormone (family G) resulted in catch-­up growth. Neuron-­specific Secisbp2 KO mice present with growth retardation, with unclear mechanistic basis.79 Selective depletion of multiple selenoproteins in murine osteochondroprogenitor cells results in epiphyseal abnormalities and chondronecrosis,80 suggesting an important, cell-­autonomous role for selenoproteins in skeletal development. However, in a single SECISBP2-­mutated case (family E), radiological skeletal survey and bone mineral density were found to be normal. Axial muscular dystrophy, leading to fatigue and muscle weakness, is a musculoskeletal phenotype highly analogous to that seen in patients with a myopathic disorder (rigid spine muscular dystrophy) due to mutations in selenoprotein N.81 Motor incoordination in some SECISBP2-­mutated patients could be due to selenoprotein N myopathy and axial muscle weakness. However, neuron-­specific Secisbp2 null mice79 also exhibit an obvious locomotor phenotype, together with degenerative loss of PV+/Gad67+ interneurons in basal ganglia, providing a possible alternative explanation for the movement disorder and raising the possibility that this phenotype is progressive. Azoospermia with spermatogenic maturation arrest described in the adult SECISBP2-­mutated patient can be linked with deficiency of three selenoproteins (mGPX4, TXNRD3, SELV), with recognized roles in spermatogenesis. Mitochondrial GPX4 (mGPX4) is a structural component of the mitochondrial capsule in the midpiece of spermatozoa,82 with murine mGpx4 KO resulting in male infertility,83 and reduced human seminal mGPX4 activity correlates with oligospermia.84 TXNRD3 is highly enriched in spermatids and may catalyze protein disulfide bond isomerization in sperm development.85 Human SELV expression is known to be testis-­restricted, but its function is unknown.86 Consistent with the role of mGPX4 and TXNRD3 in latter spermatogenic stages, testicular histology in the adult SECISBP2-­mutated patient showed maturation arrest, with preservation of early cell types (e.g., spermatogonia and spermatocytes) but lack of mature spermatids and spermatozoa. Male and female infertility are described in a mouse model of Secisbp2 deficiency.78 The bilateral high-­ frequency sensorineural hearing loss in SECISBP2-­mutated cases could be due to DIO2 deficiency, with hearing loss and retarded cochlear development being recognized features

in Dio2 null mice,11 whereas cochlear anatomy is normal and DIO2 activity only partially reduced in human SECISBP2-­mutated cases. An alternative hypothesis links hearing loss to damage mediated by elevated cellular reactive oxygen species (ROS), similar to the noise-­ induced hearing loss observed in Gpx1 null mice.87 ROS-­mediated cochlear damage can be cumulative,88 which could explain the progressive nature of hearing loss, resulting in a more severe deficit in the older, adult, SECISBP2-­mutated case. Several other recorded phenotypes (enhanced insulin sensitivity and increased adipose mass, reduced red blood cell and total lymphocyte counts, cutaneous photosensitivity, developmental delay, growth and skeletal phenotype) probably have a multifactorial basis, comprising a combination of abnormal thyroid hormone metabolism, loss of antioxidant and endoplasmic reticulum stress defense pathways, and deficiency of specific selenoprotein(s), resulting in irreparable cell and organ damage due to cumulative cellular stress. Many studies in mouse models and in humans provide a substantial body of evidence to suggest a link between selenoproteins and most of these phenotypes.67,89-­92 It is conceivable that cumulative cellular stress (oxidative, endoplasmic reticulum) and irreparable cell damage could also predispose to other phenotypes (e.g., premature ageing, cancer) that have not yet manifested in the relatively young cohort of patients identified hitherto.

Clinical Management. Oral selenium supplementation in some SECISBP2-­mutated patients raised total serum selenium levels, but without clinical74,93,94 or biochemical (circulating GPXs, SELENOP, thyroid hormone metabolism) effect.95 This is probably because the disorder involves defective incorporation of selenium into the selenoproteins, rather than deficiency of this trace element per se, although the effect could be mutation-­dependent. Treatment of some probands with T3 alone (T4 was not effective in one case) or in combination with growth hormone resulted in some improvement in growth, development, and bone maturation, suggesting that T4 or T3 treatment at younger age might be beneficial. Treatment of proband G with a combination of alpha tocopherol (vitamin E) and T3 resulted in the most promising response. Serum levels of products of lipid peroxidation decreased, FT4 and FT3 concentrations ameliorated, and circulating white blood cells and neutrophils increased, all of which were reversed after treatment withdrawal.68 These observations suggest that treatment with antioxidants, to compensate for loss of antioxidant selenoproteins, is a possible therapeutic option for this disorder. However, due to limited number of SECISBP2-­mutated cases and a paucity of evidence, clinical trials of antioxidants, perhaps in combination with T3 and growth hormone, are warranted, to develop a rational therapeutic approach in this disorder.

1124

PART 6  Thyroid

tRNA[Ser]Sec

P-Ser-tRNA[Ser]Sec

Ser-tRNA[Ser]Sec AMP + PPi

Sec-tRNA[Ser]Sec Sec

P-Ser

Ser ADP

C65

2Pi

SARS

PSTK

SEPSECS

Ser + ATP

ATP

H2SePO3−

A C

U Anticodon

OH

A

Sec

C

O

G

NH

5′ G

C

C

G

SeH

C

G

C

G

G

C

G

U

A

U

G

C

U U G

mcm5Um O

O

O

N

G U C U G G G

OH OH

G C A G G C

O NH

O

N

O OH OH

34

U U

AMP + Pi

C

A

SEPHS2 HSe−

U

C65G

C A C C

U

U A

A G U G G U U C A G A G A C C U U A G G G C U U C G C U A C A A

G U

mcm5U

O

O

A

U G A C U C C

O NH

O

ATP

C

Selenoprotein diet

Anticodon

OH

Fig. 66.7  Pathway of Sec-­tRNA[Ser]Sec synthesis and clover leaf model of Sec-­tRNA[Ser]Sec. A, Synthesis of selenocysteine (Sec) occurs using its own tRNA, starting with attachment of serine to tRNA[Ser]Sec by seryl-­tRNA synthetase (SARS). This residue is phosphorylated by phosphoseryl-­tRNA kinase (PSTK), and the phosphoserine is subsequently converted to Sec-­tRNA[Ser]SecmcmU by Sep (O-­phosphoserine) tRNA:Sec tRNA synthase (SEPSECS) using selenophosphate. The selenophosphate is synthesized from selenide, originating from diet and degradation of Sec-­containing proteins, by selenophosphate synthetase 2 (SEPHS2).70 The position of the mutated base (C65) and the anticodon bases (U34, C35, and A36) are highlighted as black spheres in Sec-­tRNA[Ser]Sec. B, The amino acid Sec is depicted, as well as a clover leaf model of Sec-­tRNA[Ser] Sec with the position of the mutated base (C65G, ringed) and the modification on uridine 34 in the anticodon (boxed) to Sec-­tRNA[Ser]Sec mcm5U or Sec-­tRNA[Ser]Sec mcm5Um by an unidentified Um34 methylase.99,100

Sec-­tRNA[Ser]Sec The amino acid Sec is different from other amino acids in that it is synthesized using its own tRNA, tRNA[Ser]Sec, encoded by TRU-­TCA1-­1, via a well-­described pathway involving SEPSECS (Fig. 66.7).70 Two major isoforms of the mature Sec-­tRNA[Ser]Sec have been identified, containing either 5-­ methoxycarbonyl-­ methyluridine (mcm5U) or its methylated form, 5-­methoxycarbonylmethyl-­2’-­O-­methyluridi ne (mcm5Um), at position 34 (Fig. 66.7). Uridine 34 is located in the anticodon loop, and its methylation may contribute to stabilization of the codon–anticodon interaction. These Sec-­tRNA[Ser]Sec subtypes have a role in controlling the hierarchy of selenoprotein synthesis, with production of essential, cellular housekeeping selenoproteins (e.g., TXNRDs, GPX4) being dependent on the mcm5U isoform and cellular, stress-­related selenoprotein (e.g., GPX1, GPX3) synthesis requiring the mcm5Um isoform.96,97

A single patient homozygous for a single nucleotide change (C65G) in TRU-­TCA1-­1 has been identified (Fig. 66.7)98 and exhibits a similar clinical and biochemical phenotype (abdominal pain, fatigue and muscle weakness, raised FT4, normal T3, raised rT3 and TSH, low plasma selenium concentrations) to that seen in patients with SECISBP2 deficiency. However, the pattern of cellular expression of selenoproteins in the two disorders differs, with synthesis of housekeeping selenoproteins (e.g., TXNRDs, GPX4) being preserved in the TRU-­TCA1-­1 case, while levels of cellular stress–related selenoproteins (e.g., GPX1, GPX3) were similarly reduced in both disorders. In the proband with the TRU-­TCA1-­1 C65G mutation, lower total tRNA[Ser]Sec expression with disproportionately greater diminution in Sec-­tRNA[Ser]Sec mcm5Um levels were found. The mechanism of this is unresolved, with possibilities including effects of the mutation on stability or posttranscriptional maturation of Sec-­tRNA[Ser]Sec or the

CHAPTER 66  Thyroid Hormone Transporters and Metabolism stability of the Sec-­tRNA[Ser]Sec–SEPSECS complex. Similar to differential preservation of selenoprotein synthesis seen in murine tRNAsec mutant models,70,96 it is thought that the low levels of tRNA[Ser]Sec seen in the proband are sufficient for normal synthesis of housekeeping selenoproteins, whereas diminution of Sec-­tRNA[Ser]Sec mcm5Um levels accounts for reduced synthesis of stress-­related selenoproteins. Given that systemic selenium status is known to influence relative proportions of the tRNA[Ser]Sec isoforms, with Sec-­ tRNA[Ser] Sec mcm5Um being low in selenium deficiency but enriched in the selenium-­replete state,96,99,100 it is possible that altering the selenium status of the patient with selenium supplementation to restore particular selenoprotein deficiencies, with careful monitoring for toxicity, could represent a rational therapeutic approach. KEY POINTS  • Selenium is an essential micronutrient incorporated into at least 25 human selenoproteins as the amino acid selenocysteine (Sec), and SECISBP2 is an obligate and limiting factor for their biosynthesis. Sec incorporation is also dependent on its own transfer RNA (tRNA[Ser]Sec), encoded by TRU-­ TCA1-­1. • Individuals with SECISBP2 defects exhibit a multisystem phenotype including growth retardation, fatigue and muscle weakness, sensorineural hearing loss, increased whole-­body fat mass with enhanced insulin sensitivity, azoospermia, and cutaneous photosensitivity. Characteristic biochemical abnormalities in patients are raised FT4, normal to low FT3, raised rT3, and normal/slightly high thyroid-­stimulating hormone, reflecting abnormal thyroid hormone metabolism due to deficiency of all three Sec-­containing deiodinase enzymes and low plasma selenium levels due to deficiency of circulating SELENOP and GPX3. • A single individual with a TRU-­TCA1-­1 mutation exhibits the same biochemical phenotype and some clinical features as patients with SECISBP2 defects. However, selenoprotein expression in this patient’s cells differ, with relative preservation of essential housekeeping versus stress-­related selenoproteins.

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PART 6  Thyroid

34. Visser WE, de Rijke YB, van Toor H, et al. Thyroid status in a large cohort of patients with mental retardation: the TOP-­R (Thyroid Origin of Psychomotor Retardation) study. Clin Endocrinol. 2011;75:395–401. 35. Vancamp P, Demeneix BA, Remaud S. Monocarboxylate transporter 8 deficiency: delayed or permanent hypomyelination? Front Endocrinol. 2020;11:283. 36. Braun D, Schweizer U. Thyroid hormone transport and transporters. Vitam Horm. 2018;106:19–44. 37. Friesema EC, Jansen J, Heuer H, et al. Mechanisms of disease: psychomotor retardation and high T3 levels caused by mutations in monocarboxylate transporter 8. Nat Clin Pract Endocrinol Metab. 2006;2:512–523. 38. Frints SG, Lenzner S, Bauters M, et al. MCT8 mutation analysis and identification of the first female with Allan-­Herndon-­Dudley syndrome due to loss of MCT8 expression. Eur J Hum Genet. 2008;16:1029–1037. 39. Liao XH, Di Cosmo C, Dumitrescu AM, et al. Distinct roles of deiodinases on the phenotype of Mct8 defect: a comparison of eight different mouse genotypes. Endocrinology. 2011;152:1180–1191. 40. Wirth EK, Rijntjes E, Meyer F, et al. High T3, low T4 serum levels in Mct8 deficiency are not caused by increased hepatic conversion through type I deiodinase. Eur Thyroid J. 2015;4:87–91. 41. Trajkovic-­Arsic M, Muller J, Darras VM, et al. Impact of monocarboxylate transporter-­8 deficiency on the hypothalamus-­pituitary-­thyroid axis in mice. Endocrinology. 2010;151:5053–5062. 42. Di Cosmo C, Liao XH, Dumitrescu AM, et al. Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion. J Clin Invest. 2010;120:3377–3388. 43. Di Cosmo C, Liao XH, Ye H, et al. Mct8-­deficient mice have increased energy expenditure and reduced fat mass that is abrogated by normalization of serum T3 levels. Endocrinology. 2013;154:4885–4895. 44. Herrmann B, Harder L, Oelkrug R, et al. Central hypothyroidism impairs heart rate stability and prevents thyroid hormone-­induced cardiac hypertrophy and pyrexia. Thyroid. 2020;30:1205–1216. 45. Mayerl S, Muller J, Bauer R, et al. Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis. J Clin Invest. 2014;124:1987–1999. 46. Vatine GD, Al-­Ahmad A, Barriga BK, et al. Modeling psychomotor retardation using iPSCs from MCT8-­deficient patients indicates a prominent role for the blood-­brain barrier. Cell Stem Cell. 2017;20:831–843.e5. 47. Lopez-­Espindola D, Garcia-­Aldea A, Gomez de la Riva I, et al. Thyroid hormone availability in the human fetal brain: novel entry pathways and role of radial glia. Brain Struct Funct. 2019;224:2103–2119. 48. Mayerl S, Heuer H, Ffrench-­Constant C. Hippocampal neurogenesis requires cell-­autonomous thyroid hormone signaling. Stem Cell Reports. 2020;14:845–860. 49. Lopez-­Espindola D, Morales-­Bastos C, Grijota-­Martinez C, et al. Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination. J Clin Endocrinol Metab. 2014;99:E2799–E2804. 50. Refetoff S, Pappa T, Williams MK, et al. Prenatal treatment of thyroid hormone cell membrane transport defect caused by MCT8 gene mutation. Thyroid. 2020:713-720. 51. Wemeau JL, Pigeyre M, Proust-­Lemoine E, et al. Beneficial effects of propylthiouracil plus L-­thyroxine treatment in a patient with a mutation in MCT8. J Clin Endocrinol Metab. 2008;93:2084–2088. 52. Visser WE, Vrijmoeth P, Visser FE, et al. Identification, functional analysis, prevalence and treatment of monocarboxylate transporter 8 (MCT8) mutations in a cohort of adult patients with mental retardation. Clin Endocrinol (Oxf). 2013;78:310–315. 53. Braun D, Schweizer U. Efficient activation of pathogenic DeltaPhe501 mutation in monocarboxylate transporter 8 by chemical and pharmacological chaperones. Endocrinology. 2015;156:4720–4730. 54. Iwayama H, Liao XH, Braun L, et al. Adeno associated virus 9-­based gene therapy delivers a functional monocarboxylate transporter 8, improving thyroid hormone availability to the brain of Mct8-­deficient mice. Thyroid. 2016;26:1311–1319. 55. Barez-­Lopez S, Hartley MD, Grijota-­Martinez C, et al. Sobetirome and its amide prodrug Sob-­AM2 exert thyromimetic actions in Mct8-­deficient brain. Thyroid. 2018;28:1211–1220.

56. Kersseboom S, Horn S, Visser WE, et al. In vitro and mouse studies supporting therapeutic utility of triiodothyroacetic acid in MCT8 deficiency. Mol Endocrinol. 2014;28:1961–1970. 57. Di Cosmo C, Liao XH, Dumitrescu AM, et al. A thyroid hormone analog with reduced dependence on the monocarboxylate transporter 8 for tissue transport. Endocrinology. 2009;150:4450–4458. 58. Zada D, Tovin A, Lerer-­Goldshtein T, et al. Pharmacological treatment and BBB-­targeted genetic therapy for MCT8-­dependent hypomyelination in zebrafish. Dis Model Mech. 2016;9:1339–1348. 59. Verge CF, Konrad D, Cohen M, et al. Diiodothyropropionic acid (DITPA) in the treatment of MCT8 deficiency. J Clin Endocrinol Metab. 2012;97:4515–4523. 60. Groeneweg S, Peeters RP, Moran C, et al. Effectiveness and safety of the tri-­iodothyronine analogue Triac in children and adults with MCT8 deficiency: an international, single-­arm, open-­label, phase 2 trial. Lancet Diabetes Endocrinol. 2019;7:695–706. 61. Stromme P, Groeneweg S, Lima de Souza EC, et al. Mutated thyroid hormone transporter OATP1C1 associates with severe brain hypometabolism and juvenile neurodegeneration. Thyroid. 2018;28:1406–1415. 62. Mayerl S, Visser TJ, Darras VM, et al. Impact of Oatp1c1 deficiency on thyroid hormone metabolism and action in the mouse brain. Endocrinology. 2012;153:1528–1537. 63. Admati I, Wasserman-­Bartov T, Tovin A, et al. Neural alterations and hyperactivity of the hypothalamic-­pituitary-­thyroid axis in Oatp1c1 deficiency. Thyroid. 2020;30:161–174. 64. Dumitrescu AM, Liao XH, Abdullah MS, et al. Mutations in SECISBP2 result in abnormal thyroid hormone metabolism. Nat Genet. 2005;37:1247–1252. 65. Dumitrescu AM, Refetoff S. The syndromes of reduced sensitivity to thyroid hormone. Biochim Biophys Acta. 2013;1830:3987–4003. 66. Silwal A, Sarkozy A, Scoto M, et al. Selenoprotein N-­related myopathy: a retrospective natural history study to guide clinical trials. Ann Clin Transl Neurol. 2020;7:2288–2296. 67. Schoenmakers E, Agostini M, Mitchell C, et al. Mutations in the selenocysteine insertion sequence-­binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans. J Clin Invest. 2010;120:4220–4235. 68. Saito Y, Shichiri M, Hamajima T, et al. Enhancement of lipid peroxidation and its amelioration by vitamin E in a subject with mutations in the SBP2 gene. J Lipid Res. 2015;56:2172–2182. 69. Zoidis E, Seremelis I, Kontopoulos N, et al. Selenium-­dependent Antioxidant Enzymes: Actions and Properties of Selenoproteins. Antioxidants (Basel). 2018;7 doi:10.3390/antiox7050066. 70. Labunskyy VM, Hatfield DL, Gladyshev VN. Selenoproteins: molecular pathways and physiological roles. Physiol Rev. 2014;94:739–777. 71. Sunde RA, Raines AM. Selenium regulation of the selenoprotein and nonselenoprotein transcriptomes in rodents. Adv Nutr. 2011;2: 138–150. 72. Papp LV, Lu J, Holmgren A, et al. From selenium to selenoproteins: synthesis, identity, and their role in human health. Antioxid Redox Signal. 2007;9:775–806. 73. Fu J, Korwutthikulrangsri M, Gonc EN, et al. Clinical and molecular analysis in 2 families with novel compound heterozygous SBP2 (SECISBP2) mutations. J Clin Endocrinol Metab. 2020;105:e6-e11. 74. Di Cosmo C, McLellan N, Liao XH, et al. Clinical and molecular characterization of a novel selenocysteine insertion sequence-­binding protein 2 (SBP2) gene mutation (R128X). J Clin Endocrinol Metab. 2009;94:4003– 4009. 75. Bubenik JL, Driscoll DM. Altered RNA binding activity underlies abnormal thyroid hormone metabolism linked to a mutation in selenocysteine insertion sequence-­binding protein 2. J Biol Chem. 2007;282:34653– 34662. 76. Zhao W, Bohleber S, Schmidt H, et al. Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations. J Biol Chem. 2019;294:14185–14200. 77. Seeher S, Atassi T, Mahdi Y, et al. Secisbp2 is essential for embryonic development and enhances selenoprotein expression. Antioxid Redox Signal. 2014;21:835–849.

CHAPTER 66  Thyroid Hormone Transporters and Metabolism 78. Fu J, Fujisawa H, Follman B, et al. Thyroid hormone metabolism defects in a mouse model of SBP2 deficiency. Endocrinology. 2017;158: 4317–4330. 79. Seeher S, Carlson BA, Miniard AC, et al. Impaired selenoprotein expression in brain triggers striatal neuronal loss leading to co-­ordination defects in mice. Biochem J. 2014;462:67–75. 80. Downey CM, Horton CR, Carlson BA, et al. Osteo-­chondroprogenitor-­ specific deletion of the selenocysteine tRNA gene, Trsp, leads to chondronecrosis and abnormal skeletal development: a putative model for Kashin-­Beck disease. PLoS Genet. 2009;5:e1000616. 81. Moghadaszadeh B, Petit N, Jaillard C, et al. Mutations in SEPN1 cause congenital muscular dystrophy with spinal rigidity and restrictive respiratory syndrome. Nat Genet. 2001;29:17–18. 82. Ursini F, Heim S, Kiess M, et al. Dual function of the selenoprotein PHGPx during sperm maturation. Science. 1999;285:1393–1396. 83. Schneider M, Forster H, Boersma A, et al. Mitochondrial glutathione peroxidase 4 disruption causes male infertility. FASEB J. 2009;23:3233– 3242. 84. Foresta C, Flohe L, Garolla A, et al. Male fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase. Biol Reprod. 2002;67:967–971. 85. Su D, Novoselov SV, Sun QA, et al. Mammalian selenoprotein thioredoxin-­glutathione reductase. Roles in disulfide bond formation and sperm maturation. J Biol Chem. 2005;280:26491–26498. 86. Kryukov GV, Castellano S, Novoselov SV, et al. Characterization of mammalian selenoproteomes. Science. 2003;300:1439–1443. 87. McFadden SL, Ohlemiller KK, Ding D, et al. The influence of superoxide dismutase and glutathione peroxidase deficiencies on noise-­induced hearing loss in mice. Noise Health. 2001;3:49–64. 88. Riva C, Donadieu E, Magnan J, et al. Age-­related hearing loss in CD/1 mice is associated to ROS formation and HIF target proteins up-­ regulation in the cochlea. Exp Gerontol. 2007;42:327–336. 89. Liao C, Carlson BA, Paulson RF, et al. The intricate role of selenium and selenoproteins in erythropoiesis. Free Radic Biol Med. 2018;127:165–171.

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90. Verma S, Hoffmann FW, Kumar M, et al. Selenoprotein K knockout mice exhibit deficient calcium flux in immune cells and impaired immune responses. J Immunol. 2011;186:2127–2137. 91. Sengupta A, Lichti UF, Carlson BA, et al. Selenoproteins are essential for proper keratinocyte function and skin development. PLoS One. 2010;5:e12249. 92. Schweikert K, Gafner F, Dell’Acqua G. A bioactive complex to protect proteins from UV-­induced oxidation in human epidermis. Int J Cosmet Sci. 2010;32:29–34. 93. Azevedo MF, Barra GB, Naves LA, et al. Selenoprotein-­related disease in a young girl caused by nonsense mutations in the SBP2 gene. J Clin Endocrinol Metab. 2010;95:4066–4071. 94. Catli G, Fujisawa H, Kirbiyik O, et al. A novel homozygous selenocysteine insertion sequence binding protein 2 (SECISBP2, SBP2) gene mutation in a Turkish boy. Thyroid. 2018;28:1221–1223. 95. Schomburg L, Dumitrescu AM, Liao XH, et al. Selenium supplementation fails to correct the selenoprotein synthesis defect in subjects with SBP2 gene mutations. Thyroid. 2009;19:277–281. 96. Carlson BA, Yoo MH, Tsuji PA, et al. Mouse models targeting selenocysteine tRNA expression for elucidating the role of selenoproteins in health and development. Molecules. 2009;14:3509–3527. 97. Shetty SP, Copeland PR. Selenocysteine incorporation: a trump card in the game of mRNA decay. Biochimie. 2015;114:97–101. 98. Schoenmakers E, Carlson B, Agostini M, et al. Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis. J Clin Invest. 2016;126:992–996. 99. Hatfield D, Lee BJ, Hampton L, et al. Selenium induces changes in the selenocysteine tRNA[Ser]Sec population in mammalian cells. Nucleic Acids Res. 1991;19:939–943. 100. Diamond AM, Choi IS, Crain PF, et al. Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec). J Biol Chem. 1993;268:14215–14223. 101. Kelley LA, Sternberg MJ. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc. 2009;4:363–371.

67 Thyroid Hormone Action Gregory A. Brent and Anthony N. Hollenberg

OUTLINE Components of Thyroid Hormone Action, 1128 Regulation of Ligand Availability in Development and the Adult, 1128 Thyroid Hormone Binding to Serum Proteins, 1129 Local Regulation of Thyroid Hormone Availability, 1129 Thyroid Hormone Receptors and Their Response Elements, 1130 Thyroid Hormone Receptor Coregulatory Proteins, 1132 Positive And Negative Gene Regulation By Thyroid Hormone, 1134 Thyroid Hormone Receptor Posttranslational Modifications, 1135 Nongenomic Thyroid Hormone Action, 1135 Thyroid Hormone Tissue-­Specific Actions, 1135 Thyroid Hormone Receptor Isoform–Specific Expression, 1135 Growth, 1136 Stem Cell Proliferation, 1136

Thyroid Hormone and Somatic Growth, 1136 Brain, 1136 Heart and Blood Vessels, 1137 Skeletal Muscle, 1137 Skeletal System, 1137 Small Intestine, 1137 Metabolic Regulation and Actions in Liver, 1137 White and Brown Adipose Tissue, 1138 Skin, 1138 Lessons from Resistance to Thyroid Hormone Receptor Beta and Alpha, 1138 Resistance to Thyroid Hormone Beta, 1139 Resistance to Thyroid Hormone Alpha, 1139 Summary and Future Directions, 1139

 

COMPONENTS OF THYROID HORMONE ACTION Thyroid hormone (TH) action is mediated by the two principal THs, thyroxine (T4) and triidothyronine (T3), that are synthesized within thyroid follicular cells such that the human adult thyroid synthesizes and releases THs in a ratio of approximately 85% T4 and 15% T3. TH signaling is regulated at multiple levels (Fig. 67.1). Circulating serum binding proteins deliver T4 and T3 to tissues, and binding proteins help to ensure consistent hormone delivery throughout the body. Higher hormone levels are generally correlated with increased TH response. However, there are differences in availability of hormone in peripheral tissues that are the consequence of variations in cellular transport and local conversions of T4 to T3 by the deiodinase enzymes.4 In some tissues, as best characterized in the brain, this tissue uptake is regulated by specific TH transporters.2,3 TH receptors (TRs) mediate the majority of TH signaling by nuclear actions and regulate gene expression. Tissue sensitivity is further adjusted by expression of coregulatory proteins that interact with TRs. The relative activity of these pathways, and the variation by tissue type, means that the correlation between plasma hormone levels and response is not absolute, consistent with the variety of clinical manifestations of thyroid disease. All components of TH signaling will be described, as well as the associated defects that have been reported with the various TH signaling pathways.

*Portions of this chapter were taken from an earlier edition, authored by Dr. Fredric E. Wondisford, and these contributions are acknowledged.

1128

KEY POINTS  • Thyroid hormone signaling is influenced by ligand availability, tissue uptake, nuclear receptor isoform type, and interaction with corepressors and coactivators.

REGULATION OF LIGAND AVAILABILITY IN DEVELOPMENT AND THE ADULT TH is required for normal brain and somatic development, and regulation of ligand availability occurs at multiple levels. While TH synthesis is covered in another chapter, it is important to note that the fetal thyroid (in particular the follicular cells) develops from anterior endoderm and is functional in context of TH synthesis in the late stages of the first trimester.5 Until that time, the developing fetus is dependent upon maternal THs, which cross the placenta in small amounts. The importance of the maternal transfer of TH for development is best delineated by syndromes of congenital hypothyroidism, where TH synthesis is not possible.6,7 As long as TH therapy is begun at birth, fetal development, including neurologic development, is normal. In contrast to the ligand deficiency, which is compensated by maternal TH, fetuses with rare defects in the TH transporter (monocarboxylate transporter 8 [MCT8]) are born with significant irreversible neurologic deficits due to an inability of maternal or fetal T4 to impact the brain at critical stages of development.8

CHAPTER 67  Thyroid Hormone Action

Bloodstream

T3

T3

T3

Albumin T4 T4

MTC8/10 OATP1

T3

Dio1/Dio3 Dio1/Dio2 T4

T2

TR

T4

Nucleus

RX R

T3

1129

TRE

Dio1/Dio3 T4

Dio1/Dio2 rT3

TBG

R TT

R T TR TT

TR T4 T T4

AAAAA

mRNA

Protein

Fig. 67.1  Triiodothyronine (T3) modulates gene expression in virtually every vertebrate. Thyroxine (T4)/ T3 circulates attached to serum proteins, including thyroxine-­binding globulin (TBG), transthyretin (TTR), and albumin. A small fraction of circulating T4 is free to be transported into the cytoplasm, where it is activated to T3 by outer-­ring deiodination catalyzed by type 1 deiodinase (Dio1) or type 2 deiodinase (Dio2). The resulting T3 is thought to be diffused into the nucleus to bind the thyroid hormone receptors (TRs). Upon binding to the TRs, T3 modulates the rate of mRNA synthesis, and ultimately the protein levels of thousands of genes in virtually every cell. RXR, Retinoid X receptor; TRE, thyroid hormone response element. (From Mendoza A, Hollenberg AN. New insights into thyroid hormone action. Pharmacol Ther. 2017;173:135–145.)

THYROID HORMONE BINDING TO SERUM PROTEINS TH circulates predominantly bound to serum protein, which provides a significant reservoir for distribution to target tissues (Fig. 67.1). Both major forms of TH (T4 and T3) are transported in the circulation in complex with plasma proteins.1 The ratio of total T4 to T3 in plasma is approximately 60:1. This is higher than the 15-­fold ratio of T4 to T3 that is initially secreted by the thyroid gland, because of greater plasma binding of T4 versus T3, resulting in greater clearance rates of T3. Approximately 99.98% of total circulating T4 and 99.7% of T3 form noncovalent interactions with serum proteins: mostly thyroxine-­ binding globulin, but to a lesser extent transthyretin (also referred to as thyroxine-­binding prealbumin) and albumin. The fact that less T3 circulates in complex with plasma proteins means that the ratio of free T4 to T3 is around 4-­fold, with typical circulating free hormone levels around 20 picomolar (pM) and 6 pM, respectively. The free fraction is considered biologically active and can enter target tissues.

LOCAL REGULATION OF THYROID HORMONE AVAILABILITY As outlined, thyroid follicular cells synthesize primarily T4 but also some T3. Critical, however, to the development of certain tissues is the amount of locally available T3, which is the primary driver of the physiologic response to the THs due to its genomic signaling capabilities. The regulation of local cellular ligand availability is mediated by the availability of circulating of THs in conjunction with: 1) cellular transporters that allow for the entry of T4 and T3 into cells, and 2)

deiodinases that determine the amount of intracellular T3 made available for genomic and nongenomic signaling (Fig. 67.1). The importance of specific cell surface TH transporters came to light in 2003 when MCT8 was identified, in cloning experiments performed with Xenopus oocytes, as being able to transport T4 and T3 across cell membranes through a channel specific to the iodothyronines.9 Soon after the identification of the MCT8, its relevance in humans was quickly identified based upon the fact that relevant mutations that impaired its function were discovered in patients with the Allan–Herndon–Dudley syndrome (AHDS), a rare X-­linked syndrome leading to a severe intellectual and motor delay syndrome in affected males.10,11 Indeed, besides the severe clinical syndrome often resulting in early mortality in this syndrome, AHDS patients had long been characterized by the presence of relatively normal thyroid-­ stimulating hormone (TSH) with a low T4 and high T3. Extensive work on the function of the MCT8 suggests that the syndrome is the result of severe neurologic hypothyroidism mediated by the inability of THs to cross the blood–brain barrier and neurons in utero, leading to the clinical syndrome after birth. The peripheral TH abnormalities are at least partially explained by a central resistance to feedback at the level of the hypothalamus and pituitary in the regulation of TSH production, coupled with the requirement of the MCT8 for the movement of T4 out of the thyroid follicular cell via the basolateral membrane into the circulation, leading to enhanced thyroidal T3 production and release. In addition, the consumption of available T4 by the type 1 deiodinase (Dio1; see later) in the liver may also play a role in its low levels and the elevated T3 levels, and there may also be a role for enhanced renal metabolism in AHDS patients.12 Importantly, if TH analog therapy is used in patients with AHDS,

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the circulating levels of T3 fall with proper regulation of central TSH production.13 While MCT8 is the best-­characterized TH transporter, other family members exist, and their cell-­specific expression can certainly help to determine cellular TH levels. Currently, many other TH transporters have been identified, including monocarboxlate transporter 10 (MCT10), organic anion transporting polypeptide 1C1 (OATP1C1), and SLC17A4. Interestingly, a mutation in OATP1C1 has been identified in a teenager with neurocognitive and motor deficits. Its further importance as a transporter became apparent in mouse studies, where MCT8 knockout mice were found to have a normal neurologic phenotype likely because of the presence of OATP1C1 that allowed for TH transport into the brain.13a Importantly, OATP1C1 does not appear to play a developmental role in TH transport in humans. Significantly, MCT8/OATP1C1 double knockout mice develop severe neurologic hypothyroidism akin to that seen in AHDS.14 In addition to their role as transporters of T4 and T3 into cells, it is likely that TH transporters also play a role in the efflux of THs from cells, and this in particular may be the role of MCT10. Much work remains to be done in order to elucidate the cell-­specific mechanisms of TH transport. Once TH reaches a particular cell type, the resulting T3 availability for signaling is mediated by the deiodinase family. Also, as reviewed in another chapter, the deiodinases can be classified into activating and deactivating subtypes in context of their ability to produce T3. While Dio1, which is preferentially expressed in liver, can metabolize T4 to either T3 or the inactive rT3, its action is primarily to produce T3. Similarly, the type 2 deiodinase (Dio2) plays a significant role in T3 production in many tissues, including neurons, the cochlea, and skeletal muscle (Fig. 67.1). In contrast, the type 3 deiodinase (Dio3) inactivates T4 to rT3 and T3 to T2 in a variety of tissues, especially developmentally, but is also expressed ectopically in the syndrome of infantile hepatic hemangiomatosis, leading to “consumptive hypothyroidism” and demonstrating its role. Certainly the fine tuning of expression of intracellular deiodinases play a significant role in T3 availability for signaling.15

THYROID HORMONE RECEPTORS AND THEIR RESPONSE ELEMENTS The majority of the actions of the THs are mediated by the interactions of T3 with its cognate nuclear receptors, the thyroid hormone receptor (TR) isoforms (Fig. 67.2). While the bulk of T3 action is mediated by the genomic actions of the TRs, evidence is certainly accumulating that there are fast-­acting nongenomic actions of T3 that are mediated by the TRs via their actions in the cytoplasm. Evidence of the nuclear actions of T3 first came into view in the 1960s, when Tata demonstrated the significant action of T3 on RNA synthesis in the liver and the requirement for RNA synthesis for the metabolic effects of T3.16 Subsequent work by Oppenheimer and Samuels in the 1970s demonstrated specific nuclear binding sites for T3 in rat liver, paving the way for the isolation and cloning of the TRs. Interestingly, the pioneering biochemical work in the 1960s and through the 1980s played little role in the first identification of a functional TH receptor. In 1985, the glucocorticoid receptor was the first nuclear receptor identified, and its sequence seemed analogous to the v-­erb-­A gene of the avian erythroblastosis virus.17 Subsequently, the Vennstrom laboratory identified human versions of v-­erb-­A, termed c-­erb-­A, which is now known as thyroid receptor α, or Thra, in humans.18 At the same time, the Evans laboratory identified a homologous receptor that is now known as the thyroid receptor β, or Thrb, in humans.19 Both receptors were shown at the time to be nuclear and to bind T3 with high affinity. Importantly, the functional concept of similar receptors engaging with a variety of steroid and THs

Activation function-1

DNA binding

A

1

1

B

52

52

Hinge C

120

Ligand binding/ Dimerization D/F

410

120

TRα1

492

370

TRα2

1

94

174

147

227

461 TRβ1

1

514 TRβ2

Fig. 67.2  Schematic representation of the thyroid hormone receptor isoforms. Thyroid hormone receptors (TRs) are encoded by the THRA and THRB genes, which produce multiple isoforms. Depicted are the major isoforms that modulated the actions of triiodothyronine (T3), with exception of TRα2, which does not bind T3 but acts as negative dominant by competing for the thyroid hormone response element with other TRs. TR isoforms share high sequence homology within their functional domains. (From Mendoza A, Hollenberg AN. New insights into thyroid hormone action. Pharmacol Ther. 2017;173:135–145.)

brought about a new era in physiology and therapeutics that continues through this day based on the ability to develop compounds that acts as antagonists or agonists to these nuclear receptors. Soon after the identification of the original TR isoforms it subsequently became clear that both human Thra and Thrb were subject to splicing and to the creation of different isoforms (Fig. 67.2). The TRβ gene, located on chromosome 3, produces two major isoforms termed TRβ1 and TRβ2 that differ structurally in their N-­termini. In contrast, the TRα gene undergoes splicing in its C-­terminus to generate a nonbinding TH receptor isoform termed TRα2, while the principal TH receptor produced from this gene is TRα1. Other isoforms of the TRs have been detected in rodents but are not found or are not functional in humans.20,21 Importantly, the tissue of expression more than likely determines the role of the TH receptor isoform, rather than the isoform itself. However, the cell-­specific expression of each of the TR isoforms has been difficult to delineate because of their relatively low levels of expression, and the ability of isoform-­specific antibodies to identify in vivo expression is not strong. Recent genetic approaches to tagging the endogenous alleles in mice has been successful and will yield important information in the future.22,23 Most of our knowledge of the actions of individual isoforms comes from pioneering mouse genetic studies coupled with the human syndrome of resistance to TH (RTH). TRβ1 is specifically expressed in the liver, where it is a key regulator of cholesterol metabolism and hepatic fat content. Indeed, TRβ1 has long been sought as and continues to be a drug target for the treatment of metabolic diseases, including hypercholesterolemia and nonalcoholic fatty liver disease and its complications. TRβ2 functions specifically in the hypothalamus and the pituitary to regulate the central components of the thyroid axis, including thyrotropin-­releasing hormone (TRH) expression in the paraventricular nucleus of the hypothalamus and TSH α and β subunit expression in the pituitary (Fig. 67.3). Interestingly, the exact mechanism by which TRβ2 regulates TRH remains unclear. It is presumed that TRβ2 is expressed in the same neurons as TRH, but because of the lack of reagents available for detecting cell-­specific TRβ2, this remains unclear and is an area of important investigation. In addition to its role centrally, TRβ2 plays a key role in the retina, where it controls the proper differentiation of rods and cones to allow for color vision. In contrast to their separate roles, TRβ1 and TRβ2

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CHAPTER 67  Thyroid Hormone Action

Hypothalamus

Brain Cognitive function, mood and movement

TRH

TRβ

TRβ2

Pituitary Thyrotrophs

Adipose Muscle TSH

T3 Dio1 Dio2

Thyroid Gland T4

Cholesterol metabolism, SHBG synthesis

Liver

Bone

Intestine

Strength, recovery from injury, regulation of ion channels and myosn

Lipolysis, lipogenesis

TRα Heart

Skeletal development, bone resorption and formation Development, absorption, motility

Chronotropy, inotropy, regulation of ion channels and myosin

Fig. 67.3  Thyroid hormone receptor (TR) isoforms and tissue-­specific actions. Tissues with predominant TRα or TRβ expression and effects of thyroid hormone signaling. SHBG, Sex hormone–binding globulin; Dio1, type 1 5’-­deiodinase; Dio2, type 2 5’-­deiodinase.

appear to collaborate in the regulation of hearing, based on their function in the cochlea.24 TRα1, while present in the liver and pituitary, plays little role in isolation in these tissues. Its major roles include actions in the atrium and ventricle of the heart, as well as in bone, where its expression in a variety of cell types in that tissue is likely to be more than 10-­fold more than that of the TRβ isoform.25 In addition, TRα1 has principal actions in the gastrointestinal tract, where it plays a strong role in function and cell renewal. Additionally, TRα1 is important in the regulation of many actions of THs in the brain, but again, cell-­specific actions are limited by available tools. While TRα2 has always been viewed as a potential dominant inhibitor of TRα1, its role in vivo does not appear to be paramount in determining cell-­ specific TH actions. Taken together, it is clear that each of the isoforms have cell-­specific functions, likely based on their ratio of expression. However, it remains to be determined in future work whether the isoforms’ structural differences delineate a unique genomic response. This would ideally require experiments where TRβ1 would replace TRα1 in a particular target tissue. In experiments performed in cell culture where TRβ1 and TRα1 were expressed at a similar level, there was evidence of isoform specificity in terms of target activation.26 As outlined, the TRβ and TRα isoforms are distinct at the nucleic acid and amino acid levels but share a high degree of structural homology that is also shared with other members of the nuclear receptor superfamily. The TRs all possess both a similar DNA-­ binding domain in the central region of the protein, followed by a ligand-­binding domain that is specific for T3 at nM concentrations (with TRα1 having a slight degree of enhanced sensitivity for T3) and overlaps with the activating function 2 domain. What differentiates the TR isoforms are unique N-­terminal domains (also referred to as activating function 1 [AF-­1] domains) that are relatively unstructured but may allow for differential DNA binding and also possess independent transcriptional properties. The DNA-­binding domains of the TR isoforms consist of two zinc finger protein domains that mediate contact with DNA and allow for receptor binding. The specificity of TR binding to target DNA elements is encoded for by the P-­box in the first zinc finger. The importance of the P-­box is

DR-4

4

IP-6

6

Pal Half-site GH trimer Multiple TRE

4 4

4

4 6 Fig. 67.4  The diverse organization of thyroid hormone response element (TREs). A schematic of orientations (arrows) and spacing (numbers between arrows) of AGGTCA half-­sites observed in natural TREs. With multiple TREs, the orientations can differ between elements.

highlighted by experimental mutations in mouse models that have allowed investigators to interrogate the role of the TR in the absence of its ability to bind DNA.27 The second zinc finger, in conjunction with the C-­terminal extension (or hinge region), also plays a critical role in DNA response element recognition of the TR isoforms and in their ability to heterodimerize with the retinoid X receptor (RXR) isoforms, their preferred partners when bound to DNA response elements. Importantly, this interaction with RXR in all cases has RXR bound 5’, or upstream, to the TR. This interaction via the DNA-­ binding domain and C-­terminal extension and hinge region with the TR and RXR, confirmed in structural studies, likely explains the preference of the RXR/TR heterodimer for response elements that contain two classic nuclear receptor half-­sites (AGGTCA) spaced by four nucleotides (Fig. 67.4). This is in contrast to other nuclear receptor heterodimer pairs such as the vitamin D receptor with RXR and the retinoic acid receptor isoforms with RXR, which prefer elements that have spacings of three and five base pairs, respectively.28

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PART 6  Thyroid

The majority of TR isoform binding is likely as a heterodimer with RXR, but it remains possible that the TR can also function to bind DNA as a homodimer, or even as a monomer. To exerts its actions, the TR must engage T3 via its ligand-­binding domain (LBD). Structural studies of the TR LBD demonstrate that it, like other nuclear receptors, contains a ligand-­binding pocket that is surrounded by 12 alpha helices29 (Fig. 67.5). That it exerts functions both in the presence and absence of T3, helix 12 (H12) demonstrates an ability to be in an open position in the absence of ligand. When T3 moves into the ligand-­ biding pocket, H12 shifts and closes over the pocket.30 This movement of H12 creates two distinct grooves to allow for separate groups of TR-­ interacting proteins to engage the receptor, either in the presence of or absence of T3. Most likely, in the presence of low levels of T3, the unliganded structure is preferred, while in the presence of excess ligand the liganded structure is preferred (Fig. 67.5). The importance of a variety of these helices in forming the ligand binding pocket for T3 is shown by the many mutations found RTH syndrome, where mutations in the LBD usually lead to a reduced or absent ability to bind T3.31 Importantly, structural and biochemical studies have determined the nature of the RXR/TR heterodimer and its ability to engage with target response elements in vitro and in cell culture models. Indeed, these techniques identified a variety of T3 response elements such as the DR4 element described previously, as well as palindromic and inverted palindromic arrangements of the AGGTCA half-­site. However, identification of the true response elements for the TR in vivo was not possible until the

LBD

LBD

Hydrophobic cleft

Hydrophobic cleft H12

H12

ID motif (corepressor) NR box (coactivator) lxxllxxLM LxxLL Binds extended Binds upper part of hydrophobic cleft hydrophobic cleft + H12 Fig. 67.5  Helix 12 position and coregulator binding. Thyroid hormone receptor TR C-­terminal helix 12 is displaced away from the hydrophobic cleft without bound hormone, exposing a large hydrophobic cleft, which serves as an interaction site for α-­helical motifs from corepressors (IDs). With hormone, H12 packs over the lower part of the corepressor binding surface, blocking corepressor binding and creating a new binding site for coactivator NR boxes (LxxLL). LBD, Ligand-­binding domain. Tbl1x

CBP

O

T3

H3C

SRC1

O

MED1

TR

TR

R

Beginning in the 1990s it became clear that the TR recruited activity that was dependent upon the presence or absence of ligand. In these

HDAC3

NCoR1 RX

Thyroid Hormone Receptor Coregulatory Proteins

R

Liganded

RX

Unliganded

development of in vivo chromatin immunoprecipitation, which allows for the identification of DNA sequences that engage the TR directly. Experiments performed in mouse liver using an overexpressed TRβ1 isoform that can be biotinylated in vivo, and thus precipitated by streptavidin, demonstrated thousands of binding sites for the TR, with many of the sites located at great distances from regulated genes or within introns. Classically regulated T3 targets, such as the Dio1 gene, was shown to have TR binding sites in its first intron, while the spot 14 and fatty acid synthase genes had more traditional sites within 10 kB of their transcription start sites. Importantly, using motif enrichment tools, the most common binding site seen was the DR+4 element, and there was good overlap with previously known RXR binding sites, demonstrating the action of the heterodimer in vivo. However, not all TR binding sites were also occupied by RXR, raising the possibility that the TR homodimer and monomer could also play a role in TH action. While DR+4 sites were associated with genes induced or positively regulated by T3, there were fewer binding sites associated with genes negatively regulated by T3.32 Similar results were seen in experiments in mouse liver utilizing an antibody against the TR itself, again with the DR+4 motif predominating.33 Interestingly, in both of these studies a percentage of TR-­binding sites were enhanced in the presence of T3, suggesting that the traditional view of the TR being engaged on DNA response elements irrespective of the concentration of ligand is not correct for every response element. The significance of the DR+4 motif in TR binding has been further supported by cell culture studies using tagged TRs that could either be biotinylated or immunoprecipitated. Finally, a recent mouse model utilized a strategy whereby the endogenous TRβ1 allele was tagged at its N-­terminus by hemagglutinin (HA) sequences and would thus be expressed with a tag that could be immunoprecipitated by an antibody directed against HA. In liver chromatin, this model confirmed the importance of the DR+4 element as the primary TRβ1 binding site but also demonstrated a role for the DR+4 binding site in mediating negative regulation or the downregulation of genes in the presence of T3. Interestingly, this novel model also suggested that T3-­mediated recruitment of the receptor did play a significant role across the genome.23 Thus, these in vivo unbiased approaches to examining TR binding to DNA are beginning to articulate a picture whereby the DR+4 element is paramount for TR binding, especially in liver. Further work will be required to demonstrate that the binding sites identified (especially in the context of negative regulation) actually mediate regulation of the gene in closest proximity. Indeed, it remains possible that distinctly different binding sites that are less frequently seen and are at great distances from target genes may play critical roles in regulation.

GTF

CH3 RNAPol2

Fig. 67.6  CoRepressors and CoActivators are recruited to the TR based on the presence of ligand. In the absence or in the presence of limiting amounts of T3 the TR recruits the nuclear corepressors (ie NCoR1) as part of a multiprotein complex to repress transcription. In the presence of T3 the corepressor complex is dismissed and coactivators including SRC1 are recruited to activate trasncription. Coregulators, corepressors, and coactivators harbor nuclear receptor–interacting domains. NCoR1 and SMRT harbor three nuclear receptor interacting domains; N3 and N2 interact preferentially with unliganded thyroid hormone receptor (TR), whereas steroid receptor coactivator (SRC) harbors an RID region that interacts with the TR upon adoption of the liganded conformation. (From Mendoza A, Hollenberg AN. New insights into thyroid hormone action. Pharmacol Ther. 2017;173:135–145.)

CHAPTER 67  Thyroid Hormone Action studies the TR-­LBD was fused to a heterologous DNA-­binding domain (in this case yeast Gal4), and its function could be altered by the presence of a full-­length TR that would compete for the activity allowed for by the Gal4-­TR hybrid.34 Central to these experiments was the long-­ held observation that, in cellular transcriptional assays on artificial DNA-­binding elements, the unliganded TR was a potent repressor of gene expression for positive target genes, while it activated the expression of the regulatory elements of negatively regulated target genes. With the addition of T3, the TR then activated positively regulated genes and repressed negatively regulated genes, demonstrating that the TR possessed repressing and activating functions, depending upon the presence of ligand. The importance of the function of the unliganded TR was further highlighted by genetic studies demonstrating that mice could survive the deletion of all TRs, while mice that lacked TH at birth that was not replaced could not survive.35 To identify proteins that could mediate differential functions of nuclear receptors and the TR based on the presence or absence of ligand, pivotal experiments were performed initially using an immobilized estrogen receptor LBD to screen radiolabeled cellular lysates for target-­interacting proteins or yeast-­two hybrid assays using the LBD of a nuclear receptor as bait to identify interacting proteins either in the presence or absence of ligand. The results of these experiments and many others that followed was the identification of groups of proteins that could interact with the TR based on ligand availability, and as a field include hundreds of proteins. Strikingly, as will be outlined later, a central quality of the majority of these interacting proteins was an ability to enzymatically alter histone acetylation or methylation, implying that the TR functions as an epigenetic switch to modify chromatin in the presence or absence of its ligand. Two general classes of proteins that interact with the TR are fundamental to its actions and are termed corepressors and coactivators (Fig. 67.6).36,37 As their names suggest, they offer distinct regulatory functions based on their recruitment. Importantly, the structure of the TR-­LBD, with the location of H12, predicts how they may be differentially recruited. Indeed, coactivators, which include members of the steroid receptor coactivator (SRC-­1, 2, and 3) family, CREB-­binding protein (CBP)/p300 and coactivator-­ associated arginine methyltransferase 1 (CARM1), and protein arginine N-­methyl transferase 1 (PRMT1) all contain motifs termed nuclear receptor–interacting domains, which are specified by leucine-­rich motifs (LxxLL) that dock in a groove created by a closed H12 as T3 binds.38 In contrast, the corepressors heralded by nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of the retinoic acid and TH receptor (SMRT or NCoR2) possess in their C-­terminus isoleucine-­rich domains (LxxH/ IxxII), also termed CoRNR boxes, which bind to the groove in the unliganded TR created by the open position of H12.39,40 It is likely that, in the presence of limiting amounts of T3 or in the hypothyroid state, the majority of TRs exist in the unliganded state, and as T3 concentration increases, the liganded-­conformation is favored, corepressors are dismissed, and coactivators are recruited. However, mouse genetic experiments and human genome-­wide association study (GWAS) data suggest that the amount of specific coregulator present dictates the ability of the TR to be activated or repressed. For example, in humans with excess NCoR1, T4 levels must be higher to achieve normal TSH levels. Similarly, mice that lack functional NCoR1 have lower TH levels with a normal TSH. Taken together, these data suggest that the corepressor/coactiviator ratio in vivo regulates sensitivity to TH.41,42 As discussed, the principal coactivators of the TR include members of the SRC family, CBP/P300, the methyltransferases, and also members of the Mediator complex. There are likely others also that play a role, depending on the target gene and cell type where T3-­dependent regulation is occurring. The SRC family is recruited to the T3-­bound

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RXR/TR heterodimer via their nuclear receptor–interacting domains, which demonstrate specificity to particular nuclear receptors. It is likely that CBP/p300 and other coactivators are scaffolded on the principal SRC–TR interaction, and thus play an essential role. The Mediator complex appears to link the activated TR complex to RNA polymerase. It has also been suggested in biochemical assays that SRC-­1 makes contact with TR isoforms via the N-­terminal or AF-­1 domain, which could play a role in T3-­mediated negative regulation. Genetic analysis of coactivator function in mice has demonstrated a critical role for SRC-­1 in T3 action both in the liver and centrally, where these mice display a phenotype of resistance to TH, implying that SRC-­1 is required for determining the set point of the hypothalamic-­pituitary-­ thyroid (HPT) axis.43 When deleted in conjunction with individual TR isoforms, SRC-­1 has also been shown to play a role in growth and body weight that could be T3-­dependent. However, SRC-­1 may not play a role in the regulation of heart rate by T3. In contrast to SRC-­1, murine knockout studies of SRC-­2 and SRC-­3 demonstrate no specific defect in T3 action, though they have numerous other metabolic phenotypes. Much work remains to be done to ascertain the specific coactivators that allow for the cell-­specific actions of TR isoforms.36 The principal TR corepressors NCoR1 and SMRT also form a multiprotein complex that mediates histone deacetylation and transcriptional repression. The large, 270-­kD proteins are paralogs and share homology in terms of their domain structure but have specificity in terms of their ability to interact with nuclear receptors, including the TR. The N-­terminal regions of NCoR1 and SMRT contain repression domains that recruit proteins such as G protein pathway suppressor (GPS2) and transducin β-­like 1 (TBL1 or TBL1X) and its homolog, TBL-­related 1 (TBLR1or TBL1XR1), which all together form the core repression complex, together with HDAC3, which is recruited and activated by a specific domain (deacetylase activation domain [DAD]) present in both NCoR1 and SMRT. Mice that lack a functional DAD domain can no longer mediate deacetylation but still have repressive properties, indicating that NCoR1 and SMRT have activity independent of their ability to recruit and activate HDAC3. The C-­termini of NCoR1 and SMRT contain three nuclear receptor–interacting domains that mediate interactions with nuclear receptors. Early biochemical studies suggested a preference of NCoR1 for the TR, but testing of this was hampered in vivo by the fact that global deletion of NCoR1 or SMRT is embryonic-­lethal in the mouse. To get around this lethality, a mouse model was developed that created a hypomorphic NCoR1 allele, termed NCoRΔID, that encoded the full NCoR1 protein except for the two interacting domains that are required for interactions with the TR.44 Using this targeted allele, tissue-­specific or global models could be built that would live and express an NCoR1 protein that was nonfunctional in terms of TR action. In each model developed where NCoRΔID was expressed in lieu of NCoR1, mice displayed evidence of increased sensitivity to T3. For example, in mice where NCoRΔID was expressed in all tissues, both T4 and T3 levels were significantly low in the sitting of normal TSH, yet energy expenditure was increased, growth was normal, and T3-­mediated gene expression was normal, all of which is consistent with increased sensitivity to T3. Further proof of this concept was provided by mice with the PV mutation causing RTH present in their Tβ or TRα alleles. When crossed with NCoRΔID mice, the syndrome of RTH was corrected in both models, again consistent with NCoR1 mediating sensitivity to T3.45 Similarly, in NCoRΔID mice that lacked SRC-­1, increased sensitivity to T3 was reversed, confirming the notion that the balance between available corepressors and coactivators determines sensitivity to T3.46 Further support for this hypothesis comes from the identification of a variety of NCoR1 splice variants in vivo that, in turn, alter hormone sensitivity.47 The importance of this observation likely carries

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PART 6  Thyroid

TABLE 67.1  Thyroid Hormone Action Classification Action Type

Category

Description

Type 1 Type 2

TR-­dependent signaling of TH with direct binding to DNA TR-­dependent signaling of TH with indirect binding to DNA

Type 3

TR-­dependent signaling of TH without DNA binding

Type 4

TR-­independent TH signaling

Canonical TH signaling, TR binds directly to consensus TRE TR indirectly modifies gene expression by chromatin remodeling, possibly tethered to DNA by other transcription factors Cytoplasmic TR can activate kinases and promote membrane signal transduction pathways TH interaction with membrane receptors, including integrins and actin polymerization

TR, Thyroid hormone receptor; TH, thyroid hormone; TRE, thyroid hormone receptor response element. From Flamant F, Cheng SY, Hollenberg AN, et al. Thyroid hormone signaling pathways: time for a more precise nomenclature. Endocrinology. 2017;158:2052–2057.

through to humans, where GWAS and quantitative trait locus studies demonstrate that increased free T4 levels are associated with increased NCoR1 in order to overcome the decreased sensitivity due to higher levels of NCoR1.42 Interestingly, human mutations in TBLX1 have also been described, and in these patients TH levels are low and the TSH is normal. Currently, they are classified as having central hypothyroidism, but it remains possible that, if the corepressor complex is dysfunctional, they would in fact have increased sensitivity to TH.48 The specificity of NCoR1 for the TR has been further confirmed in mouse models that either delete SMRT action in the liver or globally in adulthood to get around the embryonic lethality of early SMRT deletion. In both of these models, SMRT had no effect on the regulation of T3 target genes or in the systemic regulation of the HPT axis.49,50

POSITIVE AND NEGATIVE GENE REGULATION BY THYROID HORMONE The majority of TH’s actions are mediated by its genomic regulation via the TR isoforms and their ability to recruit coregulators to modify the histone environment to stimulate or repress target genes, KEY POINTS  • The structure of the thyroid hormone receptor isoforms ligand-­binding domain changes secondary to the amount of ligand present, which determines the type of coregulator complex recruited and confers tissue-­level sensitivity.

referred to as type 1 action. While each of the players are now known, it is likely that multiple mechanisms regulate target genes. Indeed, the regulation of TH action by the TR has been classified into three distinct subtypes: 1) direct regulation of target genes by bound TR; 2) regulation of gene expression by tethered TR, i.e., via another transcription factor; and 3) regulation that is independent of TR recruitment or due to nongenomic mechanisms (Table 67.1).51. A number of recent studies have addressed positive regulation using genetic mouse models and genome-­wide chromatin assays to clarify the mechanism by which T3 induces gene expression, particularly in the liver, where hundreds of genes are both upregulated and downregulated by T3.23,52,53 To date, most positive regulation by the TR is explained via its direct binding to genomic response elements (Fig. 67.1). TR action occurs at many regulated genes, whereby, in the absence of T3, there is decreased histone acetylation (H3K27 and H3K9) in regions in proximity to the regulated gene that correspond to a TR binding site. The acetylation status of these sites is controlled by the recruitment of

NCoR1 and HDAC3, and the presence of T3 leads to hyperacetylation via the accrual of coactivators such as SRC-­1 and CBP, leading to enhanced histone acetylation. Examples of such classically regulated targets include spot14, fatty acid synthase, and malic enzyme. Interestingly, many direct targets of the TR are repressed independently of NCoR1, such as the Dio1 or cyp17a1 genes, suggesting that either repression is mediated by a separate corepressor, or repression of these targets is mediated by a lack of TR binding that only occurs when T3 is present, which then leads to the recruitment of coactivators that mediate histone acetylation and gene expression. Importantly, chromatin immunoprecipitation analysis of histone acetylation discloses many areas of the genome that are acetylated in response to T3 but that are not near a TR binding site. It remains possible that remote TR binding sites act from a great distance to regulate these areas of histone acetylation and potentially target genes. While the mechanisms underlying positive regulation have become clearer with genome-­ wide techniques, the mechanisms underlying negative regulation by TH remain in question. Understandably, many studies have focused on how the TRβ isoform regulates TRH and TSH subunit gene expression, given the importance of this in determining the set point of the thyroid axis. The regulation of the central axis is known to be dependent on TRβ2 via its ability to bind to DNA, as mouse models that either lack the isoform or express a TRβ isoform that cannot bind DNA have evidence of central resistance to TH, with elevated TRH mRNA expression and increased TSH subunit gene expression. Furthermore, the coactivator SRC-­1 also appears to play a role in the regulation of the central axis, as its deletion leads to central resistance to TH. In contrast, expression of NCoRΔID in the absence of SRC-­1 reestablishes the proper set point of the axis, implying that TRH and TSH subunit gene expression is mediated by the TRβ2 isoform via its interactions with NCoR1 and SRC-­1. However, studies to date have not defined this at the cellular level in terms of demonstrating intact DNA-­binding sites for the TR in the hypothalamus or pituitary. Early experiments using cotransfection assays in heterologous cell lines identified TR binding sites in regions close to the transcriptional start site of the TRH and TSHβ genes. Indeed, site 4 in the TRH promoter recruits the RXR/TR heterodimer, while a strong monomeric TR binding site exists just downstream of the transcriptional start site of the TSHβ gene. However, the importance of these sites in vivo has been difficult to test because of the difficulty of isolating hypothalamic and pituitary cell types for genome-­wide analyses utilizing chromatin immunoprecipitation and other such techniques. Recent work, therefore, has focused on the liver, which is more accessible to in vivo approaches, to define the mechanism.

CHAPTER 67  Thyroid Hormone Action As outlined earlier, hundreds of genes are regulated negatively by T3 in the liver and follow the classic example of gene activation in hypothyroidism and then repression in hyperthyroidism. Deletion of TRβ isoforms in hepatocytes alone demonstrates its requirement for negative regulation in the vast majority of cases. Analysis of the requirement of TRβ binding in the vicinity of negatively regulated targets suggests that its binding is important for a significant number of genes, but not all. Furthermore, motif enrichment analysis has suggested the presence of either a DR+0 motif, or more recently a DR+4 motif, that would recruit the TR.23,32 While direct TR binding is likely important, there is also clearly a role for negative regulation via independent actions of the TR in context of inducing another pathway (i.e., microRNA regulation) or interacting with another transcription factor.54 The roles of the corepressors and coactivators are less clear, as other SRC isoforms appear to compensate for the loss of SRC-­1 when it is ablated, as negative regulation is preserved in the liver of SRC-­1 knockout mice. When NCoR1 is disrupted in the liver, negative regulation is also preserved, and SMRT appears to play little compensatory role. Still, recent work that demonstrates DR+4 TRβ binding sites in enhancers near negatively regulated genes suggests that it is the ratio of coactivators to corepressors recruited that determines target gene regulation, such that, in the hypothyroid state, there is enhanced coactivator recruitment leading to histone acetylation and enhanced gene expression, while the presence of T3 leads to enhanced corepressor recruitment and gene repression. Further in vivo work will be required to clarify the exact mechanisms of negative regulation.

Thyroid Hormone Receptor Posttranslational Modifications KEY POINTS  • Most positive regulation by the thyroid hormone receptor (TR) is explained via its direct binding to genomic response elements. TR action occurs at many regulated genes, whereby, in the absence of triidothyronine (T3), there is decreased histone acetylation (H3K27 and H3K9) in proximity to the regulated gene. The acetylation status of these sites is controlled by the recruitment of NCoR1 and HDAC3, and the presence of T3 leads to hyperacetylation via the accrual of coactivators such as SRC-­1 and CBP, leading to enhanced histone acetylation.

While the TR isoforms are strongly influenced by ligand both in terms of coregulator recruitment and DNA binding, it is also clear that other signaling pathways impact their function. The TRα isoform is known to possess a protein kinase A site within its DNA-­ binding domain that may interact with its ability to bind to DNA, though this has not been shown in vivo. Additionally, TRβ can be phosphorylated, but the significance is not known.55 Most interestingly, both TR isoforms can be sumoylated, with TRα having two sites and TRβ three.56 The mutation of sumoylation sites on either isoform impairs their ability to mediate ligand-­dependent gene activation or repression, potentially through aberrant or diminished coregulator recruitment. Furthermore, active introduction of TR isoform sumoylation mutants interfered with the normal differentiation of preadipocytes into adipocytes. Importantly, the sumoylation sites present on TRβ appear to govern distinct pathways involved in adipogenesis, including Wnt signaling and nuclear receptor signaling via coregulator recruitment.57

NONGENOMIC THYROID HORMONE ACTION While the majority of TH action is mediated by T3 engaging its cognate receptor in the nucleus, there have been significant advances

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in understanding rapid or nongenomic actions of T3 via a cytoplasmic TR isoform (Table 67.1). However, some actions of T3 are felt to occur too quickly to be explained by a genomic process. Indeed, in vitro and in vivo, when present in the cytoplasm, the unliganded TRβ can interact with p85, the regulatory subunit of PI3 kinase. This interaction, mediated by one of the zinc fingers in the DNA-­binding domain, is blocked by T3, and PI3 kinase signaling is induced. If the site necessary for PI3 kinase interaction is mutated in vivo in a mouse knockin model, normal nuclear TRβ signaling is preserved in terms of the HPT axis and induction liver target genes. However, synaptic strength and plasticity in hippocampal neurons studied in slices was significantly less in the mutant mice, implying that PI3 kinase activation via the TRβ isoform is critical.58 Further support for rapid nongenomic effects of the TR isoforms comes from more recent experiments, where in vivo mouse models were developed that possessed mutations in the first zinc finger of both the TRα and TRβ isoforms that prevented DNA binding, and T3 signaling was compared with wild-­type controls. Strikingly, TRβ mutant mice could still selectively regulate glucose and triglyceride lowering, and additionally temperature elevation, while TRα mutant mice could still regulate heart rate in response to in vivo circulating T3 levels. Importantly, the effects in the TRβ DNA-­binding mutant mice on glucose, triglycerides, and temperature were blocked in the mouse model where T3-­mediated PI3 kinase activation was inhibited. Taken together, these data suggest that it is likely that the TR isoforms can have noncanonical effects (so called type 3 signaling) that impact physiology and are likely regulated through the activation of PI3 kinase.58

THYROID HORMONE TISSUE-­SPECIFIC ACTIONS Thyroid Hormone Receptor Isoform–Specific Expression As described previously, TRs are expressed in essentially all tissues, although well-­defined tissues, such as liver, heart, and brain, express higher levels of TRs than other organs21,59 (Fig. 67.3). The estimates of TR isoform content across tissues have varied depending on the tool that is used to detect TR, which have included T3 binding, TR isoform mRNA levels, western blot with antibody to native TR protein, or introduction of specific immunogenic “tags” into the TR protein for detection by a standard antibody that has a high affinity for that tag. There are limitations to all of the approaches used, which result in wide variations in estimates of TR isoform content, especially those relying on mRNA measurement rather than protein. The affinity and specificity of antibodies for TR isoforms also varies, and there are additional differences in tissue TR content based on the species that is studied. A “knockin” strategy involves introducing a standard HA tag sequence into the Thra and Thrb genes in mice and then measuring protein content in various tissues with a high-­affinity antibody that recognizes the HA tag.22 TRα was the predominant form in all tissue, except liver, where TRβ was predominant. In metabolically-­ responsive tissues, including fat and muscle, TRα protein levels were 10 times higher than those of TRβ protein. In the brain, there were low levels of TRα and TRβ, but high levels of TRα2, which does not bind T3. TRβ2 was very high in the pituitary, as has been previously reported, but an interesting sex difference was seen, with TRβ2 content greater in females than males, and females had lower serum TSH and Tshb mRNA. TR isoform expression can also vary within a tissue. The heart has predominantly TRα in the atria, mediating the chronotropic actions of TH, and the ventricle contains more TRβ. The phenotypes of individuals with RTH beta and alpha have largely been validated as being associated with TR isoform–specific functions, which have also been identified in mouse, Xenopus, and zebrafish models.60 A further variation is TR intracellular

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PART 6  Thyroid

distribution. Although TR resides predominantly in the nucleus, there is rapid shuttling between the cytoplasm and nucleus that varies by specific TR isoform.61

Growth TH is necessary for proper growth and development of the fetus, as well as for normal child development, and exerts widespread influences on multiple aspects of metabolism and organ function in adults. TH regulation of gene expression in development is complex, and several important underlying principles have been identified.21,62 First, developmental defects that arise in hypothyroid mothers are not reversed by later hormone replacement, whereas adults with TH imbalances exhibit metabolic disturbances that in most cases can be reversed by restoring TH to correct levels. TH triggers key developmental events in defined temporal windows, with abnormalities seen with both deficient and excess TH, especially as has been shown in sensory development of the inner ear and retina.63 These developmental windows are regulated by specific expression of TR isoforms and the Dio2 enzyme, which activates T4 to T3, and the Dio3 enzyme, which converts T4 to the inactive rT3. TH-­regulated genes important for organ function in the adult, such as those involved in metabolic regulation and brain function, remain sensitive to alterations in hormone levels, and gene expression is linked to thyroid status. Gene expression patterns reveals that some genes are induced in multiple tissues, whereas other target genes are regulated in a manner that is highly tissue-­and gene-­specific. Another additional level of regulation is TH action on expression of microRNAs, an emerging area of TH regulation.64 Thus, TH is a primary regulator of some genes, but the hormone must cooperate with other factors to induce expression of other genes. An example is TH metabolic regulation, which involves “crosstalk” with other metabolic regulators, including peroxisome proliferator-­activated receptor (PPAR)α and PPARγ, as well as PPARγ coactivator 1-­α (PGC-­1α).65,66 Specific effects of TH will be discussed, arranged by the tissue or site of action.

Stem Cell Proliferation In most tissues, TH stimulates stem cell proliferation as well as differentiation, in contrast to morphogens, like retinoic acid, that predominantly promote differentiation.67 Direct TH stimulation of stem cell proliferation, predominantly mediated by TRα, includes stem cells in the intestine, skeletal muscle, pancreatic islets, heart, liver, adipose tissue, bone, and neurons67,68 (Table 67.2). In many tissues, such as skeletal muscle and pancreatic islet cells, the stimulation of stem cells requires TR, but is also regulated by local production of T3, by actions of Dio2 and Dio3.69 Stimulation of stem cell proliferation is important in development, but also to mediate regeneration after injury, as has been shown in skeletal muscle, cardiac muscle, and brain.70 These findings have led to an interest in TH treatment in a variety of conditions to promote regeneration after injury, such as traumatic brain injury, stroke, myocardial infarction, and muscle injury. KEY POINTS  • Stimulation of stem cell proliferation is important in development, but also to mediate regeneration after injury, as has been shown in skeletal muscle, cardiac muscle, and brain.

Thyroid Hormone and Somatic Growth TH deficiency in both the mother and fetus results in reduced somatic growth. Normal maternal thyroid status is sufficient to compensate for

fetal thyroid deficiency in congenital hypothyroidism, with maternal TH crossing the placenta from high to low levels in the fetus. Combined maternal and fetal hypothyroidism, as seen in profound iodine deficiency, results in irreversible somatic growth deficit. Hypothyroidism in children is associated with reduced height. TH directly stimulates production of growth hormone in some species, including mice and rats. In children with deficiency of both growth hormone and TH, treatment with both hormones works to restore normal growth.

Brain TH is required for normal brain development, initially from transport of maternal TH across the placenta and then from TH produced by the human fetus, at around 13 weeks, when the thyroid gland develops.71 Maternal TH can compensate for defective fetal thyroid gland development in the condition referred to as congenital hypothyroidism. The infant, however, must be identified by screening at birth and started promptly on TH replacement to support normal brain development. The combination of maternal and fetal TH deficiency, as is seen with profound iodine deficiency during development, is associated with profound and irreversible abnormalities of brain and nervous system development. TRα1 is the predominant TR isoform in most of the brain, although TRβ2 is the TR isoform expressed in the pituitary and hypothalamus21,22 (Fig. 67.3). TH is important for neural stem cell proliferation and expression of both neurons and glial cells.72,73 TH promotes neurogenesis and oligodendrocyte differentiation and maturation.74-­76 A number of TH-­stimulated gene targets have been identified in the cortex, hippocampus, and cerebellum. TH induction of a number of genes in the brain is antagonized by the expression of chicken ovalbumin upstream transcription factor 1 (COUPTF1), which has a developmental pattern of expression and blunts the expression of neural differentiation in key developmental windows.77 TH induction of neural proliferation is not only important developmentally, but has also been shown to play a role in recovery after brain injury. TH treatment reduces hypoxia-­induced damage and promotes recovery.78 TH treatment within a few hours of traumatic brain injury in rodent models significantly reduces the extent of injury and promotes recovery. Genes have been identified in rodent models that are TH-­responsive and important in neurogenesis, including T-­box brain transcription factor 1 (Tbr1), Sox2, and neurogenin1.79,80 A TH gene target in the hippocampus, and area important for memory and emotion, is calcium calmodulin ATPase IV (CamKIV). CamKIV is upregulated by T3, but stimulation is inhibited by COUPTF1.81 Oligodendrocyte differentiation is promoted by TH, and a number of gene targets have been identified. Myelination is promoted by the TH-­stimulated, and TH analog-induced expression of myelin basic protein has been reported.82 Another TH gene target is glial fibrillary acidic protein (GFAP), which is expressed in astrocytes. GFAPs form the intermediate filament and play an important role in neural support. TH and TH analog promotion of myelination have been used to promote myelination in models of demyelinating illness, such as multiple sclerosis. In addition to stimulating myelination, TH and thyromimetics have been shown to act to inhibit myelin and axonal degeneration and oligodendrocyte loss in models of autoimmune encephalitis.83 Mouse models with THRA1 mutations have various defects, depending on the mutation, but most have shown some defects in brain development, neurological impairment, and behavioral abnormalities.84 Defects include those of neuronal and glial cell lines. TH regulates brain development, and hypothyroidism results in intellectual disability and multiple neurologic defects.59 Slow mentation and other central nervous system disturbances characterize

CHAPTER 67  Thyroid Hormone Action

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TABLE 67.2  Thyroid Hormone Receptors and Proliferation/Differentiation Cell Type

Proliferation

Primary Receptor

Genes/Pathways

Response Modulated

Hepatocyte

Increased

TRβ

Cyclin D1

Pancreatic β cells

Increased

TRα, TRβ

Intestinal epithelial cells

Increased

TRα

Cardiomyocytes

Increased

TRα

Skeletal muscle cells

Increased

TRα

Cyclin D1/CDK/Rb/E2F PI3K/Akt Wnt/β-­catenin Notch, BMP MHCα, ANF, SERCA2, β-­1 adrenergic receptors, K+ channels PI3K/AKT MyoD and contractile genes

Liver hyperplasia, liver regeneration, regression of neoplastic nodules Cell proliferation and survival during pancreatic development Intestinal maturation and renewal Contractility, heart rate, cardiac hypertrophy, cardiomyocyte maturation Muscle contraction, repair in response to injury

TR, Thyroid hormone receptor. From Pascual A, Aranda A. Thyroid hormone receptors, cell growth and differentiation. Biochim Biophys Acta. 2013;1830:3908–3916.

hypothyroidism. In hyperthyroidism, there can be episodes of anxiety and even psychosis. CamKIV, a TH-­responsive gene expressed in the hippocampus, is important in learning and memory, and reduced expression may be responsible for some of the cognitive deficit seen in patients with hypothyroidism. KEY POINTS  • Thyroid hormone (TH) is important for neural stem cell proliferation and expression of both neurons and glial cells. TH promotes neurogenesis and oligodendrocyte differentiation and maturation. TH and TH analog promotion of myelination have been used to promote myelination in models of demyelinating illness and recovery from brain injury.

Heart and Blood Vessels THs have multiple effects on the cardiovascular system.85 One action is to increase cardiac output via an increase in cardiac contractility and heart rate. TH regulates genes involved in cardiac contractility; there is induction of the sarcoplasmic reticulum Ca2+ ATPase 2, involved in calcium reuptake during the diastolic phase, and α myosin heavy chain (MHC), a fast ATPase required for heart contractility that is expressed in adult heart. Conversely, TH inhibits expression of βMHC, a slow ATPase expressed in embryonic heart and upregulated in stress conditions. Excess TH, in contrast, is associated with atrial arrhythmias, as well as development of high-­output heart failure. TH and its analogs have been used to increase cardiac output in heart failure patients and after coronary artery revascularization, with modest improvement.

Skeletal Muscle THs promote muscle catabolism and increase skeletal muscle energy expenditure in adults.15 TH also induces the insulin-­sensitive glucose transporter in muscle and promotes fat burning. These effects may help to sensitize muscle to insulin response. Accordingly, human Dio2 gene polymorphisms are correlated with insulin resistance in human populations, although this finding remains controversial.86 TH has a specific role in promoting proliferation of satellite cells and skeletal muscle stem cells, during development and during regeneration following injury.70 TH activation, T4 to T3 conversion by Dio2,87 and the presence of the TRα are required for normal development and response to injury.

Skeletal System TH is required for normal bone growth and maturation.88 Juvenile hypothyroidism causes delayed bone formation and short stature,

whereas thyrotoxicosis leads to increased growth and advanced skeletal development. In adults, however, TH excess promotes bone resorption. TH excess can lead to osteoporosis, especially in postmenopausal women.

Small Intestine TH is required for normal maturation of the small intestine. A prominent phenotype of mice with TRα deletions and mutations is abnormal development of the small intestine and malabsorption. TRα1 is required for proliferation of intestinal epithelial progenitor cells via induction of the β-­catenin protooncogene and underlies TH action.89

Metabolic Regulation and Actions in Liver One of the most important effects of TH involves metabolic regulation, including changes in basal metabolic rate (BMR) in multiple tissues (Table 67.3).66 THs stimulate oxygen consumption (indicative of enhanced metabolism) in multiple locations, including skeletal muscle, liver, kidney, and intestine. TH potentiates adrenergic signaling.90 Increases in BMR are probably partly related to increased mitochondrial activity and number, but details of how TH mediates these effects remain unknown. Importantly, THs also induce expression of uncoupling proteins (UCPs), which are mitochondrial membrane proteins that dissipate the proton gradient as heat, rather than allow the gradient to be used to generate adenosine triphosphate. TH does not always enhance BMR. For example, TH does not enhance BMR in most regions of the brain, and the hormone actually suppresses metabolic activity in the pituitary gland. The TH receptors in these tissues are functional, suggesting that key mediators of TH regulation of BMR are blocked or absent. THs exert multiple effects on the liver and are potent mitogens in this tissue, especially in growing animals. TH also influences multiple metabolic processes such as fatty acid β-­oxidation and gluconeogenesis, both key aspects of the fasting response. However, TH can also stimulate expression of enzymes involved in lipogenesis and generation of NADPH-­reducing equivalents that are required for fat synthesis and protection against reactive oxygen species. Because it is thought that fat oxidation and synthesis do not occur simultaneously, these processes are probably separated spatially or temporally, and TH must cooperate with other signaling mechanisms to regulate these effects. TR interacts directly with PPARα and PPARγ in regulation of some genes and may be a mediator for an indirect influence of thyroid status on metabolism.66 TH plays a major role in regulation of cholesterol metabolism in liver in rodents, and studies of patients with thyroid excess and deficiency states suggest that some of these pathways are also TH-­regulated

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PART 6  Thyroid

TABLE 67.3  Thyroid Hormone Regulation of Metabolism Tissue

Process

TR Isoform

Impact

BAT BAT WAT Liver/WAT Liver Liver Liver Liver

Potentiation of adrenergic stimulation of BAT activity Stimulation of UCP1 Direct and adrenergic potentiation, induction of lipolysis Lipogenesis Coupling of autophagy to mitochondrial fatty acid oxidation Reverse cholesterol transport Increase expression of LDL Receptor Reduced hepatic fat deposition

TRα TRβ TRα/TRβ TRβ TRβ TRβ TRβ TRβ

Increased thermogenesis Increased thermogenesis Reduce lipid stores, increased energy expenditure Restore lipid stores Reduce hepatic lipid stores Reduce serum cholesterol Reduce serum cholesterol Reduce hepatic lipid stores

BAT, Brown adipose tissue; WAT, white adipose tissue; TR, thyroid hormone receptor. From Sinha RA, Singh BK, Yen PM. Direct effects of thyroid hormones on hepatic lipid metabolism. Nat Rev Endocrinol. 2018;14:259–269 and Mullur R, Liu YY, Brent GA. Thyroid hormone regulation of metabolism. Physiol Rev. 2014;94:355–382.

in humans.91 TH induces expression of enzymes involved in cholesterol synthesis but also increases levels of the low-­density lipoprotein (LDL) cholesterol receptor, which promotes cholesterol uptake from the circulation. Likewise, TH increases expression of apolipoprotein A1, a key protein component of high-­density lipoprotein (HDL), and increases expression of an HDL receptor (SR-­B1). Consequently, TH excess may promote increased cholesterol flux from the plasma to the liver through both the LDL and HDL pathways. Additionally, TH stimulates cholesterol efflux and cholesterol-­to–bile acid conversion. More recently, TH has been shown to directly stimulate autophagy. Collectively, these mechanisms account for the net reductions of serum cholesterol levels that are observed with TH and the net reverse cholesterol transport reflected by an increased flow of bile acids into the gut. TH activation of lipolysis, reduction in serum cholesterol, as well as autophagy in the liver and reduction in liver fat storage, are attractive targets to treat a range of metabolic disorders.91 TH analogs with more selective activation of TRβ and hepatic action were very effective in lowering cholesterol in statin failure patients, but use was limited by action on cartilage in animal models. TH and analogs have been effective for regression of hepatic steatosis in animal models and some clinical studies and remain an active area of clinical development. KEY POINTS  • Thyroid hormone induces expression of the low-­density-­lipoprotein cholesterol receptor, apolipoprotein A1, a key protein component of high-­density lipoprotein (HDL), and increases expression of an HDL receptor (SR-­B1), with a net reduction of serum cholesterol levels and net reverse cholesterol transport, reflected by an increased flow of bile acids into the gut.

T3 via induction of Dio2, shown to be due to a ubiquitination/deubiquitination, where adrenergic signaling and low T4 promote deubiquitination of Dio2 and increase local Dio2 activity. Thus, T3 cooperates with norepinephrine outputs from the sympathetic nervous system to induce UCPs that promote dissipation of the mitochondrial protein gradient as heat. There is a TR isoform specificity, with TRβ mediating TH stimulation of UCP1 gene expression,92 and TRα potentiating TH-­ mediated adrenergic signaling.93 Whereas previous reports suggested that adaptive thermogenesis was only important for temperature regulation in human newborns and in small mammals, studies have suggested that brown fat may also be important in adult humans.

Skin TH is important for skin function, and TH imbalances are often first manifested in changes in appearance.94 Hypothyroidism leads to cold, dry, and thickened skin. There is also increased hair loss. Conversely, hyperthyroidism leads to warm, moist, and smooth skin with fine, soft hair. Hormone effects are a combination of inhibitory changes in keratin expression, sterol biosynthesis, diminished sebaceous gland secretion, and increased collagen breakdown.95 TH, when applied topically, has been shown to promote hair growth and wound repair.94,96 TH has specific action on hair follicle stem cells and mediates the effects seen on hair changes as a result of changes in thyroid status.97 The role of TH metabolism by Dio2 and Dio3 in the regulation of skin cancer growth and differentiation has been reported, including in melanoma and in basal and squamous cell carcinoma. T3 has been shown to be a significant inducer of the epithelial to mesenchymal transition in squamous cell carcinoma of the skin.98 An increase in local T3 from Dio2 action, and reduction of inactivation by Dio3, promotes tumor growth and invasion.

White and Brown Adipose Tissue

LESSONS FROM RESISTANCE TO THYROID HORMONE RECEPTOR BETA AND ALPHA

THs promote differentiation of precursors into white fat and induces lipogenic enzymes in preadipocytes from young rats and cell lines, but they also increase lipolysis in animals and humans.66 However, the lipolytic effects predominates, because there is a net loss of fat in thyrotoxic states and a gain in hypothyroidism. It is thought that the lipogenesis is in response to the lipolysis in order to restore fat deposits. Hyperthyroidism is associated with loss of both fat and lean body mass, in most patients proportional to the elevation in TH levels. In contrast, hypothyroidism is generally associated with only a modest increase in fat mass. TH stimulates adaptive thermogenesis in brown adipose tissues. In cold or in response to overeating, there is increased local production of

The majority of studies that have identified TR isoform–specific actions were based on models with introduced mutations or TR gene deletions in mice, zebrafish, and frogs. Although these approaches have provided important insights, ultimately, the phenotypes of humans with mutations in genes important in TH signaling are most informative for understanding TH action. The genetic defects of TH signaling involving TR genes are now classified as RTH beta, associated with mutations in the Thrb gene, and RTH alpha, due to mutations in the Thra gene. These syndromes are discussed in detail elsewhere (see Chapter 81), but the connection between genotype and phenotype in individuals with RTH will be briefly discussed for the insights provided into tissue-­ specific actions of TH and the role of TR isoforms.

CHAPTER 67  Thyroid Hormone Action

Resistance to Thyroid Hormone Beta RTH beta is due to mutations in the THRB locus and is inherited in an autosomal dominant fashion. In most cases, patients are heterozygous for Thrb mutations, and the clinical signs and symptoms suggest that there is inhibition of endogenous functional TRs by the mutant receptor through a “dominant negative effect.” This explains the lack of negative feedback in the HPT axis, mediated by TRβ, which results in elevation of serum TSH and circulating TH levels. Since the central feedback regulation is blunted, circulating serum T4 and T3 are elevated, producing a “compensation” for reduced signaling at tissues with TRβ predominance. In tissues with TRα predominance, such as the heart, then there are signs of excess TH, with tachycardia. Metabolic regulation is also predominantly mediated by TRα, and RTH beta individuals have increased metabolism, higher resting energy expenditure, and a higher calorie intake compared with unaffected matched controls, without an increase in weight.99 In a range of genetic models, there was a recapitulation of the primary biochemical phenotype of RTH, elevated blood levels of T4 and T3, and a high, nonsuppressible TSH. The high TSH levels in mice induces goiter, which is also a common clinical sign found in RTH beta patients.

Resistance to Thyroid Hormone Alpha Thra mutations have no significant alterations in circulating TSH or T4 levels, suggesting that central TH feedback at the level of the hypothalamus and pituitary is not impaired. RTH alpha patients have an increase in serum T3 concentrations, increased T3/T4 ratios, and reduced serum reverse T3 concentrations, consistent with a reduction in Dio3 activity. This finding supports an earlier study that showed that upregulation of Dio3 by T3 is mediated by TRα.100 Patients with Thra mutations present with signs suggesting hypothyroidism: short stature, delayed bone age, and clinical findings of dry skin and slow reflexes, and evidence for reduced TH action in key tissues, including delay in linear growth and tooth eruption, low serum insulin-­like growth factor-­1 levels, reduced muscle tone, impaired fine motor coordination, and severe constipation.84 In vitro analysis of these TRα mutations reveals very low T3 binding affinity, defective activation from canonical TH response elements (TREs), repression of basal promoter activity, irreversible binding of corepressors, and a clear dominant negative effect. The response of these clinical manifestations of hypothyroidism to treatment with levothyroxine have been variable, with some improvement in growth if initiated in children and improvement of the metabolic and some cognitive manifestations in adults.84 KEY POINTS  • Findings from animal models regarding thyroid hormone (TH) receptor (TR) isoform specificity is confirmed by individuals with resistance to TH (RTH). In RTH beta, central feedback regulation is blunted and circulating serum T4 and T3 are elevated, producing a “compensation” for reduced signaling at tissues with TRβ predominance. In tissues with TRα predominance, there are signs of excess thyroid hormone, such as tachycardia and hypermetabolism. Patients with Thra mutations do not have this “compensation” and have manifestations of reduced TH action, short stature, delayed bone age, low serum insulin-­like growth factor-­1 levels, impaired fine motor coordination, and severe constipation.

SUMMARY AND FUTURE DIRECTIONS TH is essential for normal development and regulates the function of a wide range of tissues in the adult. The use of animal models, including mice, zebrafish, and Xenopus, as well as analysis of human genetic

1139

disorders of thyroid signaling, has led to the recognition of multiple sites of regulation of TH action. The HPT axis closely regulates TH production and secretion from the thyroid gland. Additional sites of regulation include local tissue activation of the prohormone, T4, to the active form, T3, TH transporters in specific tissues, the amount and relative distribution of TR isoforms, and an important role of TR coactivators and corepressors. Newer areas of the mechanism of regulation that have been identified include posttranslational modification of TR by phosphorylation and sumoylation, subcellular localization of TR isoforms, and TH regulation of microRNAs. In addition to nuclear action, nongenomic actions of TH through membrane integrin receptors as well as modification of intracellular signaling pathways have been reported. A core action of TH is metabolic regulation, and many of these actions involve crosstalk with other metabolic signaling pathways, including PPARα and PPARγ. Genome-­wide approaches have confirmed previous findings, such as the classic DNA TRE sequence, but have also shown indirect effects of TR, especially on chromatin remodeling and actions in regions without consensus TREs. TH signaling has a central role in metabolism, stem cell proliferation in development and regeneration after injury, brain development and function, retinal and ear development, cardiovascular function, and cancer. Greater understanding of these complex factors that regulate TH signaling will permit more detailed description of these interacting pathways, with the potential for selective therapeutic targets that impact these important processes.

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CHAPTER 67  Thyroid Hormone Action 61. Anyetei-­Anum CS, Roggero VR, Allison LA. Thyroid hormone receptor localization in target tissues. J Endocrinol. 2018;237:R19–R34. 62. Brent GA. Mechanisms of thyroid hormone action. J Clin Invest. 2012;122:3035–3043. 63. Ng L, Kelley MW, Forrest D. Making sense with thyroid hormone-­-­the role of T(3) in auditory development. Nat Rev Endocrinol. 2013;9:296– 307. 64. Aranda A. MicroRNAs and thyroid hormone action. Mol Cell Endocrinol. 2021;525:111175. 65. Liu YY, Brent GA. Thyroid hormone crosstalk with nuclear receptor signaling in metabolic regulation. Trends Endocrinol Metab. 2010;21:166– 173. 66. Mullur R, Liu YY, Brent GA. Thyroid hormone regulation of metabolism. Physiol Rev. 2014;94:355–382. 67. Pascual A, Aranda A. Thyroid hormone receptors, cell growth and differentiation. Biochim Biophys Acta. 2013;1830:3908–3916. 68. Verga Falzacappa C, Mangialardo C, Raffa S, et al. The thyroid hormone T3 improves function and survival of rat pancreatic islets during in vitro culture. Islets. 2010;2:96–103. 69. Aguayo-­Mazzucato C, Zavacki AM, Marinelarena A, et al. Thyroid hormone promotes postnatal rat pancreatic beta-­cell development and glucose-­responsive insulin secretion through MAFA. Diabetes. 2013;62:1569–1580. 70. Milanesi A, Lee JW, Kim NH, et al. Thyroid hormone receptor alpha plays an essential role in male skeletal muscle myoblast proliferation, differentiation, and response to injury. Endocrinology. 2016;157:4–15. 71. Liu YY, Brent GA. Thyroid hormone and the brain: mechanisms of action in development and role in protection and promotion of recovery after brain injury. Pharmacol Ther. 2018;186:176–185. 72. Bernal J, Guadano-­Ferraz A, Morte B. Thyroid hormone transporters-­ functions and clinical implications. Nat Rev Endocrinol. 2015;11:690. 73. Wallis K, Dudazy S, van Hogerlinden M, et al. The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons. Mol Endocrinol. 2010;24:1904–1916. 74. Lemkine GF, Raj A, Alfama G, et al. Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor. FASEB J. 2005;19:863– 865. 75. Billon N, Tokumoto Y, Forrest D, et al. Role of thyroid hormone receptors in timing oligodendrocyte differentiation. Dev Biol. 2001;235:110– 120. 76. Morte B, Manzano J, Scanlan TS, et al. Aberrant maturation of astrocytes in thyroid hormone receptor alpha 1 knockout mice reveals an interplay between thyroid hormone receptor isoforms. Endocrinology. 2004;145:1386–1391. 77. Teng X, Liu YY, Teng W, et al. COUP-­TF1 modulates thyroid hormone action in an embryonic stem-­cell model of cortical pyramidal neuronal differentiation. Thyroid. 2018;28:667–678. 78. Li J, Abe K, Milanesi A, et al. Thyroid hormone protects primary cortical neurons exposed to hypoxia by reducing DNA methylation and apoptosis. Endocrinology. 2019;160:2243–2256. 79. Kapoor R, Desouza LA, Nanavaty IN, et al. Thyroid hormone accelerates the differentiation of adult hippocampal progenitors. J Neuroendocrinol. 2012;24:1259–1271. 80. Attardo A, Fabel K, Krebs J, et al. Tis21 expression marks not only populations of neurogenic precursor cells but also new postmitotic neurons in adult hippocampal neurogenesis. Cereb Cortex. 2010;20:304–314. 81. Liu YY, Brent GA. A complex deoxyribonucleic acid response element in the rat Ca(2+)/calmodulin-­dependent protein kinase IV gene 5’-­flanking

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region mediates thyroid hormone induction and chicken ovalbumin upstream promoter transcription factor 1 repression. Mol Endocrinol. 2002;16:2439–2451. 82. Hartley MD, Banerji T, Tagge IJ, et al. Myelin repair stimulated by CNS-­ selective thyroid hormone action. JCI Insight. 2019;4:e126329. 83. Chaudhary P, Marracci GH, Calkins E, et al. Thyroid hormone and thyromimetics inhibit myelin and axonal degeneration and oligodendrocyte loss in EAE. J Neuroimmunol. 2021;352:577468. 84. Moran C, Agostini M, McGowan A, et al. Contrasting phenotypes in resistance to thyroid hormone alpha correlate with divergent properties of thyroid hormone receptor alpha1 mutant proteins. Thyroid. 2017;27:973–982. 85. Ahmadi N, Ahmadi F, Sadiqi M, et al. Thyroid gland dysfunction and its effect on the cardiovascular system: a comprehensive review of the literature. Endokrynol Pol. 2020;71:466–478. 86. Mentuccia D, Proietti-­Pannunzi L, Tanner K, et al. Association between a novel variant of the human type 2 deiodinase gene Thr92Ala and insulin resistance: evidence of interaction with the Trp64Arg variant of the beta-­3-­adrenergic receptor. Diabetes. 2002;51:880–883. 87. Marsili A, Tang D, Harney JW, et al. Type II iodothyronine deiodinase provides intracellular 3,5,3’-­triiodothyronine to normal and regenerating mouse skeletal muscle. Am J Physiol Endocrinol Metab. 2011;301:E818– E824. 88. Wojcicka A, Bassett JH, Williams GR. Mechanisms of action of thyroid hormones in the skeleton. Biochim Biophys Acta. 2013;1830:3979–3986. 89. Godart M, Frau C, Farhat D, et al. The murine intestinal stem cells are highly sensitive to the modulation of the T3/TRalpha1-dependent pathway. Development. 2021 148:dev194357. 90. Silva JE, Bianco SD. Thyroid-­adrenergic interactions: physiological and clinical implications. Thyroid. 2008;18:157–165. 91. Sinha RA, Singh BK, Yen PM. Direct effects of thyroid hormones on hepatic lipid metabolism. Nat Rev Endocrinol. 2018;14:259–269. 92. Ribeiro MO, Bianco SD, Kaneshige M, et al. Expression of uncoupling protein 1 in mouse brown adipose tissue is thyroid hormone receptor-­ beta isoform specific and required for adaptive thermogenesis. Endocrinology. 2010;151:432–440. 93. Ribeiro MO, Carvalho SD, Schultz JJ, et al. Thyroid hormone-­-­ sympathetic interaction and adaptive thermogenesis are thyroid hormone receptor isoform-­-­specific. J Clin Invest. 2001;108:97–105. 94. Safer JD. Thyroid hormone action on skin. Curr Opin Endocrinol Diabetes Obes. 2012;19:388–393. 95. Mancino G, Miro C, Di Cicco E, et al. Thyroid hormone action in epidermal development and homeostasis and its implications in the pathophysiology of the skin. J Endocrinol Invest. 2021 44:1571–1579. 96. Safer JD. Thyroid hormone and wound healing. J Thyroid Res. 2013;2013:124538. 97. Contreras-­Jurado C, Lorz C, Garcia-­Serrano L, et al. Thyroid hormone signaling controls hair follicle stem cell function. Mol Biol Cell. 2015;26:1263–1272. 98. Miro C, Di Cicco E, Ambrosio R, et al. Thyroid hormone induces progression and invasiveness of squamous cell carcinomas by promoting a ZEB-­1/E-­cadherin switch. Nat Commun. 2019;10:5410. 99. Mitchell CS, Savage DB, Dufour S, et al. Resistance to thyroid hormone is associated with raised energy expenditure, muscle mitochondrial uncoupling, and hyperphagia. J Clin Invest. 2010;120:1345–1354. 100. Barca-­Mayo O, Liao XH, Alonso M, et al. Thyroid hormone receptor alpha and regulation of type 3 deiodinase. Mol Endocrinol. 2011;25:575– 583.

68 Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine) James D. Faix

OUTLINE Introduction, 1142 Thyrotropin, 1142 Immunoassay for Thyrotropin, 1142 Thyrotropin Reference Intervals, 1143 Standardization of Thyrotropin Immunoassays, 1144 Free Thyroid Hormones, 1145 Indirect Estimation of Free Thyroid Hormones, 1145

Free Thyroid Hormones by Equilibrium Dialysis, 1145 Immunoassay of Free Thyroid Hormones, 1146 Free Thyroid Hormone Reference Intervals, 1147 Standardization of Free Thyroid Hormones, 1147 Total Thyroxine and Triiodothyronine, 1148 Interference in the Measurement of Thyroid Hormones, 1149 Future Directions, 1149

 

INTRODUCTION Thyroid disease is common, and the signs and symptoms of thyroid disease may resemble those of other disorders. Therefore, physicians frequently request so-­called thyroid function tests even when they have a low suspicion of thyroid disease. Although these may include tests that directly assess thyroid gland activity, the most commonly requested thyroid function tests are those which determine the levels of thyrotropin (thyroid stimulating hormone [TSH]) and/or the individual thyroid hormones thyroxine (T4) and triiodothyronine (T3). TSH is the key thyroid function test, because it is at the center of the hypothalamic-­pituitary-­thyroid axis. TSH is elevated if the thyroid gland fails to produce adequate thyroid hormone and suppressed if the thyroid gland produces too much. But there are other causes of an elevated or suppressed TSH, and measurement of thyroid hormone levels is often needed as well to verify that the cause of the abnormal TSH result is thyroid disease. Originally, this meant determining the total levels of T4 and T3, but the establishment of the so-­called free hormone hypothesis––that the free or unbound hormone concentration, not the total, reflects thyroid hormone activity––resulted in assays designed to estimate the free T4 and free T3 fractions. There still is a role for measuring total T4 and T3, but free T4 is the most commonly requested test when the need to confirm the TSH level result arises. This chapter describes measurement of the major thyroid function tests: TSH and free T4, as well as total T4 and T3 and free T3. The other laboratory tests that help in the diagnosis of thyroid disease (thyroglobulin and autoantibodies against the TSH receptor, thyroid peroxidase [TPO], and thyroglobulin) are described in the relevant chapters discussing the disorders they help diagnose.

THYROTROPIN TSH is a glycoprotein hormone with a structure similar to other anterior pituitary glycoprotein hormones. They are noncovalently linked heterodimers of a common (alpha) chain and a unique (beta) chain, which contains the specific hormone’s biological activity. TSH is considered

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the primary thyroid function test.1 It is a useful screen for evidence of thyroid disorders and helps to monitor thyroid hormone replacement in patients with hypothyroidism, as well as thyroid hormone suppressive therapy in patients with thyroid carcinoma. KEY POINTS  • Measurement of thyrotropin is the key thyroid function test, because it is at the center of the hypothalamic-­pituitary-­thyroid axis.

Immunoassay for Thyrotropin The major advances in TSH testing involved moving from competitive to noncompetitive immunoassay approaches (Fig. 68.1). Routine measurement of TSH in clinical practice initially used competitive immunoassay techniques, in which TSH in the patient’s serum competed with labeled TSH for anti-­TSH antibody. Once separation of the bound from the unbound label was accomplished, the amount of label bound to antibody was inversely proportional to the concentration of TSH in the sample. These first-­generation assays were useful for detecting elevated TSH levels, but they could not differentiate euthyroid from hyperthyroid patients with very low TSH levels. Noncompetitive immunoassays introduced in the 1980s achieved a level of analytical sensitivity that accomplished this differentiation (Fig. 68.2). These assays use one antibody to capture TSH and another, which is labeled, to bind to this complex. Once the unbound labeled antibody is removed, the amount of label bound to the solid phase is directly proportional to the concentration of TSH in the sample. This approach not only enhanced the ability to measure lower levels of TSH, but it expanded the measuring range on the high end as well. These second-­generation TSH immunoassays quickly replaced the older ones. And by increasing the signal of the label on the labeled antibody and washing the solid-­ phase more efficiently, even greater analytical sensitivity was possible. Soon, so-­called third-­generation (and even fourth-­generation) TSH assays were introduced. Today, almost all commercially available TSH immunoassays are capable of third-­generation performance. Fourth-­ generation assays, including so-­called single molecule immunoassays, are not widely used clinically.

CHAPTER 68  Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine) Non-competitive

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fourth-­generation assays detect TSH with this degree of reproducibility down to levels of approximately 0.1, 0.01, and 0.001 mU/L, respectively. However, precision in the very low range for commercially available TSH immunoassays may not be as robust as it appears when functional sensitivity is initially evaluated. Also, many laboratories probably do not regularly monitor their performance in this range, and few proficiency programs regularly challenge laboratories at the limit of third-­generation performance. Nonetheless, although the ability to better differentiate euthyroid from hyperthyroid patients is a useful feature, third-­generation TSH assays are useful because the precision at higher levels (0.1–0.4 mU/L), where the results often impact clinical decisions, is markedly improved.

concentration

Fig. 68.1  Competitive versus noncompetitive immunoassay. The first immunoassays for thyrotropin (TSH) used a competitive approach (left) in which labeled TSH competes with the patient’s hormone for a limited number of binding sites. The bound label is separated from the unbound label and the bound label (top) is inversely proportional to the patient’s hormone concentration. Using a non-competitive approach (right) in which patient hormone is captured by bound and labeled antibodies, the bound label is proportional to the TSH concentration. This approach allows for measurement over a wider range and, more importantly, the ability to reliably detect lower levels of TSH is improved.

Fig. 68.2  Thyrotropin immunoassay generations. The first-­generation assays were competitive immunoassays with limited analytical sensitivity. Differentiation of euthyroid (Euth) from hyperthyroid (Toxic) individuals was possible only when noncompetitive approaches were used. Enhancements improved the sensitivity of these second-­ generation assays, producing so-­called third and even fourth generations, with even better separation.

KEY POINTS  • Today, almost all commercially available thyrotropin (TSH) immunoassays are capable of measuring TSH, with a reliable analytical sensitivity of 0.01 mU/L.

Performance of noncompetitive immunoassays for TSH is defined by so-­called functional, rather than analytical, sensitivity. More modern terms for these distinctions are “limit of detection” (LOD, analogous to analytical sensitivity) and “limit of quantitation” (LOQ, analogous to functional sensitivity). The LOD is the lowest level of the TSH that can be distinguished from the background noise of the assay. The LOQ is the lowest level of TSH that can be measured with some degree of reliability, usually defined as reproducibility or precision over an extended period of time. The original LOQ target for TSH was the level below which the precision of the assay exceeded a coefficient of variability of 20%. By convention, second-­, third-­, and

Thyrotropin Reference Intervals Determining reference intervals for TSH is not straightforward. TSH does show diurnal variation, and significantly higher values have been recorded during the late evening and early night. However, because outpatient measurements are usually made during the day, this should have no significant impact on testing. Age does have an effect on TSH levels. Although only minimal changes occur during early adult life, observations that TSH levels rise with advanced age (without evidence of thyroid dysfunction) have raised the question of whether age-­specific reference intervals should be implemented. In the absence of pregnancy, no significant gender differences have been observed. However, within these defined groups of euthyroid individuals, TSH levels do show significant interindividual variability. There appears to be a consensus that the lower limit of the euthyroid reference interval for TSH should be approximately 0.4 mU/L. Therefore, the improved reproducibility in TSH measurement just below this cutoff is helpful. This range, representing potentially mild thyroid hormone excess but not overt hyperthyroidism, is an important one for clinical purposes. It may help to identify patients with subclinical hyperthyroidism and, when monitoring thyroid hormone replacement after thyroid ablation, to detect overtreatment with increased risk of atrial fibrillation. Significant hyperthyroidism due to Graves disease, functioning adenoma, or other causes is likely when the TSH level is truly “suppressed.” This usually means less than 0.01 mU/L, because most commercially available TSH assays cannot reliably measure lower than this. TSH assays are also especially important when hypothyroidism is suspected, because the inverse logarithmic relationship between TSH and T4 means that TSH levels will rise long before free T4 concentrations fall. Experts have disagreed with regard to the correct upper limit. The cutoff for an elevated TSH with first-­generation TSH assays was approximately 10 mU/L. This fell to approximately 5 mU/L with the introduction of second-­and third-­generation assays. The major reason for this change was probably the reduced crossreactivity afforded by the use of monoclonal antibodies. A major problem with early TSH competitive immunoassays was crossreactivity with gonadotropins (luteinizing hormone, follicle-­ stimulating hormone, and human chorionic gonadotropin [hCG]), which share a common alpha subunit with TSH. For many years, most physicians have considered TSH levels greater than 10 mU/L as evidence of thyroid failure and levels between 5 and 10 mU/L as evidence of mild (or subclinical) hypothyroidism. During the past decade, however, there has been debate about the correct upper limit of the reference interval for TSH, as well as the approach to patients with subclinical hypothyroidism. An analysis of thyroid function test results from a large survey of individuals chosen to represent the population of the United States (National Health and Nutrition Examination Survey) revealed that the mean TSH level in the general population was approximately 1.5 mU/L, with only a small degree of variability.2 This finding prompted

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PART 6  Thyroid

TABLE 68.1  Discrepancies Between Thyrotropin (TSH) and Free Thyroid Hormone Levels Elevated TSH Level Without Low FT4 or FT3 Levels

Low TSH Levels Without Elevated FT4 or FT3 Levels

Drugs that increase TSH concentration (cimetidine, dopamine antagonists, lithium, spironolactone, etc.) Laboratory artifact (i.e., human antimouse antibody) Nonthyroidal illness Recent increase in thyroid hormone replacement therapy Subclinical hypothyroidism Thyroid hormone resistance syndrome

Drugs that decrease TSH concentration (dopamine glucocorticoids, opiates, somatostatin, etc.) Laboratory artifact (i.e., biotin ingestion) Nonthyroidal illness Recent decrease in suppressive thyroid hormone replacement Recent treatment for hyperthyroidism Subclinical hyperthyroidism

FT3, Free triiodothyronine; FT4, free thyroxine.

organizations to call for lowering the upper limit of TSH to 4.0 mU/L, 3.0 mU/L, or even lower. These calls were resisted by many who worried that a significant number of patients would be unnecessarily labeled as having thyroid dysfunction, especially given the fact that there was no evidence that treatment of these individuals would provide any benefit. Because of concern that the population used to determine the range of TSH may have included individuals with occult thyroid disease, the data from the survey were reanalyzed to better clarify the relationship of TSH and antibodies to thyroid peroxidase (TPO), a recognized marker of autoimmune thyroid disease. TSH levels correlated with anti-­TPO positivity, and the investigators found that TSH reference intervals determined with a population from which patients with occult autoimmune thyroid disorders had been excluded would indeed support the lower upper limit.3 The issue of the upper limit for TSH remains controversial, especially because several studies since have demonstrated that most individuals with results slightly above these lower cutoffs have no evidence of thyroid disease.4 KEY POINTS  • There appears to be a consensus that the lower limit of the euthyroid reference interval for thyrotropin should be approximately 0.4 mIU/L, but experts have disagreed with regard to the correct upper limit.

Therefore, many other factors, including diet and nutrition, weight and body composition, genetic polymorphisms, and stressful stimuli apparently influence set points for the hypothalamic-­pituitary-­thyroid axis. A recently recognized factor, discussed later, that may account for some of the variability is the performance of the commercially available immunoassays for TSH. Several special situations have focused attention on other factors that influence TSH levels. One area that has always been problematic is pregnancy, which has a significant effect on thyroid function. TSH levels fall during the first trimester due to the thyroid-­stimulating effect of hCG and then rise to baseline as hCG levels fall in the third trimester. Guidelines for the diagnosis and management of thyroid disease during pregnancy issued by the American Thyroid Association recommend trimester-­specific reference intervals for TSH, as well as TSH targets for the diagnosis and treatment of thyroid disease during pregnancy.5 As neonatal screening programs shift from T4 to TSH screening for congenital hypothyroidism, the appropriate cutoff for this indication is the subject of much debate. If screening is done during the first 24 hours after birth, programs must account for the neonatal surge in TSH production during this period. Also, even programs that screen at the traditional 2 to 4 days after birth are lowering the cutoff used in order to detect cases that would otherwise be missed.6 Finally, a variety of stressful stimuli may suppress TSH production, including depression and other psychiatric diseases, sleep disorders, and the effects of severe systemic illness and/or major surgery (so-­called nonthyroidal illness). Recovery from the latter may

also result in a temporary elevation of TSH levels. Consequently, measurement of TSH in severely ill hospitalized patients should be done with caution.7 The inverse relationship between TSH and free T4 is log-­linear in most individuals,8 and this allows TSH to be considered as a single-­test screen of thyroid function. In patients with primary hypothyroidism of whatever cause, levels should be elevated. The magnitude of serum TSH elevation correlates with the severity and, in part, the duration of thyroid hormone deficiency. Similarly, in patients with hyperthyroidism of whatever cause, levels should be lowered. If levels are below 0.01 mU/L (or the lowest limit reported by the laboratory), significant hyperthyroidism is probably present. TSH abnormalities may need to be further investigated using free T4 because of the variety of conditions that may cause TSH to be abnormal in the absence of either primary hypothyroidism or significant hyperthyroidism (Table 68.1). KEY POINTS  • Thyrotropin (TSH) abnormalities may need to be further investigated using free T4, because a variety of conditions may cause TSH to be abnormal in the absence of either primary hypothyroidism or significant hyperthyroidism.

The hypothalamic tripeptide thyrotropin-­ releasing hormone (TRH) plays a central role in the regulation of pituitary TSH secretion. The TRH test measures the increase of pituitary TSH in serum in response to the administration of synthetic TRH. A direct correlation between basal serum TSH values and the maximal response to TRH has been observed. It could be useful in some situations, such as when there is suspicion of central hypothyroidism or a TSH-­secreting pituitary adenoma. However, the use of modern sensitive immunoassays for TSH, plus the fact that TRH testing is no longer available in many areas, has decreased the need to perform this test.

Standardization of Thyrotropin Immunoassays All strategies for defining abnormal TSH levels in different patient populations will suffer if all TSH immunoassays do not produce comparable results. Variability in the performance of TSH immunoassays has been apparent for several years. Data from proficiency testing programs have revealed poor correlation between observed method bias and the reference intervals recommended by the manufacturers (which are largely equivalent). Possible sources of variability include the specificity of the antibodies used, interferences from patient antibodies against the mouse monoclonal antibodies used, interferences from other endogenous substances such as biotin (for immunoassays that use biotin-­streptavidin binding), and the TSH material used for calibration. TSH has three sites for the addition of oligosaccharides, and carbohydrate may constitute up to 25% of its mass. The pituitary releases a heterogeneous mixture of TSH glycoforms with regard to these side chains, and its nature changes when thyroid disease is

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FREE THYROID HORMONES Thyroid hormones in the blood are bound to serum protein carriers, thus leaving only a minute fraction of free hormone in the circulation that is capable of mediating biological activities. A reversible equilibrium exists between bound and unbound hormone, and it is the latter that represents the fraction of the hormone capable of traversing cellular membranes to exert its effects on body tissues. This free hormone hypothesis forms the basis of the clinical approach to a number of endocrine disorders but this is especially true for thyroid disease. Most (approximately 99%) of the total T4 present in blood is bound, the majority to thyroxine-­binding globulin (TBG). Even though it is present in a much lower concentration than the other major thyroid hormone–binding proteins (transthyretin and albumin), TBG has a significantly higher affinity for T4. Most experts agree that, in euthyroid individuals, the free T4 concentration is held constant, even when the concentration of the binding proteins change, while in thyroid disease it is abnormal. But estimating or measuring the very low concentration of free T4 has proved difficult over the years, and approaches have evolved.

5 mIU/L

CHAPTER 68  Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine)

–45

–30

–15 0 Median deviations (%)

15

Fig. 68.3  Harmonization of thyrotropin (TSH) immunoassays. Median deviations of commercially available TSH immunoassays before (black) and after (red) recalibration using targets derived from a factored analysis of all of the results. The improvement is shown for specimens with low, normal, and high TSH levels. (From Thienpont LM, Van Uytfanghe K, De Grande LAC, et al. Harmonization of serum thyroid-­stimulating hormone measurement paves the way for the adoption of a more uniform reference interval. Clin Chem. 2017;63:1248–1260. Reproduced with permission.)

Indirect Estimation of Free Thyroid Hormones The first attempts to measure the free T4 concentration relied on an estimate of the number of binding sites available on the serum binding proteins. This was accomplished by adding labeled thyroid hormone to serum and, after incubation, using an absorbent material of some kind to collect all of the unbound labeled hormone. Labeled T3, for which TBG has a lower affinity, was used rather than labeled T4 in order to prevent the labeled hormone from displacing T4 bound to the protein. The so-­called “T3 uptake” (T3U) test produced a result that correlated with the saturation of TBG with T4. If TBG saturation was high (as would be the case in hyperthyroidism), the uptake of labeled hormone by the absorbent would be high; if TBG saturation was low (as would be the case in hypothyroidism), the uptake of labeled hormone by the absorbent would be low. However, when the total T4 level was increased or decreased because of changes in TBG concentration, a baseline level of TBG saturation would have been achieved, and the uptake of the labeled hormone would be in the opposite direction. Calculation of a unitless figure called the free thyroxine index (FTI), the mathematical product of the total T4 (originally the protein-­ bound iodine) and the T3U, could thus be used to normalize changes in total T4 due to increases of decreases in TBG. Currently, labeled T4 analogs are used in place of T3 (so-­called “T-­uptake” assays), and another approach relies on measurement of TBG, with calculation of the T4/TBG ratio. Although FTI determinations were once commonly requested laboratory tests, they have been supplanted by more direct approaches, including equilibrium dialysis (or ultrafiltration) and immunoassay.

Free Thyroid Hormones by Equilibrium Dialysis Early attempts to directly measure free T4 used equilibrium dialysis to physically separate free T4 from bound T4. But the initial approach was not direct but indirect. In direct equilibrium dialysis, free hormone in the specimen moves into a second compartment on the other side of a semipermeable membrane that excludes the protein-­bound hormone. After an equilibrium between the two compartments is reached, the concentration of hormone in the second compartment is measured. However, because the free T4 is such a small percentage of the total, T4 concentrations in the second compartment were far

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PART 6  Thyroid indirect ED labeled T4 binding protein T4

add radioactivelabeled hormone

binding protein T4

binding protein T4

perform dialysis

measure radioactivelabeled hormone in dialysate

direct ED binding protein T4

binding protein T4

perform dialysis

measure hormone in dialysate

Fig. 68.4  Equilibrium dialysis (ED) measurement of free thyroxine (T4). The first approach to equilibrium dialysis for free T4 used an indirect approach (top) in which radioactively labeled T4 was added to the sample, and the amount of label in the dialysate was used along with the total T4 measured separately to estimate the free T4. In the modern direct approach (bottom), the T4 in the dialysate is measured using mass spectrometry. Note that the amount of protein-­bound T4 in actual samples would be much greater than the percentage shown, so disruption of the equilibrium between bound and free T4 would not be significantly disturbed.

below the ability of early immunoassays to measure it. Consequently, most investigators took an indirect approach by adding a trace amount of radioactive-­labeled T4 to the specimen (Fig. 68.4). This labeled T4 equilibrated with the respective bound and free endogenous T4 and the free fraction could be calculated from the proportion of labeled hormone in the dialysate. An equilibrium method that used immunoassay to directly measure T4 in the dialysate was introduced in the 1990s. Unfortunately, this product is no longer widely commercially available. More recently, a direct equilibrium method was developed and proposed as an RMP. The details of this procedure include strict requirements for the buffer used, the pH of the specimen, the dialysate membrane cutoff size, the size of the two compartments, and the temperature. The T4 in the dialysate is measured by isotope dilution–liquid chromatography/tandem mass spectrometry (LC-­MS/MS), not immunoassay. Other alternative direct approaches, such as ultrafiltration followed by LC-­MS/ MS, have also been described. There is debate regarding which is the best approach, but either is probably too laborious to be implemented for routine clinical use. Therefore, most clinical laboratories rely on commercially available immunoassays.11 KEY POINTS  • A direct equilibrium method for measurement of free thyroxine has been developed and proposed as a reference measurement procedure.

Immunoassay of Free Thyroid Hormones There have been three formats used for free T4 immunoassay. All utilize competitive immunoassay with a solid support that can be washed, removing unbound labeled reagent. But each is very different (Fig. 68.5). In the so-­called two-­step method, free hormone in the specimen interacts with antihormone antibody on a solid support, which becomes occupied by endogenous free hormone to a smaller or larger extent based on the free hormone concentration. The specimen is then removed, and excess labeled hormone is added, which fills any empty antibody binding sites. This is also often referred to as “back-­ titration.” In the labeled analogue approach (also commonly referred to as a “one-­step” method), free hormone in the specimen interacts

binding protein T4

solid-phase anti-T4

labeled T4

labeled T4 analogue

two-step one-step analogue

solid-phase anti-T4 labeled anti-T4

one-step labeled antibody solid-phase T4

Fig. 68.5  Free thyroxine (T4) immunoassay. All of the commercially available immunoassays for free T4 use one of these three approaches (described in the text).

with antihormone antibody on a solid support in the presence of labeled hormone. In order to prevent interaction of labeled hormone with binding proteins, the T4 used as the labeled hormone is altered in a way that is meant to interfere with binding to endogenous proteins but not with antibody binding. The nature of these so-­called hormone “analogs” is proprietary. Finally, the “labeled antibody” method (which is also a “one-­step” assay) avoids the use of labeled hormone entirely. Free hormone in the specimen interacts with labeled antibody in the presence of solid-­phase hormone. Some critics have pointed out that this solid-­phase hormone is also an “analog,” but the fact that it is not able to enter the free hormone pool in the same way as the soluble analog probably limits its ability to compete with the endogenous free hormone for endogenous binding sites. For all three formats, the amount of labeled reagent left on the solid-­phase after final washing is inversely proportional to the original concentration of free hormone in the specimen.12

CHAPTER 68  Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine) There are significant discrepancies between different commercially available immunoassays for the measurement of free T4. Although different calibration may explain some of the overall differences (see later), the ways in which different assays influence the equilibrium between protein-­ bound and free hormone in individual patients may also explain many of the discrepancies. Although the labeled T4 analog used in one-­step analog assays should not bind to any of the endogenous binding proteins, it initially appeared that some analogs did exhibit significant binding to albumin. Free T4 concentrations in patient specimens with albumin levels different from those of the calibrators used in the assay would either be overestimated or underestimated. Because patients who are critically ill often have low albumin levels, free T4 assays frequently underestimated the true free T4 concentration. However, most manufacturers have addressed this problem, either by adding albumin to the reagent (so that any variation in the patient’s albumin level will be compensated) or by improving the behavior of the T4 analog. Another problem is the influence of the total concentration of protein-­bound T4, especially the fraction bound to TBG, because of its high affinity for T4, as well as the fact that it may be present in a wide range of concentrations. Generally speaking, immunoassays have been shown to underrecover free T4 when TBG levels are elevated, although the validity of the design of these investigations (using artificial T4 solutions with added T4-­binding proteins) has been criticized. This limitation has often been cited as a reason to question the accuracy of free T4 concentrations in pregnant women, especially in the third trimester (see later). Finally, for both one-­ step analog and two-­ step back-­ titration immunoassays, consideration must also be given to the amount of anti-­T4 antibody used on the solid phase. It is very important that binding of free T4 to the anti-­T4 antibody occur without significantly disturbing the equilibrium between free T4 and protein-­bound T4. If this happens, protein-­bound T4 may dissociate. This phenomenon is called sequestration. The antibody reagent should not remove more than 1% of the total T4 in the specimen. Even though this represents more than the free moiety, it represents only a small percentage of the total T4 present and is unlikely to significantly disturb the equilibrium. Dilution has been traditionally used to gauge the degree to which a free T4 immunoassay is susceptible to this effect, either because of the amount of reagent antibody present or the affinity of the antibody used. Assays are considered robust as long as this effect is kept to a minimum.14 Commercially available immunoassays for free T3 have been developed, using approaches similar to those used for free T4, and free T3 is often requested instead of total T3. The same issues that may influence assays for free T4 can impact assays for free T3. The Clinical Laboratory Standards Institute has issued an approved guideline for the measurement of free thyroid hormones.13 Although it is unlikely that many clinical laboratories will be able to utilize the proposed RMPs for free T4 and free T3, they should be able to implement several of this guideline’s recommendations for ensuring that free thyroid hormone results using commercially available immunoassays are as accurate and precise as possible. These include adding increasing amounts of albumin and TBG to specimens from euthyroid individuals, as well as diluting specimens from euthyroid individuals using inert buffer in order to assess possible analog binding, the effect of binding proteins, and the degree of antibody sequestering. The guideline also recommends verifying the performance of the free T4 immunoassay in specimens from the following groups of patients: euthyroid; hyperthyroid; hypothyroid; patients with elevated TBG concentrations; and patients who are taking drugs known to compete for T4 binding sites on TBG and albumin.

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KEY POINTS  • Guidelines exist for ensuring that free thyroid hormone results using commercially available immunoassays are as accurate and precise as possible, but there are significant discrepancies between different commercially available immunoassays for the measurement of free thyroxine.

Free Thyroid Hormone Reference Intervals Reference intervals for both free T4 and free T3 vary with the manufacturers of commercially available assays, but are approximately 0.8 to 2.0 ng/dL (10–26 pmoL/L) for free T4 and 0.2 to 0.4 ng/dL (3-6 pmol/L) for free T3. With few exceptions, the free T4 and free T3 concentrations are high in hyperthyroidism, low in hypothyroidism, and normal in euthyroidism, even in the presence of changes in TBG concentration. The condition that has raised the most concern about free thyroid hormone reference intervals is pregnancy. The influence of elevated TBG on immunoassays for free T4 is especially pronounced in pregnancy. Although so-­ called hypothyroxinemia of pregnancy does exist and needs to be recognized, euthyroid reference intervals in the second and third trimesters should probably be lower than previously thought. Guidelines of the American Thyroid Association for management of thyroid disease during pregnancy recommend that the laboratories develop trimester-­specific reference intervals for the free T4 assay used. However, because of concerns about the performance of free T4 immunoassays, they also include a recommendation to use either equilibrium dialysis or ultrafiltration. Because neither direct approach is likely to be widely available, the guidelines suggest alternatives such as the use of total T4 (with trimester-­specific reference intervals) or the free thyroxine index using T-­uptake assays.5 However, the use of either alternative suggested by the guidelines may not properly classify pregnant patients.15 Immunoassays have been shown to correlate with equilibrium dialysis; although levels using immunoassay may be lower, the trending is similar,16 but variability between different commercially available assays may classify patients differently.17 KEY POINTS  • There is probably still a lack of consensus regarding the use of free thyroxine assays in pregnancy.

Standardization of Free Thyroid Hormones The same group that looked at harmonization of TSH measurements also examined commercially available immunoassays for free T4 and free T3, working in partnership with the same manufacturers. Except for one, the results were all considerably lower than those using the equilibrium dialysis RMP. However, the good news was that most of the bias appeared not to be related to any of the assay issues discussed previously, but rather to the calibration employed by the individual manufacturers. Mathematical recalibration using the regression of the results by each assay to the target values determined by the equilibrium dialysis RMP was able to remove most of the aforementioned considerable systematic bias (Fig. 68.6). This approach to standardization was pursued extensively for free T4 immunoassays in a similar fashion to that used for TSH and culminated with a similar study showing that removal of assay-­specific bias may be able to align the different reference intervals to that of the RMP. The ability to share the RMP reference interval was not initially fully successful using a stringent requirement for the location of the upper and lower cutoffs to be within the confidence limits of the RMP. This was attributed to the susceptibility of free hormone immunoassays to a variety of individual

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PART 6  Thyroid

100

Difference of the means to ED-ID-LC-MS/MS (%)

Difference of the means to ED-ID-LC-MS/MS (%)

100

50

0

–50

–100

A

50

0

–50

0

20 40 60 80 100 120 140 160 180 FT4ED-ID-LC-MS/MS (pmol/L) - Pre-recalibration

–100

B

0

20 40 60 80 100 120 140 160 180 FT4ED-ID-LC-MS/MS (pmol/L) - Post-recalibration

Fig. 68.6  Standardization of free thyroxine (T4) immunoassays. Comparison of commercially available immunoassays for free T4 to the equilibrium dialysis reference measurement procedure before (left) and after (right) recalibration. The initially most discrepant assays (high or low) are shown in red and blue, respectively. Although there was still significant variability for some of the assays, recalibration shifted the overall percentiles (15th and 18th) to within the bias expected based on biological variation (red dotted lines). ED-­ID-­LC-­MS/ MS, isotope dilution–liquid chromatography/tandem mass spectrometry; FT4, free thyroxine. (From De Grande LAC, Van Uytfanghe K, Reynders D, et al. Standardization of free thyroxine measurements allows the adoption of a more uniform reference interval. Clin Chem. 2017;63:1642–1652. Reproduced with permission.)

sample related effects and/or variability in the lots of the reagents used by the manufacturers. However, using a slightly less stringent requirement, the hope for an eventual uniform reference interval for free T4 appeared within reach.18

TOTAL THYROXINE AND TRIIODOTHYRONINE Although the primary focus of the use of thyroid function tests is the combination of TSH and free T4, there are still situations where the traditional measurement of the total level of T4 and T3 is helpful. The major secretory product of the thyroid gland is T4 in a ratio favoring T4 over T3 by 10-­to 20-­fold. Because they are present in higher concentrations and are not subject to the same specimen issues as free hormones, total T4 and T3 are easier to measure. Assays for total T4 and T3 use competitive immunoassay and include a reagent such as 8-­anilinonaphthalene sulfonic acid that blocks T4 and T3 binding to

serum proteins, so that total hormone is available for competition with the assay antibody. The usual concentration of total T4 in adults ranges from 5 to 12 microg/dL (64–154 nmol/L). The usual concentration of total T3 in adults ranges from 80 to 190 ng/dL (1.2–2.9 nmol/L). Although levels of total T4 and T3 above and below these ranges are usually associated with hyperthyroidism and hypothyroidism, respectively, the total levels do not always correspond to the free hormone concentrations. When concentrations are below or above these ranges in the absence of thyroid dysfunction, they are usually the result of an abnormal level of serum TBG. Such abnormalities are commonly seen during pregnancy or the administration of estrogen-­containing compounds, which result in a significant elevation of serum total T4 levels in euthyroid individuals. Similarly, total T4 levels are low in conditions that are associated with decreased TBG concentrations, but the free T4 is usually normal. The total T4 concentration in serum may be also be altered

CHAPTER 68  Thyroid Function Testing (Thyrotropin, Triiodothyronine, and Thyroxine) by alterations in T4 binding to TBG or compensatory changes due to defects in the conversion of T4 to T3. Although changes in transthyretin concentration rarely give rise to significant alterations in total T4 concentration, the presence of a variant serum albumin in patients with familial dysalbuminemic hyperthyroxinemia with high affinity for T4 produce apparent elevations in the measured total T4 concentration, whereas the free T4 levels remains normal. The ratio of total T3 to total T4 may sometimes be helpful in the evaluation of patients with hyperthyroidism. A low ratio may favor thyroiditis or iodine-­induced hyperthyroidism over Graves disease. A high ratio may be a helpful prognostic marker after initial of antithyroid treatment of Graves disease. KEY POINTS  • Although the primary focus of the use of thyroid function tests is the combination of thyrotropin and free thyroxine (T4), there are still situations where the traditional measurement of the total level of T4 and triiodothyronine is helpful.

INTERFERENCE IN THE MEASUREMENT OF THYROID HORMONES When there is discrepancy between the results of a laboratory test and the patient’s clinical presentation, interferences with the assay itself must be considered. Traditional problems such as crossreactivity of the antibody used have largely been eliminated. Some interferences specific for TSH and free T4 immunoassays have been discussed, but there are several categories that may influence the results of all assays. Endogenous antibodies that react with any of the assay reagents may cause a falsely low or high result, depending on the format of the assay. This category includes human antimouse antibody, antibodies against the hormone being measured, or rheumatoid factor. Other endogenous substances may interfere in other ways. A recent focus has been the effect of biotin, used in the treatment of rare metabolic disorders but also taken in high amounts as a nutritional supplement. Immunoassays that use the binding of biotin to streptavidin in order to measure the bound label may produce erroneous results in patients with high levels, again in a way that is dependent on the format of the assay.19 Finally, a large number of drugs may produce discrepant results. These effects are less likely to be assay interferences, but rather alterations in thyroid hormone physiology. These include agents that inhibit thyroid hormone synthesis and secretion, affect thyroid hormone binding proteins, or alter thyroid hormone metabolism.20 If some assay interference is suspected, a good first step is to repeat the testing using another manufacturer’s method that has a different assay format. Diluting the specimen in the hopes of diluting out the effect of the interfering substance without affecting the measuring range of the assay or attempting removal of immunoglobulin in case the interference is due to an endogenous antibody may also help. When the presence of a drug interfering with endogenous thyroid function or metabolism is suspected, it may be necessary to temporarily withhold the drug and repeat the testing.21

FUTURE DIRECTIONS TSH will continue to be the key thyroid function test when screening for thyroid dysfunction, and the efforts directed at harmonization of the commercially available assays will be successful. This will help investigators continue to evaluate the relationship between TSH levels

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and thyroid hormone activity in the general population and may allow the controversy regarding the appropriate upper limit of the reference interval to be resolved. Free T4, rather than total T4 or either total T3 or free T3, will continue to be used to confirm abnormal TSH results. Some have advocated more routine use of equilibrium dialysis for free T4 measurement, but this will not be feasible in the near future, and laboratories will continue to use commercially available immunoassays. The efforts directed at standardization of free T4 immunoassays will be hampered by several factors, including the various formats used, the differences in the influence of specimen-­specific effects, and the fact that most of the current methods produce results that are significantly lower than the equilibrium dialysis RMP. Although standardization will allow for better patient care, a major education initiative will be needed if it is accomplished. Finally, trimester-­specific reference intervals for thyroid function tests in pregnancy will be widely adopted.

REFERENCES 1. Schneider C, Feller M, Bauer DC, et al. Initial evaluation of thyroid dysfunction – are simultaneous TSH and fT4 tests necessary? PLoS One. 2018;13:e0196631. 2. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES II). J Clin Endocrinol Metab. 2002;87:489–499. 3. Spencer CA, Hollowell JG, Kazarosyan M, et al. National Health and Nutrition Examinations Survey III. Thyroid-­stimulating hormone (TSH)-­ thyroperoxidase antibody relationships demonstrate that TSH upper limit reference limits may be skewed by occult thyroid dysfunction. J Clin Endocrinol Metab. 2007;92:4236–4240. 4. Feldt-­Rasmussen U. Laboratory measurement of thyroid-­related hormones, proteins and autoantibodies in serum. In: Braverman LE, Cooper DS, Kopp PA, eds. Werner & Ingbar’s the Thyroid: A Fundamental and Clinical Text. Philadelphia: Walters Kluwer; 2021:267–299. 5. Alexander EK, Pearce EN, Brent GA, et al. 2017 Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the post-­partum. Thyroid. 2017;27:315–389. 6. Kaplowitz PB. Neonatal thyroid disease. Pediatr Clin N Am. 2019;66:343– 352. 7. Van den Berghe G. Non-­thyroidal illness in the ICU: a syndrome with different faces. Thyroid. 2014;24:1456–1465. 8. Rothacker KM, Brown SJ, Hadlow NC, et al. Reconciling the log-­linear and non-­log-­linear nature of the TSH-­free T4 relationship: Intra-­ Individual analysis of a large population. J Clin Endocrinol Metabol. 2016;101:1151–1158. 9. Stockl D, Van Uytfanghe K, Van Aelst S, et al. A statistical basis for harmonization of thyroid stimulating hormone immunoassays using a robust factor analysis model. Clin Chem Lab Med. 2014;52:965–972. 10. Thienpont LM, Van Uytfanghe K, De Grande LAC, et al. Harmonization of serum thyroid-­stimulating hormone measurement paves the way for the adoption of a more uniform reference interval. Clin Chem. 2017;63:1248–1260. 11. Thienpont LM, Van Uytfanghe K, Poppe K, et al. Determination of free thyroid hormones. Best Pract Res Clin Endo Metabol. 2013;27:689–700. 12. Faix JD. Principles and pitfalls of free hormone measurements. Best Pract Res Clin Endo Metabol. 2013;27:631–645. 13. Thienpont L, Bunk DM, Christofides ND, et al. Measurement of Free Thyroid Hormones: Approved Guideline. Clinical and Laboratory Standards Institute (CLSI) document C45-­A; Wayne, PA 2004. 14. Christofides ND. Free analyte immunoassay. In: Wild D, ed. The Immunoassay Handbook. 4th ed. Oxford: Elsevier; 2013:123–137. 15. Geno KA, Reed MS, Cervinski MA, et al. Evaluation of thyroid function in pregnant women using automated immunoassays. Clin Chem. 2021;67:772–780.

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16. Anckaert E, Poppe K, Van Uytfanghe K, et al. FT4 immunoassays may display a pattern during pregnancy similar to the equilibrium dialysis ID-­LC/tandem MS candidate reference measurement procedure in spite of susceptibility towards binding protein alterations. Clin Chim Acta. 2010;411:1348–1353. 17. Andersen SL, Christensen PA, Kronsgaard L, et al. Classification of thyroid dysfunction in pregnant women differs by analytical method and type of thyroid function tests. J Clin Endo Metabol. 2020;105:e4012–e4022. 18. De Grande LAC, Van Uytfanghe K, Reynders D, et al. Standardization of free thyroxine measurements allows the adoption of a more uniform reference interval. Clin Chem. 2017;63:1642–1652.

19. Bowen R, Benavides R, Colon-­Franco JM, et al. Best practices in mitigating the risk of biotin interference with laboratory testing. Clin Biochem. 2019;74:1–11. 20. Burch HB. Drug effects on the thyroid. New Eng J Med. 2019;381:749–761. 21. Favresse J, Burlacu MC, Maiter D, et al. Interferences with thyroid function immunoassays: clinical implications and detection algorithm. Endocrine Rev. 2018;39:830–850.

69 Thyroid Imaging Jennifer A. Sipos

OUTLINE Ultrasonography, 1151 Indications for Sonographic Assessment, 1151 Physics of Ultrasonography, 1151 Diffuse Thyroid Disorders, 1152 Primary Thyroid Lymphoma, 1153 Subacute Thyroiditis, 1153 Sonographic Risk Stratification Systems, 1154 American Thyroid Association Sonographic Risk Stratification System, 1154 Korean Thyroid Imaging Reporting and Data System, 1156 American College of Radiology Thyroid Imaging Reporting and Data System, 1156

Diagnostic Performance of Sonographic Risk Stratification Systems, 1157 Specialized Sonographic Techniques, 1157 Scintigraphy, 1159 Thyroid Nodules, 1159 Diffuse Thyroid Disorders, 1160 Congenital Hypothyroidism, 1161 Perchlorate Discharge Test, 1161 Computed Tomography And Magnetic Resonance Imaging, 1161 Incidentalomas, 1162 Computed Tomography and Magnetic Resonance Imaging, 1162 (18F)-Fluorodeoxyglucose–Positron Emission Tomography, 1162 Future Directions, 1163

 

ULTRASONOGRAPHY Indications for Sonographic Assessment Thyroid nodules are a common clinical entity, with up to 70% of patients over the age of 70 years harboring this neoplasm.1 While the majority of nodules represent a benign neoplasm, and most malignant lesions are associated with an excellent prognosis, up to a quarter of patients with carcinoma have larger, higher-­stage tumors at initial diagnosis.2 Thus, it is incumbent upon the clinician to triage nodules efficiently and effectively. Current guidelines, therefore, uniformly recommend sonography as the initial step in the evaluation of a patient suspected of having a thyroid nodule.3-­5 The physical examination of the neck is an insensitive means of identification of thyroid nodules. Palpation alone only identifies nodules in approximately 5% of patients,1 whereas screening ultrasound (US) may reveal a nodule in over 37% of patients, with incidence escalating with age.6 When a nodule is suspected on exam, US is essential to confirm the exam finding. One retrospective study of patients with suspected thyroid nodules identified on palpation found no corresponding lesion in 16% of patients. Further, another nodule that was not palpated was identified elsewhere in the gland in 23% of patients.7 In this particular study, the US findings changed the management plan in 63% of patients.7

Physics of Ultrasonography It is important to have at least a basic understanding of the verbiage used in thyroid sonography and the physics behind the findings. Echogenicity is essentially describing the color of a nodule or the thyroid itself with specific modifiers to compare to surrounding normal anatomy. Isoechoic describes echogenicity that is of the same color as normal thyroid tissue. The term hypoechoic is used to describe a

lesion or gland that is darker than normal thyroid tissue, whereas hyperechoic is brighter. Echotexture is a term used to describe the pattern of tissue being examined; a normal thyroid is defined as having a homogeneous echotexture (Fig. 69.1A). In contrast, the echotexture of a gland involved with lymphocytic thyroiditis may have a heterogeneous echotexture (see Fig. 69.1B). This concept will be discussed in greater detail in the section later on diffuse thyroid disorders. The sonographic appearance of a normal thyroid is one of a uniformly medium gray echotexture that is midway in hue between the lighter fascia and the darker musculature surrounding the gland (Fig. 69.e1). A healthy thyroid appears relatively bright on US due to its follicular architecture, which is composed of lakes of colloid surrounded by a thin layer of follicular cells. The degree of reflected sound waves directly impacts the brightness of the resulting image; greater reflection results in a brighter image. As the US wave travels through soft tissues of any type, a portion of the sound wave is reflected back to the probe when it encounters the interface of two tissues with unequal acoustic impedance (or resistance to the US wave traveling through a tissue). There is a dramatic mismatch in acoustic impedance between follicular cells and colloid; consequently, a large fraction of the US wave is reflected back to the probe when a sound wave encounters a follicle. Because there are innumerable follicles in a healthy thyroid, there is a significant amount of reflection of the US wave back to the probe. The result is a relatively bright-­appearing thyroid. In contrast, tissue that is relatively homogeneous in histologic composition will have minimal reflection of the US wave back to the probe. The resultant image is uniformly dark. Understanding the physics behind this phenomenon of acoustic impedance is important when examining thyroid tissue and nodules, as it provides insight into the underlying pathophysiology of the disease process (Fig. 69.e2).

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Carotid

Trachea

1151.e1

Carotid

A

B

Fig. 69.e1  A, Diagnostic image of an ultrasound of a normal thyroid gland on transverse imaging with labels identifying the surrounding anatomic landmarks. B, Diagnostic image of an ultrasound of a normal thyroid gland in the sagittal view.

200 µm

Fig. 69.e2  Photomicrograph of normal thyroid follicular architecture. The larger, blue arrow represents ultrasound waves traveling from the probe. When it encounters the follicle, some of the US wave is reflected back to the probe due to the change in acoustic impedance represented by the smaller, black arrow.

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PART 6  Thyroid

A

B Fig. 69.1  A, Diagnostic image of a normal thyroid ultrasound in the sagittal view. Notice the medium gray color of the gland compared to the surrounding musculature and fascia. B, Diagnostic image of a thyroid ultrasound showing Hashimoto’s thyroiditis. The thyroid is diffusely heterogeneous in echotexture. This diffuse micronodular appearance has been called a “giraffe pattern.”

in an overall heterogeneous echotexture of the gland (see Fig. 69.1B). As the disease progresses, the entirety of the gland is encompassed by the inflammatory process and may become diffusely hypoechoic (Fig. 69.e3A–C).

Hashimoto’s Thyroiditis. US of the neck can have an important role

Fig. 69.2  Diagnostic image of a thyroid ultrasound showing Hashimoto’s thyroiditis. The gland is heterogeneous with a microcystic echotexture due to small areas of germinal centers.

Diffuse Thyroid Disorders

Lymphocytic Thyroiditis. Lymphocytic thyroiditis is readily visible on sonography; the identification of diffuse heterogeneity in thyroid echotexture is a cardinal feature. Additional descriptors that may be reported in lymphocytic thyroiditis include a micronodular or microcystic echotexture (Fig. 69.2). The lymphocytic infiltrate produces a change in the histologic architecture of the normal thyroid tissue described earlier. This destruction of the normal follicular unit and replacement with sheets of lymphocytes alters the dynamics of US waves as they travel through the gland. The lymphocytic infiltrates appear as dark areas on US because the acoustic interface of follicular cells and colloid is replaced with a more uniform inflammatory process. Consequently, the US wave is not reflected back to the probe with the same intensity, and the lymphocytic infiltrate appears dark. In the early stages of the disease, the spared areas of the gland appear isoechoic, while the germinal centers appear dark, resulting

in the diagnosis of Hashimoto’s thyroiditis. Reliance on clinical and biochemical findings alone to diagnose Hashimoto’s thyroiditis would miss half of patients with disease; including the sonographic analysis significantly improves the diagnostic sensitivity and specificity.8,9 The characteristic sonographic appearance of Hashimoto’s thyroiditis includes a diffusely heterogeneous parenchyma with hypoechoic areas interspersed with normal thyroid tissue; identification of this US feature has a positive predictive value of 95%10 (see Fig. 69.1B). The vascularity of the gland may be variable, but is often increased early in the disease course. In the later stages of the disease, there may be a reduction in the vascularity with eventual avascularity as the degree of fibrosis increases (Fig. 69.e4). Echogenic septations, caused by fibrosis of the parenchyma, may be seen throughout the gland. Another important feature associated with Hashimoto’s thyroiditis includes surrounding reactive or hypertrophic lymphadenopathy. These nodes appear plump and may have a characteristic hilar stripe. They are typically seen anterior to the trachea and inferior to the thyroid gland, though they may also be seen in the lateral neck at levels II and IV (Fig. 69.e5). KEY POINTS  • Hashimoto’s thyroiditis is easily identified on ultrasound. The gland is marked by varying degrees of hypoechogenicity, with pseudonodules and septations. The presence of surrounding reactive adenopathy is also a clue to the underlying diagnosis of Hashimoto’s.

Graves Disease. The sonographic appearance of Graves disease is similar to that seen in Hashimoto’s thyroiditis but tends to be more diffusely involved; the micronodular pattern is less commonly seen (Fig. 69.3A). Generally, the gland is enlarged with a diffuse hypoechoic echotexture. Increased vascularity is a prominent component of the examination, particularly in untreated glands. This sonographic

CHAPTER 69  Thyroid Imaging

A

1152.e1

B

C Fig. 69.e3 A, Diagnostic image of an ultrasound demonstrating early stage Hashimoto’s thyroiditis with a microcystic or “moth-­eaten” pattern marked by the small white arrows. B, Diagnostic image of an ultrasound of the thyroid illustrating progression of lymphocytic thyroiditis. Notice the more diffuse involvement of the lobe and the microcystic pattern. C, Diagnostic image of an ultrasound of a thyroid gland with chronic lymphocytic thyroiditis (Hashimoto’s thyroiditis). The gland is diffusely hypoechoic with bright linear bands (white arrow), which represent fibrotic septations. Anteriorly an area of hyperechogenicity represents a pseudonodule with an area of more ‘normal appearing’ thyroid parenchyma (blue arrow).

Fig. 69.e4  Image of Hashimoto’s thyroiditis demonstrating reduced vascular flow.

Fig. 69.e5  Diagnostic image of a thyroid ultrasound showing a hypertrophic delphian node in Hashimoto’s thyroiditis (white arrow). Notice the hypoechogenicity of the thyroid gland and its similarity in color to the surrounding strap musculature.

CHAPTER 69  Thyroid Imaging

A

1153

Fig. 69.4  Diagnostic image of a thyroid ultrasound showing Graves disease. The gland is diffusely hypoechoic and mildly enlarged with a prominent inferior thyroidal vein (white arrow).

sonography are subcentimeter tumors that have questionable clinical significance,16 and the diagnostic evaluation can lead to increased morbidity if surgery is pursued.19,20

PRIMARY THYROID LYMPHOMA

B Fig. 69.3  A, Diagnostic image of a thyroid ultrasound showing lymphocytic thyroiditis involving the right lobe of the thyroid. Examination of the left lobe reveals marked enlargement and diffuse hypoechogenicity of the gland. Findings are consistent with primary thyroid lymphoma. B, Diagnostic image of a thyroid ultrasound demonstrating Graves disease. The gland is diffusely heterogeneous with markedly increased Doppler flow, also called thyroid inferno.

finding explains the bruit that often may be heard on physical examination and has been termed “thyroid inferno” when seen on US (see Fig. 69.3B).11 Thyroid nodules may be identified in up to 35% of patients with Graves disease when screening US is performed.12 There is significant debate regarding whether nodules harbor an increased risk of malignancy in the setting of coexistent autoimmune thyroiditis.13,14 Similar controversy exists regarding the prognostic significance of those nodules that are malignant; some studies suggest that the cancer is more aggressive with simultaneous Graves disease,15 while others found no difference in clinical outcomes.16 Although US identifies more nodules and cancers than palpation or scintigraphy,17 screening US examination for all patients with Graves disease currently is not recommended.18 The majority of cancers identified with screening

Primary thyroid lymphoma is a rare malignancy that represents less than 2% of all thyroid carcinomas.21 It is more commonly seen in White women over the age of 60 years with a prior history of Hashimoto’s thyroiditis.22 Fortunately, most cases are diagnosed at an early stage and are associated with an excellent prognosis, but it is important to recognize the cardinal sonographic features of primary thyroid lymphoma, as a delay in diagnosis can negatively impact survival.22 The clinical features are generally the first clue to the diagnosis; patients describe a several-­week history of rapidly enlarging neck mass that may cause worsening dysphagia and a pressure sensation. Some patients also report “B symptoms” such as night sweats, fever, and weight loss.23 Largely occurring in the setting of preexisting Hashimoto’s thyroiditis, the US appearance can appear as an extreme manifestation of lymphocytic thyroiditis. A uniform, markedly hypoechoic gland with significant posterior acoustic enhancement that is significantly enlarged should increase the suspicion for primary thyroid lymphoma (Fig. 69.4). Depending on the stage and extent of disease, all or part of the gland may be involved. In higher-­stage disease, the presence of enlarged locoregional adenopathy may also be seen.24

Subacute Thyroiditis Subacute thyroiditis is a self-­limited inflammatory disease that occurs in response to an antecedent viral illness. Patients present with acute neck pain and sudden-­onset (often bilateral) thyromegaly, as well as fatigue and fever. In the acute phase, patients also may experience symptoms of thyrotoxicosis. Though the clinical presentation and significant anterior neck pain often is sufficiently diagnostic, US can aid in the distinction from other causes of thyrotoxicosis. The typical sonographic findings include focal, nonovoid areas of markedly

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PART 6  Thyroid

TABLE 69.1  American Thyroid Association Risk Stratification System Risk Category

Description

Malignancy Risk

Threshold for FNA

High

Solid hypoechoic nodule or solid hypoechoic component in partially cystic nodule with one or more suspicious features* Hypoechoic solid nodule with smooth margins lacking suspicious features** Isoechoic or hyperechoic solid or partially cystic with eccentric solid areas lacking suspicious features** Spongiform or partially cystic without suspicious features Purely cystic (no solid component)

>70%–90%

≥1 cm

10%–20%

≥1 cm

5%–10%

≥1.5 cm

0.5 cm selective^) ≥1 cm

3%–15% T),42 TSH-­R (rs179247 and rs12885526),43 FC receptor-­ like-­ 3 (FCRL3_3C, FCRL3_5C, FCRL3_6A),44,methylenetetrahydrofolate reductase gene (TT genotype),45 tumor necrosis factor super family-­15 ( (rs3810936 and rs4979462 polymorphisms),46 and HLA genes.47 Fig. 72.4 summarizes the key components involved in the pathogenesis of GO.

Clinical Evaluation EUGOGO recommends the assessment of both activity and severity of GO according to standardized criteria, and subsequent classification as inactive or active, and mild, moderate-­to-­severe, or sight-­threatening.12 Accurate assessment is essential in determining the most appropriate treatment strategies for GO patients. CAS is the most well-­established tool used to assess disease activity in both daily practice and clinical trial settings (Table 72.2). A CAS value of 3/7 or more or 4/10 or more indicates active GO, which necessitates immunomodulatory treatment. Disease severity is determined by five components: soft tissue involvement as reflected by 7-­point CAS value (excluding pain parameters), upper lid retraction, diplopia, proptosis, and evidence of sight-­ threatening complications (Table 72.3).

KEY POINTS  • Thyroid­stimulating hormone receptor (TSH-­R) is believed to be the primary autoantigen in Graves’ orbitopathy (GO). The loss of immune tolerance leads to orbital inflammatory infiltration and activation of orbital fibroblasts, resulting in perpetuation of inflammation, excessive glycosaminoglycan production and subsequent tissue edema, de novo adipogenesis, and ultimately tissue remodeling and fibrosis. TSH-­R–insulin-­like growth factor-­1 receptor crosstalk and their signaling pathways are central to the pathophysiology of GO.

Asymmetrical and Unilateral Graves’ Orbitopathy. GO is considered a generalized orbital disease. Asymmetrical or unilateral involvement can occur, although there is no established definition of this clinical entity. In a recent prospective cross-­sectional multicenter study involving new referrals to EUGOGO centers,48 unilateral GO was defined as one or more clinical features of GO in one eye only. Asymmetrical GO was defined as bilateral disease with one or more of the following features: (1) difference in exophthalmometer readings of 2 mm or more; (2) difference in palpebral aperture of 2 mm or more; (3) difference in eyelid swelling by one grade or more; (4) difference in eyelid erythema by one grade or more; (5) difference in conjunctival redness by

CLINICAL MANIFESTATIONS AND DIAGNOSIS GO can present with a wide spectrum of clinical features, from nonspecific to pathognomonic symptoms and signs, and from trivial complaints to disabling and sight-­threatening complications (Table 72.1).

Circulation

T cell receptor

MHC class II

Fibrocyte

B cell

T cell

ORBITAL INFLAMMATORY INFILTRATION

TSHR peptide

en

er iff

D

CD40 Ligand

TSH-R-Ab

CD40

n

tio

tia

Residential CD34orbital fibroblast

Slit2

Myofibroblast

TISSUE FIBROSIS

TSHR/IGF-1R crosstalk

Cytokines, chemokines, growth factors, adhesion molecules, etc

CD34+ orbital fibroblast

Diffe

renti

ation

Adipocyte (Do novo adipogenesis)

1199

Hyaluronan & GAG over-production

ORBITAL TISSUE EDEMA & EXPANSION

Fig. 72.4  Pathogenesis of Graves’ Orbitopathy (GO)––an Overview. TSH-­R is Believed to be the Primary Autoantigen in GO. The loss of immune tolerance generates autoreactive T-­and B-­cells, which are activated by costimulation and cytokines. Orbital fibroblasts becomes activated by various stimuli (cytokines/ growth factors, TSH-­R/IGF-­1R signaling and crosstalk after stimulation by TSH-­R–Ab, and CD40–CD40 ligand interaction with T-­cells). Orbital fibroblasts play a central role in mediating the key pathological changes in GO, including orbital inflammatory infiltration, de novo adipogenesis, hyaluronan overproduction, with resultant tissue edema, expansion, remodeling, and fibrosis. IGF-­1R, Insulin-­like growth factor-­1 receptor; MHC, major histocompatibility complex; TSHR, thyroid-­stimulating hormone receptor; TSH-­R–Ab, thyroidstimulating ­hormone receptor antibody; GAG, glycosaminoglycan.

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PART 6  Thyroid

TABLE 72.1  Clinical Features of Graves’ Orbitopathy Clinical Features

Pathophysiology and Clinical Significance

• G  ritty or foreign body sensation, light sensitivity (photophobia), excess tearing

• O  cular surface irritation resulting from increased evaporative loss and/or impaired tear film formation because of proptosis or eyelid abnormalities • May mimic other common ocular conditions, e.g., allergic conjunctivitis, dry eye disease

• • • • •

• Reflect orbital inflammation and congestion

E yelid swelling and redness Conjunctival swelling (chemosis) and redness Caruncle or plica swelling Gaze-­evoked pain or spontaneous retrobulbar pain Elevated intraocular pressure

• Upper eyelid retraction

• S ympathetic activation of Muller’s muscle • Synkinetic activity of levator palpebrae superioris with superior rectus that tries to overcome the tight inferior rectus • Inflammation, degeneration, and scarring of eyelid structures

• Lower eyelid retraction/displacement

• C orrelates with the degree of proptosis • Lower lid retractors apparently unaffected in Graves’ orbitopathy

• Lid lag

• D  efined as a static phenomenon in which the upper eyelid assumes and maintains a higher position (relative to its position in primary gaze) with the eye in downgaze

• Von Graefe’s sign

• D  efined as a dynamic phenomenon in which the upper eyelid fails to descend smoothly and lags behind the eyeball during the course of downgaze

• Lagophthalmos

• Incomplete eyelid closure because of eyelid abnormalities and significant proptosis • Predisposes to exposure keratopathy, especially when Bell’s phenomenon is defective owing to tight inferior rectus

• Diplopia and squint

• T he proliferation of orbital fibroblasts (de novo adipogenesis, secretion and accumulation of hydrophilic glycosaminoglycans) and inflammatory infiltration lead to enlargement of extraocular muscles, resulting in restrictive (instead of paralytic) strabismus

• Proptosis

• E xpanded volume of retrobulbar structures pushes the eyeball forward • Globe subluxation may occur in extreme proptosis

• Visual loss

• Watch out for sight-­threatening Graves’ orbitopathy

TABLE 72.2  Clinical Activity Score Parameters

7-­Point CAS

1. Spontaneous retrobulbar pain • Applicable to first clinical 2. Pain on attempted upward/ assessment downward/side gaze or during 3. Redness of eyelids sub­sequent 4. Redness of conjunctiva follow-­ups 5. Swelling of eyelids (parameters 6. Swelling of conjunctiva 1-7) (chemosis) 7. Swelling of caruncle or plica 8. Increase of ≥2 mm in proptosis 9. Decrease in uniocular ­excursion in any one ­direction of ≥8 degrees 10. Decrease in visual acuity equivalent to 1 Snellen line

10-­Point CAS • A  pplicable during subsequent follow-­ups (parameters 1-10)

CAS, Clinical activity score. • One point is given to each of the seven signs and symptoms in the 7-­point CAS during initial assessment. • On subsequent follow-­up, either the 7-­point or the 10-­point CAS (which includes three additional parameters, namely deterioration in ocular dysmotility, proptosis, or visual acuity) can be used.

one grade or more; (6) presence of unilateral DON. Some 58% of GO patients had symmetrical disease, whereas 31% and 11% had asymmetrical and unilateral disease, respectively. Compared with the symmetrical and unilateral groups, the asymmetrical group had an older mean age, lower female-­to-­male ratio, and more active and severe disease. Whereas asymmetry is a potential indicator of more active and severe GO than other clinical variants, unilaterality is associated with less severe disease.

Dysthyroid Optic Neuropathy. DON is the most common form of sight-­threatening GO. Direct compression of swollen extraocular muscles on the optic nerve at orbital apex is believed to be the pathogenic mechanism in over 90% of cases. However, some propose that optic nerve stretch in the presence of marked proptosis may explain a minority of cases, although this theory remains controversial. The most common symptom is visual blurring. Approximately 50% of affected patients have bilateral, but not necessarily symmetrical, involvement (simultaneous or sequential). Although there are no established diagnostic criteria validated for DON, the salient diagnostic features are summarized in Table 72.4.

Thyroid Function Test Over 95% of GO cases are associated with Graves hyperthyroidism.49,50 Although GO most commonly occurs simultaneously with or after the onset of hyperthyroidism, it precedes the onset of hyperthyroidism in 20% of patients (and most of them are diagnosed within

CHAPTER 72  Graves’ Orbitopathy

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TABLE 72.3  Assessing Severity of Graves’ Orbitopathy Mild Graves’ Orbitopathy

Moderate-­to-­Severe Graves’ Orbitopathy

General description

• M  inimal impact on daily life • Usually present ≥1 of the following signs:

• N  o sight-­threatening GO • Significant impact of daily life to justify the risks of immunosuppressants (if active) or surgical intervention (if inactive) • Usually present ≥2 of the following signs:

Soft tissue involvement Lid retraction Proptosis

Mild 5-­fold cutoff), acute or severe hyperthyroidism (high serum free T3 >3-­to 5-­fold), active smoking, recent-­onset hyperthyroidism (duration 40%)

1 + 2 = possible IgG4-­RD; 1 + 3 = probable IgG4-­RD; 1 + 2 + 3 = definite IgG4-­RD (Modified from Kottahachchi D, Topliss D. Immunoglobulin G4-­related thyroid disease. Eur Thyroid J. 2016;5:231–239.)

follicular cell degeneration, in addition to other features common to IgG4-­RD in other organs, such as elevated serum IgG4, mass-­forming fibrosis with increased IgG4-­positive plasma cells, and rapid progression to organ failure. Serum IgG4 presumably derives from IgG4+ plasma cells within the germinal center in the thyroid. In fact, circulating levels of IgG4 decrease after thyroidectomy. The peculiar and rare fibrotic variant of HT, which was initially considered a late stage of HT, is actually considered a distinct clinicopathological entity consisting of severe compressive symptoms and firm malignant-­like thyroid. It entails marked fibrotic replacement and typical inflammatory features in a lobular pattern, without extracapsular extension. The associated finding of increased IgG4+ cells and serum IgG4/IgG ratio suggests that this fibrotic variant, rather than HT as a whole, could actually be part of the spectrum of IgG4-­RD.91 The role of IgG4 in GD is not straightforward, and the prevalence of IgG4-­RD in patients with GD is presumably very low. Some particular characteristics include an older age at diagnosis, relative male predominance, a better response to antithyroid drugs, and a relatively higher rate of development of Graves’ ophtalmopathy.98,99 There is yet no strong evidence for the existence of IgG4 GD, but rather GD with a potential concomitant association with elevated IgG4. IgG4-­RD is a rather novel entity, entailing a potential underestimation of its real prevalence. For IgG4 thyroiditis, a heterogeneous prevalence has been reported, ranging from 5% to 27%. Several reasons may explain the discrepancies observed. First, there is the variability of histologic criteria and thresholds used to define increased IgG4-­positive plasma cells and/or IgG4/IgG ratios across the different studies. In addition, the number of IgG4+ plasma cells depends on disease duration; specifically, these cells are characteristically increased at the onset of the disease but decrease in favor of fibrosis in more advanced stages. Furthermore, the low need for thyroidectomy in patients with thyroiditis may jeopardize the availability of a histologic definitive diagnosis. A rather surprising issue is its predominance in males, in contrast to the higher proportion of women affected in autoimmune diseases.95,100,101 KEY POINTS  • IgG4 thyroiditis usually entails the development of thyroid failure, tumefactive enlargement, compressive symptoms and suspicion of malignancy.

Clinical Aspects IgG4-­RD may mimic malignant disease owing to its subacute onset and the frequent development of tumefactive lesions. In the specific

setting of the thyroid, it may seem similar to Riedel’s thyroiditis, with a trend for more severe compressive symptoms and/or suspicion of malignancy, and subsequent relatively rapid progression to thyroid function failure owing to chronic autoimmune inflammation. The presence of pain may also serve as an indicator of the potential existence of IgG4 thyroiditis. In addition, patients may exhibit synchronous or metachronous lesions in extrathyroidal organs.90

Diagnosis High levels of thyroid antibodies are frequently observed in patients with IgG4 thyroiditis. Ultrasonography usually reveals a hypoechogenic pattern. Diagnosis relies on several key histologic features: dense lymphoplasmacytic infiltrates rich in IgG4 plasma cells, fibrosis with a storiform (irregularly whorled) pattern, obliterative phlebitis, and eosinophilic infiltration. Phlebitis without lumen obliteration and an increased number of eosinophils are considered minor nonspecific features.97,102 IgG4 immunostaining is also important for the diagnosis.102 Indeed, there should be positive IgG4 staining within the involved tissue, for example, thyroid or orbital tissue, for a definitive diagnosis of IgG4-­ induced thyroiditis or orbitopathy/exophthalmos. All other parameters are not specific or definitive for an accurate diagnosis. In this regard, the role of high circulating levels of IgG4 does not seem to be solely sufficient for a definitive diagnosis; in fact, other conditions, such as infections, connective tissue diseases, and immunodeficient states, may also exhibit high IgG4 levels, and multiple studies have reported conflicting results regarding its predictive sensitivity. In spite of these uncertainties, today, the term “IgG4 thyroiditis” is used both when the histologic diagnosis (gold standard) is confirmed and when elevated serum IgG4 levels are present in a patient with thyroiditis, regardless of histologic confirmation, a circumstance that is rather frequent, because not many patients with thyroiditis require thyroidectomy as treatment. In an attempt to overcome this difficulty, levels of thyroid antigen–specific IgG4, rather than total serum IgG4, have been suggested to be more helpful for establishing a correct diagnosis. Clinical, serological, and histological features need to be considered as a whole and collated with the proposed 2012 criteria to decide if a given case deserves the diagnosis of IgG4 thyroiditis.100,101

Treatment and Follow-­Up First-­line therapy for IgG4-­RD usually involves glucocorticoids, but azathioprine, mycophenolate mofetil, and methotrexate have also been commonly used as an alternative, or to maintain remission after glucocorticoid administration. In cases of recurrent or refractory disease, the anti-­CD20 monoclonal antibody rituximab may be attempted with the aim of depleting B cells, as it is sometimes successful in treating Riedel’s thyroiditis. Extensive and established fibrosis will encumber effectiveness of any of the attempted medical treatments, resulting in the probable need for surgery.

SUMMARY AND FUTURE DIRECTIONS Thyroiditis comprise several forms of thyroid inflammation with variable repercussions for thyroid function. Their etiology, epidemiology, pathology, and clinical impact differ, and specific terminology has been developed accordingly. The most common forms of thyroiditis include HT, acute/infectious thyroiditis, de Quervain’s SAT, silent thyroiditis, Riedel’s thyroiditis, and IgG4 thyroiditis. Autoimmunity is the main etiopathogenic mechanism involved in thyroid disease, and its quintessential example is HT. Epidemiological data suggest an interaction between genetic susceptibility, existential factors, and exogenous environmental triggers in this breakdown of immune tolerance. Humoral

CHAPTER 73  Thyroiditis and cellular immune mechanisms are closely related and cross-­linked in AITD. Once they are triggered, they undergo subsequent feedback circuits, with combined amplification and inhibition processes, that reflect the complexity of these underlying pathways in the onset, development, and perpetuation of AITD, and that still deserve further investigation. The various forms of thyroiditis may be interrelated, and studies have confirmed the association of AITD and other specific or systemic autoimmune disorders and thyroid malignancy. Long-­term monitoring of AITD patients is required for adequate management. Further studies to clarify potential preventive or therapeutic approaches, beyond mere replacement therapy, will aid in identifying the optimal approach to treating AITD.

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43. Kim D. Low vitamin D status is associated with hypothyroid Hashimoto’s thyroiditis. Hormones (Basel). 2016;15:385–393. 44. Koehler VF, Filmann N, Mann WA. Vitamin D status and thyroid autoantibodies in autoimmune thyroiditis. Horm Metab Res. 2019;51: 792–797. 45. Guastamacchia E, Giagulli VA, Licchelli B, et al. Selenium and iodine in autoimmune thyroiditis. Endocr Metab Immune Disord -­Drug Targets. 2015;15:288–292. 46. Tomer Y, Davies TF. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev. 2003;24:694–717. 47. Lee HJ, Li CW, Hammerstad SS, et al. Immunogenetics of autoimmune thyroid diseases: a comprehensive review. J Autoimmun. 2015;64:82–90. 48. Hansen PS, Brix TH, Iachine I, et al. The relative importance of genetic and environmental effects for the early stages of thyroid autoimmunity: a study of healthy Danish twins. Eur J Endocrinol. 2006;154:29–38. 49. Brix TH, Hegedus L. Twin studies as a model for exploring the aetiology of autoimmune thyroid disease. Clin Endocrinol. 2012;76:457–464. 50. McLeod DS, Cooper DS. The incidence and prevalence of thyroid autoimmunity. Endocrine. 2012;42:252–265. 51. McLeod DS, Cooper DS, Ladenson PW, et al. Race/Ethnicity and the prevalence of thyrotoxicosis in young Americans. Thyroid. 2015;25:621– 628. 52. Simmonds MJ. GWAS in autoimmune thyroid disease: redefining our understanding of pathogenesis. Nat Rev Endocrinol. 2013;9:277–287. 53. Krassas GE, Tziomalos K, Pontikides N, et al. Seasonality of month of birth of patients with Graves’ and Hashimoto’s diseases differ from that in the general population. Eur J Endocrinol. 2007;156:631–636. 54. Aksoy DY, Kerimoglu U, Okur H, et al. Effects of prophylactic thyroid hormone replacement in euthyroid Hashimoto’s thyroiditis. Endocr J. 2005;52:337–343. 55. Radetti G, Gottardi E, Bona G, et al. The natural history of euthyroid hashimoto’s thyroiditis in children. Study group for thyroid diseases of the Italian society for pediatric endocrinology and diabetes. J Pediatr. 2006;149:827–832. 56. Wang SY, Tung YC, Tsai WY, et al. Long-­term outcome of hormonal status in Taiwanese children with Hashimoto’s thyroiditis. Eur J Pediatr. 2006;165:481–483. 57. Hadithi M, de Boer H, Meijer JW, et al. Coeliac disease in Dutch patients with Hashimoto’s thyroiditis and vice versa. World J Gastroenterol. 2007;13:1715–1722. 58. Kahaly GJ, Frommer L, Schuppan D. Celiac disease and glandular autoimmunity. Nutrients. 2018;10:814. 59. Minelli R, Gaiani F, Kayali S, et al. Thyroid and celiac disease in pediatric age: a literature review. Acta Biomed. 2018;89:11–16. 60. Sibilla R, Santaguida MG, Virili C, et al. Chronic unexplained anaemia in isolated autoimmune thyroid disease or associated with autoimmune related disorders. Clin Endocrinol. 2008;68:640–645. 61. Fiore E, Rago T, Latrofa F, et al. Hashimoto’s thyroiditis is associated with papillary thyroid carcinoma: role of TSH and of treatment with L-­thyroxine. Endocr Relat Cancer. 2011;18:429–437. 62. Lee JH, Kim Y, Choi JW, et al. The association between papillary thyroid carcinoma and histologically proven Hashimoto’s thyroiditis: a meta-­ analysis. Eur J Endocrinol. 2013;168:343–349. 63. Silva de Morais N, Stuart J, Guan H, et al. The impact of Hashimoto’s thyroiditis on thyroid nodule cytology and risk of thyroid cancer. J Endocr Soc. 2019;3:791–800. 64. Lai X, Xia Y, Zhang B, et al. A meta-­analysis of Hashimoto’s thyroiditis and papillary thyroid carcinoma risk. Oncotarget. 2017;8:62414– 62424. 65. Moon S, Chung HS, Yu JM, et al. Associations between Hashimoto’s thyroiditis and clinical outcomes of papillary thyroid cancer: a meta-­analysis of observational studies. Endocrinol Metab (Seoul). 2018;33:473–484. 66. Rhoden KJ, Unger K, Salvatore G, et al. RET/papillary thyroid cancer rearrangement in nonneoplastic thyrocytes: follicular cells of Hashimoto’s thyroiditis share low-­level recombination events with a subset of papillary carcinoma. J Clin Endocrinol Metab. 2006;91:2414–2423.

67. Moshynska OV, Saxena A. Clonal relationship between Hashimoto’s thyroiditis and thyroid lymphoma. J Clin Pathol. 2008;61:438–444. 68. Feller M, Snel M, Moutzouri E, et al. Association of thyroid hormone therapy with quality of life and thyroid-­related symptoms in patients with subclinical hypothyroidism: a systematic review and meta-­analysis. J Am Med Assoc. 2018;320:1349–1359. 69. Guldvog I, Reitsma LC, Johnsen L, et al. Thyroidectomy versus medical management for euthyroid patients with hashimoto’s disease and persisting symptoms: a randomized trial. Ann Intern Med. 2019;170:453. 446. 70. Liontiris MI, Mazokopakis EE. A concise review of Hashimoto’s thyroiditis (HT) and the importance of iodine, selenium, vitamin D and gluten on the autoimmunity and dietary management of HT patients. Points that need more investigation. Hell J Nucl Med. 2017;20:51–56. 71. Wichman J, Winther KH, Bonnema SJ, et al. Selenium supplementation significantly reduces thyroid autoantibody levels in patients with chronic autoimmune thyroiditis: a systematic review and meta-­analysis. Thyroid. 2016;26:1681–1692. 72. van Zuuren EJ, Albusta AY, Fedorowicz Z, et al. Selenium supplementation for Hashimoto’s thyroiditis: summary of a Cochrane systematic review. Eur Thyroid J. 2014;3:25–31. 73. Mazokopakis EE, Kotsiris DA. Hashimoto’s autoimmune thyroiditis and vitamin D deficiency. Current aspects. Hell J Nucl Med. 2014;17:37–40. 74. Lundin KE, Wijmenga C. Coeliac disease and autoimmune diseasegenetic overlap and screening. Nat Rev Gastroenterol Hepatol. 2015;12: 507–515. 75. Bauer M, Goetz T, Glenn T, et al. The thyroid-­brain interaction in thyroid disorders and mood disorders. J Neuroendocrinol. 2008;20:1101–1114. 76. Yalcin MM, Altinova AE, Cavnar B, et al. Is thyroid autoimmunity itself associated with psychological well-­being in euthyroid Hashimoto’s thyroiditis? Endocr J. 2017;64:425–429. 77. Cai YJ, Wang F, Chen ZX, et al. Hashimoto’s thyroiditis induces neuroinflammation and emotional alterations in euthyroid mice. J Neuroinflammation. 2018;15:299. 78. Leyhe T, Müssig K. Cognitive and affective dysfunctions in autoimmune thyroiditis. Brain Behav Immun. 2014;41:261–266. 79. Churilov LP, Sobolevskaia PA, Stroev YI. Thyroid gland and brain: enigma of Hashimoto’s encephalopathy. Best Pract Res Clin Endocrinol Metab. 2019;33:101364. 80. Castillo P, Woodruff B, Caselli R, et al. Steroid-­responsive encephalopathy associated with autoimmune thyroiditis. Arch Neurol. 2006;63:197–202. 81. Raman L, Murray J, Banka R. Primary tuberculosis of the thyroid gland: an unexpected cause of thyrotoxicosis. BMJ Case Rep. 2014;2014. 82. McAninch EA, Xu C, Lagari VS, et al. Coccidiomycosis thyroiditis in an immunocompromised host post-­transplant: case report and literature review. J Clin Endocrinol Metab. 2014;99:1537–1542. 83. Farwell AP. Infectious thyroiditis. In: Braverman LE, Utiger RD, eds. Werner & Ingbar’s the Thyroid: A Fundamental and Clinical Text. 8th ed. Philadelphia: Lippincott Williams & Wilkins; 2000:1044–1050. 84. Paes JE, Burman KD, Cohen J, et al. Acute bacterial suppurative thyroiditis: a clinical review and expert opinion. Thyroid. 2010;20:247–255. 85. Samuels MH. Subacute, silent, and postpartum thyroiditis. Med Clin North Am. 2012;96:223–233. 86. Stasiak M, Tymoniuk B, Michalak R, et al. Subacute thyroiditis is associated with HLA-­B*18:01, -­DRB1*01 and -­C*04:01-­the significance of the new molecular background. J Clin Med. 2020;16:534. 87. Görges J, Ulrich J, Keck C, et al. Long-­term outcome of subacute thyroiditis. Exp Clin Endocrinol Diabetes. 2020;128:703–708. 88. Fatourechi V, Aniszewski J, Fatourechi G, et al. Clinical features and outcome of subacute thyroiditis in an incidence cohort: olmsted County, Minnesota study. J Clin Endocrinol Metab. 2003;88:2100–2105. 89. Iyer PC, Cabanillas ME, Waguespack SG, et al. Immune-­related thyroiditis with immune check-­point-­inhibitors. Thyroid. 2018;28: 1243–1251. 90. Dahlgren M, Khosroshahi A, Nielsen GP, et al. Riedel’s thyroiditis and multifocal fibrosclerosis are part of the IgG4-­related systemic disease spectrum. Arthritis Care Res. 2010;62:1312–1328.

CHAPTER 73  Thyroiditis 91. Stan MN, Sonawane V, Sebot TJ, et al. Riedel’s thyroiditis association with IgG4-­related disease. Clin Endocrinol. 2017;86:425–430. 92. Hennessey JV. Clinical review: Riedel’s thyroiditis: a clinical review. J Clin Endocrinol Metab. 2011;96:3031–3041. 93. Falhammar H, Juhlin CC, Barner C, et al. Riedel’s thyroiditis: clinical presentation, treatment and outcomes. Endocrine. 2018;60:185–192. 94. Soh SB, Pham A, O’Hehir RE, et al. Novel use of rituximab in a case of Riedel’s thyroiditis refractory to glucocorticoids and tamoxifen. J Clin Endocrinol Metab. 2013;98:3543–3549. 95. Brito-­Zeron P, Ramos-­Casals M, Bosch X, et al. The clinical spectrum of IgG4-­related disease. Autoimmun Rev. 2014;13:1203–1210. 96. Stone JH, Zen Y, Deshpande V. IgG4-­related disease. N Engl J Med. 2012;366:539–551. 97. Deshpande V, Zen Y, Chan JK, et al. Consensus statement on the pathology of IgG4-­related disease. Mod Pathol. 2012;25:1181–1192.

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98. Takeshima K, Inaba H, Furukawa Y, et al. Elevated serum immunoglobulin G4 levels in patients with Graves’ disease and their clinical implications. Thyroid. 2014;24:736–743. 99. Bozkirli E, Bakiner OS, Ersozlu Bozkirli ED, et al. Serum immunoglobulin G4 levels are elevated in patients with Graves’ ophthalmopathy. Clin Endocrinol. 2015;83:962–967. 100. Li Y, Zhou G, Ozaki T, et al. Distinct histopathological features of Hashimoto’s thyroiditis with respect to IgG4-­related disease. Mod Pathol. 2012;25:1086–1097. 101. Rotondi M, Carbone A, Coperchini F, et al. IgG4-­related thyroid autoimmune disease. Eur J Endocrinol. 2019;180:R175–R183. 102. Kottahachchi D, Topliss D. Immunoglobulin G4-­related thyroid disease. Eur Thyroid J. 2016;5:231–239.

74 Hypothyroidism Jacqueline Jonklaas

OUTLINE Brief History, 1234 Hypothalamic-­Pituitary-­Thyroid Axis, 1235 Thyroid Physiology and Thyroid Hormone Action, 1235 Epidemiology, 1236 Clinical Manifestations, 1236 Symptoms, 1236 Signs, 1237 Etiology, 1237 Primary Hypothyroidism, 1237 Central Hypothyroidism, 1238 Peripheral Causes of Hypothyroidism, 1239 Diagnosis, 1239 Reference Intervals, 1239 Serum Thyroid-­Stimulating Hormone, 1239

Log-­Linear Relationship, 1240 Free Thyroxine Levels, 1240 Triiodothyronine Levels, 1240 Thyroid Antibody Testing, 1240 Detection of Central Hypothyroidism, 1240 Subclinical Versus Overt Hypothyroidism, 1240 Screening, 1241 Normal Thyroid-­Stimulating Hormone With Symptoms ­Overlapping With Hypothyroidism, 1241 Treatment, 1241 Levothyroxine Therapy, 1241 Therapy Other Than Levothyroxine, 1246 Summary and Areas of Future Research, 1248

 

KEY POINTS  • Thyroid hormone is important for the function of all organ systems of the body; signs and symptoms of hypothyroidism are thus diverse. • Hypothyroidism is most commonly caused by progressive autoimmune destruction of the thyroid gland. It most frequently occurs in women and older individuals and results in a lifelong requirement for thyroid hormone replacement. • Although a constellation of symptoms characterize the hypothyroid state, the lack of pathognomonic symptoms may delay recognition of the need for testing for hypothyroidism. • The laboratory hallmark of primary hypothyroidism is an elevated serum thyroid-­stimulating hormone concentration. • Treatment with synthetic thyroid hormone in the form of levothyroxine lowers serum thyroid-­stimulating hormone concentrations to the normal range and reverses signs and symptoms of hypothyroidism in most patients. • Optimization of levothyroxine treatment by facilitating adherence and avoiding overtreatment and undertreatment remains a challenge. • Combination therapy with levothyroxine and liothyronine is being actively investigated as a potential therapy for patients in whom levothyroxine alone does not reverse their symptoms. • Future advances may allow researchers and clinicians to prevent the development of autoimmune destruction of the thyroid gland or provide therapy in the form of directing progenitor cells to form functioning thyroid follicles in vivo.

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BRIEF HISTORY Thyroid diseases and their treatments have been documented in both written and pictorial form throughout history. Sixth-­century Chinese scholars were apparently aware of the ability of seaweeds and thyroid glands from deer and sheep to treat goiters that occurred in mountainous region of the county,1 the latter perhaps representing one of the earliest examples of “organotherapy.” The facies depicted in artwork as diverse as the Adena pipe figurine from before the first century AD2 and the Mona Lisa from the early 1500s3 have been attributed to hypothyroidism. Although descriptions of myxedema existed, it was not until thyroidectomy was performed in Switzerland for goiter that resection of the thyroid was clearly linked to the subsequent development of myxedema.4 Starting during the 1890s, various modes of thyroid hormone replacement have been attempted for treating hypothyroidism, including grafting of sheep thyroid glands in patients, intravenous injection of thyroid extract in dogs, and subcutaneous administration of sheep thyroid extract in patients.5 Oral administration of sheep thyroid extract was then used, with extraordinary improvement in the hypothyroid symptoms and signs of the patients, and also some descriptions of the toxicity of overreplacement. Desiccated thyroid extract (DTE) then became the standard of treatment for hypothyroidism for many years. Although thyroxine (T4) itself was isolated in 1914 and synthesized in 1927, it was not synthesized for commercial purposes until 1949. Identification, isolation and synthesis of triiodothyronine (T3) occurred from 1952 to 1953.

CHAPTER 74  Hypothyroidism

1235

T4 I HO

I O

I

CH2-CH-COOH NH2

I

D2, D1 (ORD)

T3

I HO

D3, D1 (IRD) I

I O I

HO

CH2-CH-COOH NH2

I O

I

CH2-CH-COOH NH2

rT3

D2, D1 (ORD)

D3, D1 (IRD)

I HO

I O

CH2-CH-COOH NH2 T2

Fig. 74.1  Deiodination of thyroid hormone isotopes. (From van der Spek AH, Fliers E, Boelen A. The classic pathways of thyroid hormone metabolism. Mol Cell Endocrinol. 2017;458:29–38.)

Starting in the 1950s, levothyroxine (LT4) gradually replaced DTE as standard therapy. Although LT4 remains the most prescribed therapy for hypothyroidism, use of DTE continues, and interest in such therapy has been increasing in recent years.6 This chapter discusses the diagnosis and treatment of hypothyroidism in adults.

HYPOTHALAMIC-­PITUITARY-­THYROID AXIS The hypothalamic-­pituitary-­thyroid axis is a classic negative feedback loop involving four hormones. Hypothalamic thyrotropin-­releasing hormone (TRH) stimulates the release of thyroid-­stimulating hormone (TSH) from the pituitary gland. TSH supports the synthetic machinery within the thyroid gland itself and stimulates the resorption of thyroglobulin from within the lumen of thyroid follicles. Both T4 and T3 are then released from the thyroid gland into the circulation in a ratio of approximately 14:1.7 There is a reciprocal relationship between TSH and free T4 (FT4), such that, as the concentration of FT4 increases or decreases, the response is an exponential decrease or increase in TSH concentration. Thus, an inverse relationship is seen when TSH concentrations are plotted on a logarithmic scale versus FT4 concentrations on a linear scale. However, this relationship may be more complex than a simple linear one, with the slope of this relationship being affected by sex, age, genetics, and use of LT4. The relationship may actually be curvilinear, as opposed to linear, and each individual may have TSH-­FT4 set points that differ from the population as a whole.8 The development of primary hypothyroidism can thus be detected when the serum TSH concentration rises above the upper limit of its reference interval.

THYROID PHYSIOLOGY AND THYROID HORMONE ACTION Dietary iodine in the form of iodide or iodate is absorbed by the gastrointestinal tract and distributed in the extracellular fluid. Circulating iodide is actively transported into the thyrocyte by the sodium-­iodide symporter located within the basolateral membrane. Once iodide has translocated to the apical membrane, its efflux is mediated by three apical iodide channels. A hydrogen peroxide generation system is present at the apical surface of the thyrocyte. Molecules of the enzyme thyroid

peroxidase, also present at the apical surface of the thyrocyte, are activated by hydrogen peroxide. Oxidized thyroid peroxidase can then in turn oxidize iodide. The reactive iodonium ion intermediate formed is then covalently bound to tyrosyl residues present in thyroglobulin to generate monoiodotyrosine and diiodotyrosine residues through a process known as organification. Thyroid peroxidase also catalyzes the coupling of the monoiodotyrosine and diiodotyrosine residues to generate T4 and T3 residues in thyroglobulin, which is secreted into the follicular lumen. As needed, thyroglobulin is then pinocytosed at the apical membrane, and T4 and T3 are secreted after proteolysis of thyroglobulin. Pharmacologic amounts of iodine inhibit iodide trapping, organification, and release of the thyroid hormones. Thyroid hormones are necessary for the development and metabolic homeostasis of all the tissues and organ systems of the body. Depending on the various estimates, the intact thyroid gland produces approximately 85 to 100 mcg per day of T4 and 5 to 6.5 mcg per day of T3. This results in the direct thyroidal production of T4:T3 in the previously mentioned approximately 14:1 ratio. The deiodinase enzymes, which are selenoproteins, are involved in activating and deactivating thyroid hormones. Type 1 and 2 deiodinases convert the precursor T4 into T3, the active form of thyroid hormone, by outer ring deiodination, producing another 26.5 mcg of T3 daily. Type 3 deiodinase is responsible for converting T4 and T3 into their inactive forms of reverse T3 and 3,3′-­diiodothyronine, respectively, via inner ring deiodination9 (Fig. 74.1). Although thyroid hormones are lipophilic, they nevertheless require specific thyroid hormone transporters to gain entry into cells. Three transporters from two transporter families have high specificity for thyroid hormones. These are monocarboxylate transporters 8 and 10 (MCT8 and MCT10) and organic anion transporting polypeptide 1C1.10 Deiodinases and thyroid hormone transporters are differentially expressed in different tissues and are thus essential for the tissue-­specific actions of thyroid hormones. Once it has gained entry into the nucleus of the target cell, the active ligand T3 binds to thyroid hormone receptors, which in turn bind, mainly as heterodimers with retinoid-­X receptors, to thyroid hormone response elements in target genes11 (Fig. 74.2). Two genes, THRA and THRB, encode the thyroid hormone receptor proteins (TRα and TRβ), of which there are several splice products

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or isoforms. TRα1 is predominately expressed in brain, heart, and skeletal muscle. TRβ1 is widely expressed, while TRβ2 is mostly expressed in the brain, pituitary, retina, and inner ear, and TRβ3 is mostly expressed in kidney, liver, and lung. This differential expression of TRα and TRβ is also essential for the tissue-­specific actions of thyroid hormones. Following the binding of the heterodimer complex to the thyroid hormone response element with in the target gene, there is ensuing RNA transcription and protein synthesis to generate the cellular response to thyroid hormone. Most genes are positively regulated by T3, although some are negatively regulated.12

EPIDEMIOLOGY Throughout the world, the prevalence of hypothyroidism varies from 0.25% to 4.2%.13 Prevalence rates differ in iodine-­sufficient countries versus iodine-­deficient countries. For example, in an iodine-­deficient country (Denmark), the overall prevalence of overt hypothyroidism was 0.37%, compared with an overall prevalence of overt hypothyroidism in an iodine-­sufficient area (Norway) of 0.7%. The incidence of

T3

T3 D2 T4

D3

T4

T2

D3

TR RXR TRE Nucleus mRNA

D2 rT3

Protein

Fig. 74.2  Thyroid hormone transport, metabolism, and action in a thyroid hormone target cell. TRE, T3-reponsive elements; TR, nuclear thyroid hormone receptor; RXR, retinoid-X receptor. (From Groeneweg S, van Geest FS, Peeters RP, et al. Thyroid hormone transporters. Endocr Rev. 2020;41: bnz008.)

hypothyroidism generally increases following introduction of iodine fortification programs. Prevalence rates also differ by sex and age, with more hypothyroidism in women and in older age groups. To illustrate the effect of sex, in the 1995 report of the 20-­year follow-­up analysis of the Whickham population the prevalence of hypothyroidism was 1.3% and 9.3% in men and women, respectively. To illustrate the effect of age, the prevalence of hypothyroidism ranges from 1% to 2% in some studies, compared with 7% in a study of individuals aged between 85 and 89 years. Cases of subclinical hypothyroidism (SCH) exceed those of overt hypothyroidism. For example, in a study using National Health and Nutrition Examination Survey III data the overall prevalence of hypothyroidism was 4.6%, with 0.3% being overt hypothyroidism and 4.3% being subclinical disease.14 With respect to overt hypothyroidism, the prevalence in the general population depends on the definition used and population studied and ranges from 0.2% to 5.3% in Europe and from 0.3% to 3.7% in the United States.13

CLINICAL MANIFESTATIONS The clinical manifestations of hypothyroidism are diverse and potentially emanate from the effects of thyroid hormone deficiency in any organ system of the body15 (Table 74.1). Both signs and symptoms can occur across a wide spectrum of severity, ranging from subtle to profound.

Symptoms The symptoms of hypothyroidism reflect the effect of low levels of thyroid hormone throughout the body. Symptoms may be mild and barely perceptible or can be severe. In addition, symptoms of hypothyroidism are nonspecific and can overlap with symptoms of nonthyroid origin. For example, weight gain can be associated with untreated hypothyroidism, but can also occur in euthyroid individuals. The same statement can be made concerning the presence of fatigue, constipation, memory problems, and dry skin. To provide some examples, in one study, dry skin was reported in 71% of those with hypothyroidism versus 54% of those who were euthyroid, muscle weakness was described in 21% of those with hypothyroidism versus 21% of those

TABLE 74.1  Symptoms and Signs of Hypothyroidism Presentation

Signs and Implications

General metabolism Cardiovascular

Weight gain, cold intolerance, fatigue Fatigue on exertion, shortness of breath

Neurosensory

Hoarseness of voice, decreased taste, vision, or hearing Impaired memory, paresthesia, mood impairment

Increase in body mass index, low metabolic rate, myxedema,a hypothermiaa Dyslipidaemia, bradycardia, hypertension, endothelial dysfunction or increased intima media thickness,a diastolic dysfunction,a pericardial effusion,a hyperhomocysteinemia,a electrocardiogram changesa Neuropathy, cochlear dysfunction, decreased olfactory and gustatory sensitivity

Neurological and psychiatric

Gastrointestinal Endocrine

Hemostasis and hematological

Constipation Infertility and subfertility, menstrual disturbance, galactorrhoea Muscle weakness, muscle cramps, arthralgia Bleeding, fatigue

Skin and hair Electrolytes and kidney function

Dry skin, hair loss Deterioration of kidney function

Musculoskeletal

aUncommon

Impaired cognitive function, delayed relaxation of tendon reflexes, depression,a dementia,a ataxia,a carpal tunnel syndrome and other nerve entrapment syndromes,a myxedema comaa Reduced esophageal motility, nonalcoholic fatty liver disease,a ascites (very rare) Goiter, glucose metabolism dysregulation, infertility, sexual dysfunction, increased prolactin, pituitary hyperplasiaa Creatine phosphokinase elevation, Hoffman syndrome,a osteoporotic fracturea (most probably caused by overtreatment) Mild anemia, acquired von Willebrand disease,a decreased protein C and S,a increased red cell distribution width,a increased mean platelet volumea Coarse skin, loss of lateral eyebrows,a yellow palms of the hands,a alopecia areata Decreased estimated glomerular filtration rate, hyponatraemiaa

presentation. (From Chaker L, Bianco AC, Jonklaas J, et al. Hypothyroidism. Lancet. 2017;390:1550–1562.)

CHAPTER 74  Hypothyroidism who were euthyroid, and poor memory was noted in 18% of those with hypothyroidism versus 16% of those who were euthyroid.16 This has the dual consequences that individuals with these symptoms may be more likely to be suspected of having hypothyroidism, and that individuals with these symptoms may be diagnosed with hypothyroidism because they were screened, even if hypothyroidism was not the proximate cause of their symptoms. In addition to those symptoms mentioned earlier, other symptoms may include cold intolerance, hair loss, brittle hair and nails, muscle cramps, weakness, inability to concentrate, depression, and menometrorrhagia. In the same study as the one providing the percentages earlier, a change in symptoms in those with hypothyroidism compared with those who were euthyroid (such as being colder than the previous year or being more constipated than the previous year) had a higher likelihood ratio for hypothyroidism.16 High likelihood ratios were evident for having a hoarser voice than the previous year (likelihood ratio 5.2; 95% confidence interval [CI]: 2.1–12.6) and finding math more difficult than the previous year (likelihood ratio 5.4, 95% CI: 2.2–13.1). Interestingly, these types of symptoms are prevalent in those with Hashimoto hypothyroidism, even in the setting of euthyroidism. Furthermore, higher titers of thyroid peroxidase antibodies (TPO Abs) are associated with greater symptomatology.17 For example, comparing euthyroid individuals with TPO Ab titers greater than or less than 121 IU/mL revealed statistically significant greater rates of chronic fatigue (66% vs. 49%), lack of concentration (32% vs. 19%), and nervousness (68% vs. 39%) in patients with higher titers.

Signs Mild degrees of thyroid hormone deficiency may not result in obvious signs, except perhaps a firm goiter because of lymphocytic infiltration of the thyroid gland parenchyma. Over time, and with more decrement in thyroid hormone levels, cool and dry skin, coarse hair, loss of body hair, hoarse voice, coarse facial features, facial edema, generalized edema, bradycardia, and delayed relaxation phase of the deep tendon reflexes may be seen (Fig. 74.3). Extreme manifestations of these findings may be seen in myxedema coma.18 Profound nail changes with horizontal ridges and marked hair loss may even occur with comparatively modest TSH elevations of 30.5 mIU/L and then be reversed with restoration of euthyroidism.19 Manifestations of other autoimmune diseases such as vitiligo or hyperpigmentation may coexist with those of hypothyroidism. KEY POINTS:  Clinical Manifestations • Signs and symptoms of hypothyroidism emanate from all the organ systems of the body. • Symptoms of hypothyroidism overlap with symptoms of other conditions. • Sign and symptoms of hypothyroidism extend across a wide spectrum from barely discernible to severe.

ETIOLOGY The most common cause of hypothyroidism, in which the thyroid gland itself fails, is referred to as primary hypothyroidism. Primary hypothyroidism is generally considered “overt” when the TSH is elevated and the FT4 is low. Milder degrees of hypothyroidism, also known as SCH, are defined by an elevated TSH accompanied by a FT4 concentration that has not yet fallen below the normal range. Secondary or central hypothyroidism occurs when there is deficient production of TSH and is a much less common cause of hypothyroidism. Rarer causes of hypothyroidism also exist, such as TRH deficiency, thyroid dysgenesis, and “consumptive” hypothyroidism15 (Table 74.2).

A

B

C

D

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Fig. 74.3  Appearance of a 47-­year-­old man 12 years (A), 5 years (B), and 3 years (C) before hypothyroidism secondary to atrophic myxedema (D) was diagnosed. Note the typical myxedema face characterized by puffy, nonpitting swelling of the skin and coarse facial features.

Primary Hypothyroidism

Autoimmune Destruction. Among the causes of primary hypothyroidism, autoimmune (Hashimoto) thyroiditis is the most common cause in iodine-­sufficient areas.20 This is heralded by the presence of circulating TPO Abs, lymphocytic infiltration of the thyroid gland, and increased levels of TSH. Although the presence of TPO Abs is associated with an increased risk of progression to hypothyroidism, not all individuals with such positivity develop hypothyroidism. The pathology of Hashimoto thyroiditis involves infiltration of the interstitium around thyrocytes with lymphocytes, plasma cells, and macrophages.20 The lymphocytes organize into lymphoid follicles, and lymphocytes that come into close contact with the thyrocytes are believed to mediate their destruction. There is also ongoing fibrosis, and the thyroid gland has a firm consistency upon palpation. Hashimoto thyroiditis can occur alone or in association with other autoimmune disorders, such as type 1 diabetes and pernicious anemia. Hashimoto thyroiditis is eight times more common in women than men, with a peak age of onset of 40 to 60 years. It occurs more frequently in White and Asian Americans than in Black Americans.

Drug-­Induced. The list of medications that are associated with the development of hypothyroidism is ever-­expanding. A partial list is included in Table 74.2.15 Some of these drugs can also alter the requirement for thyroid hormone in individuals already receiving LT4 replacement therapy. Generally the effect is to cause an increased requirement. With respect to inducing the de novo development of hypothyroidism, several mechanisms may be responsible, such as altering thyroid hormone metabolism, altered thyroid-­binding proteins, causing damage

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PART 6  Thyroid

TABLE 74.2  Causes of Hypothyroidism Primary Hypothyroidism • Chronic autoimmune thyroiditis (also known as Hashimoto thyroiditis) • Iodine—severe iodine deficiency, mild and severe iodine excess • Drugs—for example, amiodarone, lithium, tyrosine kinase inhibitors, interferon-­α, thalidomide, monoclonal antibodies (e.g., ipilimumab and nivolumab), antiepileptic drugs (e.g., valproate), drugs for second-­line treatment of multidrug-­resistant tuberculosis • Iatrogenic—radioiodine treatment (e.g., for Graves disease or toxic nodular disease), hemithyroidectomy, radiotherapy or surgery in the neck or head region • Transient thyroiditis—viral (de Quervain syndrome), postpartum, silent thyroiditis, destructive thyroiditis • Thyroid gland infiltrationa —infectious (e.g., mycoplasma), malignant (e.g., thyroid malignancy, lymphoma, metastasis of malignancy elsewhere), autoimmune (e.g., sarcoidosis), inflammatory (e.g., Riedel thyroiditis) • Genetica —autoimmunity-­related genes (e.g., HLA class 1 region, PTPN22, SH2B3, and VAV3 ), general and thyroid-­specific genes (e.g., F0XE1, ATXN2, and PDE86) Central Hypothyroidism • Pituitary tumors (secreting or nonsecreting) • Pituitary dysfunction (e.g., Sheehan syndrome) • Hypothalamic dysfunction (e.g., posttraumatic) • Resistance to thyroid-­stimulating hormone (TSH) or thyrotropin-­releasing hormone • Drugs (e.g., dopamine, somatostatins, glucocorticosteroids, and retinoid X receptor-selective ligands) • Increased TSH concentration because of leptin stimulationb Peripheral (Extrathyroidal) Hypothyroidism • Consumptive hypothyroidism • Tissue-­specific hypothyroidism owing to decreased sensitivity to thyroid hormone (e.g., mutations in MCT8 [also known as SLC16A2], SEQSBP2, TH RA, TH RB) aRare

cause of primary hypothyroidism. mainly from animal models. (From Chaker L, Bianco AC, Jonklaas J, et al. Hypothyroidism. Lancet. 2017;390:1550–1562.) bEvidence

to thyroid gland parenchyma or vasculature, decreasing TSH production, enhancing autoimmunity, or supplying iodine or agents with an iodine-­like action. One class of drugs that is frequently associated with the development of hypothyroidism is anti-­cancer drugs. Agents associated with the development of hypothyroidism include immune checkpoint inhibitors, tyrosine kinase inhibitors, interferon-­α, and interleukin-­2.21

thyroidism, as the second phase of a three-­phase process (hyperthyroidism, hypothyroidism, euthyroidism). Causes include granulomatous, postpartum, and silent thyroiditis. Alternatively, the thyroiditis can be more widespread and not associated with recovery, as may occur in silent thyroiditis in those who already have lymphocytic infiltration of their thyroid gland, and in Riedel thyroiditis.

elevation of serum TSH levels and development of diffuse and nodular goiter as the thyroid axis attempts to maintain adequate thyroid functioning by increasing iodine uptake, accompanied also by enhanced iodine recycling. As the degree of iodine deficiency worsens it can lead to hypothyroidism caused by failure of the compensation mechanisms leading to lack of the substrate needed for thyroid hormone synthesis. Addition of iodine to salt is an inexpensive and effective means of providing adequate dietary iodine. Because of iodine fortification programs, the areas of the world affected by iodine deficiency have decreased. Exposure to iodine, in addition to reversing hypothyroidism as described earlier, can also induce hypothyroidism in those with predisposing risk factors such as prior episodes of thyroiditis or Hashimoto thyroiditis.24,25 One way in which iodine exposure can occur is in the form of iodinated contrast media.26 With the exposure to iodine, a normal thyroid gland will exhibit decreased thyroid hormone synthesis (Wolff–Chaikoff effect), followed by resumption of normal thyroid hormone synthesis (escape from the Wolff–Chaikoff effect). However, a gland affected by Hashimoto thyroiditis may not resume normal thyroid functioning (failure to escape from the Wolff–Chaikoff effect). In the former case of the normal gland, decreased sodium-­iodine symporter expression, and possibly increased pendrin-­mediated iodine efflux,27 appear to relieve the gland of the inhibitory effects of the high intrathyroidal iodine concentration, whereas in the latter case of the gland affected by autoimmune thyroiditis, these counterregulatory mechanisms may not deploy.

Iodine Deficiency or Excess. The effect of iodine on thyroid func-

Central Hypothyroidism

Other Processes Resulting in Destruction of the Thyroid Gland. There are several iatrogenic means by which the thyroid gland can be destroyed. These include surgery, radioactive iodine given as treatment for hyperthyroidism or thyroid cancer, and radiotherapy for head and neck malignancies. In the case of a hemithyroidectomy, the relative risk of developing hypothyroidism is approximately 30%.22 Infiltration of the thyroid gland associated with a spectrum of disease processes, such as sarcoidosis, hemochromatosis, hematologic malignancies, solid malignancies, and infectious agents can impair functioning to a sufficient degree to cause hypothyroidism.

Thyroiditis. Thyroiditis can be transient and cause temporary hypo-

tion is complex. Not only is there a U-­shaped curve, with insufficient iodine causing hypothyroidism and excessive iodine causing hyperthyroidism, but also introduction of iodine supplementation programs can enhance autoimmunity and change the pattern of thyroid diseases in a country.23,24 Iodine deficiency is associated with

Secondary Hypothyroidism. Secondary hypothyroidism is characterized by insufficient TSH stimulation of a normal thyroid gland. This condition may be inherited because of several genetic defects, for which a number of candidates genes have been identified.28 The acquired forms of secondary hypothyroidism include lesions in the

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CHAPTER 74  Hypothyroidism sella turcica region (pituitary adenomas, craniopharyngioma, gliomas, etc.), pituitary damage owing to surgery or radiation, head injury, vascular accidents, drugs, and infiltrative processes (lymphocytic hypophysitis, sarcoidosis, hemochromatosis, infectious agents).29

1000

1000

100

10

hypothalamic dysfunction can also result in central hypothyroidism. Some of the candidate’s genes associated with TSH deficiency may also be associated with TRH deficiency. Similar to the case with secondary hypothyroidism, tertiary hypothyroidism may be the consequence of hypothalamic lesions or hypothalamic injury because of surgery or radiation, head injury, vascular accidents, drugs, and infiltrative processes.29

Peripheral Causes of Hypothyroidism Several rare conditions can be associated with impaired sensitivity to thyroid hormone. This was originally described as occurring with alterations in the THRA and THRB genes, but the discovery of genetic mutations and polymorphisms causing alterations in cell membrane transport (e.g., MCT8) and metabolism (e.g., selenocysteine insertion sequence– binding protein 2 and type 2 deiodinase) of thyroid hormone have led to a broader definition of impaired sensitivity to thyroid hormone that includes many defects that could interfere with the activity of thyroid hormone. Because of the different functions and tissue-­specific expression of these genes, affected patients exhibit highly variable phenotypes. Some of them are characterized by a tissue hypothyroidism with well-­ recognizable alterations in the thyroid function tests, but others display a combination of hypothyroid and hyperthyroid manifestations.30 In addition to mutations causing tissue-­specific hypothyroidism, an interesting and also rare cause of peripheral hypothyroidism is “consumptive hypothyroidism.” This is a paraneoplastic syndrome in which a tumor produces excessive levels of the type 3 deiodinase, leading to markedly increased degradation of T4 and resultant low T3 levels, high reverse T3 levels, and elevated TSH. Large doses of LT4 are generally required until the tumor can be resected. Although originally described in tumors of vascular origin (hepatic hemangiomatosis) in infants, cases have now been described in adults because of both vascular and nonvascular tumors.31

KEY POINTS:  Etiology • The commonest cause of hypothyroidism is autoimmune destruction of the thyroid gland. • Processes interfering with hypothalamic or pituitary function are less ­common causes of hypothyroidism.

DIAGNOSIS Diagnosis of thyroid “disease” is based on a thyroid parameter (TSH, FT4, T3, etc.) confirmed as being outside its reference interval, combined with the signs and symptoms that would be anticipated for these “out of range” values.

Reference Intervals As for any other analyte, the reference interval for a thyroid analyte is derived from the 95% confidence intervals for that particular analyte established in at least 120 normal volunteers.32 These reference intervals, or decision limits, are provided to try to aid in interpretation of test results. Laboratories typically generate their own reference interval based on their local population. Theoretically, this should be a healthy population, and a minimum of 120 individuals should contribute to

TSH mIU/L

Tertiary Hypothyroidism. In addition to pituitary dysfunction,

3

100

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

10 64 yrs

3 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Free thyroxine (ng/dl)

Fig. 74.4  Relationship between free thyroxine and thyroid-­ stimulating hormone (TSH) in a group of patients with hypothyroidism according to age. For all patients together the Spearman correlation coefficient = –0.795, P < .0001. The insert shows the slopes within the four age groups: dots and dashes 64 years. FT4, Free thyroxine. (From Over R, Mannan S, Nsouli-­Maktabi H, et al. Age and the thyrotropin response to hypothyroxinemia. J Clin Endocrinol Metab. 2010;95:3675–3683.)

the database. Ensuring a healthy population without thyroid disease would involve, for example, excluding individuals with TPO Abs, those taking interfering medications, and those with goiters. If a different reference interval were deemed appropriate for a particular subpopulation, for example men versus women, this would require 120 individuals for each sex. These reference intervals are then used by various agencies to make recommendations about categorization or decisions based on the reference intervals. Given the consequences that may occur depending on the exact limits of these reference intervals, it is critical for clinicians to appreciate the factors that can influence these reference intervals. The bounds of a reference interval can, for example, result in making diagnoses that have minimal consequences or missing diagnoses in which intervention would be of benefit.33 Natural variations in thyroid parameters such as circadian rhythms, body mass index or weight, season, and climate can potentially affect reference intervals. In addition, age, sex, thyroid hormone assays, race and ethnicity, and iodine status can also affect reference intervals.

Serum Thyroid-­Stimulating Hormone The most commonly measured thyroid analyte is the serum TSH concentration. Hypothyroidism and hyperthyroidism are diagnosed when the TSH is confirmed to be above and below the limits of the reference interval, respectively. The effect of age on the reference interval is a particularly important issue for TSH. Serum TSH concentrations tend to rise with age, such that the upper limit of the reference interval is higher in older individuals. This is a very important consideration, as the benefits of thyroid hormone treatment for mild TSH elevations are not well established in older age groups. TSH assays, however, may detect abnormal TSH isoforms with different amounts of glycosylation, such as those that may be associated with TSH-­secreting pituitary tumors or normal aging.34 Altered glycosylation can cause a mismatch between TSH concentrations and TSH bioactivity. Several factors can interfere with measurement of TSH. For example, consumption of biotin can cause TSH values to be falsely measured as low, whereas heterophilic antibodies can cause TSH values to be falsely measured as high.35

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PART 6  Thyroid

Euthyroid state

Mild hypothyroidism Overt hypothyroidism

Triiodothyronine Levels

Fig. 74.5  The progression of hypothyroidism. TSH, Thyroid-­stimulating hormone; FT4, free thyroxine; T3, triiodothyronine.

If measuring T3, it is generally better to measure total T3 rather than free T3 because of the low concentrations of free hormone present and the lesser performance of immunoassays at these lower concentrations. Concentrations of total T3 are affected by binding protein abnormalities, as might occur with estrogen therapy and during pregnancy, and can also be affected by autoantibodies to thyroid hormone. An accurately measured elevated total T3 that is above the upper limit of the reference interval is consistent with hyperthyroidism. A total T3 that is below the lower limit of the reference interval is not necessarily sensitive or specific for hypothyroidism (Fig. 74.5). This is because total T3 levels fall later than FT4 levels in the course of development of hypothyroidism because of enhanced T4 to T3 conversion, and also because T3 levels are lowered by illness, poor nutrition, and several drugs such as beta-­blockers and glucocorticoids.

Log-­Linear Relationship

Thyroid Antibody Testing

TSH

FT4

T3

Normal range

Measurement of serum TSH is considered an ideal screening test for thyroid dysfunction, as there is an inverse log/linear relationship between TSH and FT4. This means that a small alteration in FT4 will produce a far larger change in serum TSH, thereby signaling even mild perturbations in FT4. Thus, even though TSH measurement is an indirect assessment of FT4, it is generally more sensitive for assessing thyroid dysfunction than is measuring FT4. Reliance on TSH as an indicator of thyroid status assumes that the pituitary is functional, that the individual is not suffering from a nonthyroidal illness, and that thyroid status is stable. When thyroid status is unstable, the serum TSH concentrations may lag behind the clinical picture. As previously mentioned, the TSH–FT4 relationship may be more complex than a simple linear one, with the slope being affected by sex, age, genetics, and use of LT4. For example, younger individuals may mount a more vigorous TSH response to low FT4 values than older individuals36 (Fig. 74.4).

With the previously described Hashimoto disease, TPO Abs and thyroglobulin antibodies are generally present in the serum, precede the development of thyroid dysfunction, and signal the ongoing cellular damage to the thyroid gland that is occurring as hypothyroidism develops. Occasionally patients may have autoimmune thyroid disease with negative antibody results. TPO Abs are a risk factor for progression into hypothyroidism over time, and also for developing hypothyroidism after treatment with agents such as amiodarone, lithium, and interferon-­α. Although changes in TPO Ab titers may reflect a change in disease activity, they are not useful for monitoring treatment for hypothyroidism. The presence of TPO Abs, however, is helpful for predicting the development of hypothyroidism during pregnancy, as well as the risk of miscarriage and the failure of in vitro fertilization. The presence of TSH receptor antibodies is useful for the evaluation of patients with hyperthyroidism.

Free Thyroxine Levels

If central hypothyroidism caused by pituitary or hypothalamic dysfunction is suspected, reliance on the serum TSH values for diagnostic purposes is no longer indicated. A low or low normal TSH in the setting of a low FT4 should raise the possibility of central hypothyroidism. Further evaluation should include a careful patient history, the assessment of other pituitary axes, and imaging of the pituitary and sella area.39

FT4 measurements by immunoassay are affected by protein binding and may be less accurate at extremes of binding protein concentrations, such as may occur during pregnancy. Results of FT4 measurement may be anomalous in the presence of albumin variants (e.g., familial dysalbuminemic hyperthyroxinemia), in the presence of medications that displace thyroid hormone from thyroxine-­binding globulin (e.g., phenytoin or carbamazepine), or in the context of critical illness. FT4 may also be measured after free hormone has been separated from bound hormone using equilibrium dialysis or ultrafiltration. Such methods are more time-­consuming and generally are less readily available. Other forms of interference include interfering antibodies, a problem that can be bypassed by using tandem mass spectrometry–based assays rather than immunoassays.37 In vitro drug interference can also occur, such as the in the case of heparin causing in vitro generation of free fatty acids, displacement of thyroid hormone from binding proteins, and false elevation of FT4 values.35 FT4 levels below and above the limits of the reference limit are consistent with hypothyroidism and hyperthyroidism, respectively. Although the gold standard for assessing thyroid status has been considered to be serum TSH, some studies suggest that thyroid hormone levels may be important too. A recent systematic review found that clinical parameters representing the cardiac, bone, and metabolism systems were more associated with FT4 levels than TSH levels, suggesting that further research is needed to understand the importance of thyroid hormone levels as an indicator of thyroid status.38

Detection of Central Hypothyroidism

Subclinical Versus Overt Hypothyroidism SCH has generally been defined as an elevation in the serum TSH level above the upper limit of the reference interval, with a FT4 concentration that remains in the normal range. When making a determination of whether SCH is present, the reference interval being used clearly impacts the diagnosis. In the literature, upper limits of normal that have been used include TSH values of 3, 4, 5, and 6 mIU/L. Clearly, these chosen cutoffs are based on the reference interval for the particular TSH assay used, which is in turn defined by the laboratory that is providing the assay. The reference population employed by the laboratory may not be fully described, and standardized collection procedures are often not used.40 The statistical methods used also affect the results.41 The particular cutoff value for a normal TSH used in any one study will determine the prevalence of SCH that is identified. This choice has consequences in terms of both detection and classification of thyroid disease. Lower TSH cutoffs will have the downstream effect of identifying a greater number of individuals with SCH in whom potential treatment may be considered. An additional very important consideration before diagnosing SCH is confirming the TSH elevation.

CHAPTER 74  Hypothyroidism Many above-­range TSH values revert to normal upon follow-­up, with the rate of normalization being inversely proportional to the degree of the TSH elevation above normal.33

Screening The US Preventive Services Task Force does not recommend routine screening of adults for thyroid disease.42 Other organizations differ as to whether they recommend routine screening or case-­finding in older, asymptomatic individuals. However, with respect to criteria for population screening, hypothyroidism is prevalent and an important health problem. Moreover, diagnosis is simple and accurate, and treatment is efficacious, cost-­effective, and safe. Confounding the issue are the possibilities that screening and early diagnosis may either only detect degrees of hypothyroidism that are mild and for which the benefits of treatment are less well documented or may detect disease for which treatment results in significant benefit. Although routine screening for thyroid dysfunction has not been shown to be of benefit for asymptomatic nonpregnant adults, mounting evidence suggests metabolic, cardiovascular, and skeletal risks in populations with TSH values above the normal range.43,44 Although the benefits of treatment of overt hypothyroidism are undisputed, positive results from randomized controlled trials of treating SCH would bolster the case for screening.45

Normal Thyroid-­Stimulating Hormone With Symptoms Overlapping With Hypothyroidism Because symptoms of hypothyroidism are nonspecific and overlap with symptoms of other conditions, even if an individual is found to have hypothyroidism following screening based on symptoms, the symptoms may or may not resolve with initiation of LT4. Thus, the symptoms may have led to the diagnosis of hypothyroidism, despite the fact that hypothyroidism was not the cause. Individuals with a normal serum TSH who have symptoms overlapping with symptoms of hypothyroidism do not have resolution of these symptoms with LT4 therapy.46

1241

Initiation of Therapy. At the time of their diagnosis with hypothyroidism, individuals may have varying degrees of residual thyroid function. Based on the relationship between declining thyroid hormone levels and stimulation of TSH production, those with SCH would still have substantial endogenous thyroid reserve, whereas those with overt hypothyroidism would have remaining endogenous thyroid function ranging from a complete absence to modest amounts. With respect to initiation of LT4 therapy, this can generally be started at a full replacement dose, so that biochemical derangements are more quickly reversed. This method does not appear to be associated with adverse cardiac consequences in those without underlying cardiac disease.47 Recommended exceptions to immediate full replacement are noted later under “Specific Situations.” At least two general approaches can be taken to starting doses of LT4 for replacement, both generally leading to similar estimates. One approach is to select the starting dose based on the degree of TSH elevation. Several formulae are available for guidance, should that approach be taken.48,49 The more common approach, however, is to calculate the starting dose using a weight-­based formula. Assuming little or no residual thyroid function, weight-­based replacement doses are on the order of 1.6 to 1.8 mcg/kg.7 The purest example of this would be a patient who is athyreotic as the result of a thyroidectomy.50 More accurate estimates may be obtained if ideal body weight or lean body mass are used, rather than actual body weight.7,51 The consequence of this may be that, if actual body weight is used in an overweight or obese individual, then the dose selected may be too high. The required dose may also be affected by the TSH goal of the treatment, with higher doses needed to achieve TSH values in the lower half of the normal range.7 The effect of age is generally to decrease a patient’s anticipated requirement, and this phenomenon may be mediated through body weight.52 Pregnancy will also lead to an increased requirement for LT4, such that dosage increases are needed to maintain a normal serum TSH. Adjustment of Therapy. LT4 has a half-­life of approximately 1 week,

KEY POINTS:  Diagnosis • Primary hypothyroidism is diagnosed based on the exponential rise in thyroid stimulating hormone that signals declining thyroid hormone levels. • Accurate measurement of thyroid analytes is essential for correct diagnosis of hypothyroidism. • Mild disease may apparently have relatively few consequences if undiagnosed. • If overt hypothyroidism remains undiagnosed, significant adverse health consequence can ensue.

TREATMENT Although there are some causes of hypothyroidism that are associated with recovery from the hypothyroid condition (thyroiditis, drug-­ induced hypothyroidism, iodine deficiency), most cases of hypothyroidism are associated with a lifelong need for thyroid hormone. Moreover, despite the interest in alternative therapies such as diets and nutritional supplements, the only known treatment for hypothyroidism is provision of thyroid hormone.

Levothyroxine Therapy Use of LT4 is, in many respects, an ideal form of drug therapy. LT4 is inexpensive, chemically similar to endogenous T4 and absorbed well when administered orally and has a long enough duration of action to permit once-­daily administration. With very rare exceptions, side effects seem to be related to use of inadequate or excessive doses of LT4.

such that a new steady state with respect to thyroid parameters (TSH, FT4, T3) is generally achieved by 6 weeks after a dosage initiation or a dosage change. Based on this reassessment of thyroid status, the LT4 can be adjusted up or down and thyroid parameters rechecked in a further 6 weeks. If the TSH is outside of the desired range by a modest amount, an adjustment by one dosage increment (e.g., from 100 mcg to 112 mcg) may be sufficient. For larger deviations from the desired range, an increase of several increments may be needed. Although simple dosage regimens are preferable to facilitate adherence, some patients may require alternating doses (such as 137 mcg alternating with 150 mcg) or may need small adjustments that can be achieved by taking a half tablet or one-­and-­a-­half tablets on one day of the week, while taking the usual one tablet on the other days of the week. Once a patient has achieved the desired serum TSH level, a confirmation that the TSH remains normal at 3 or 6 months may be indicated. However, once a correct dose has been confirmed, usually monitoring of serum TSH and FT4 on an annual basis is sufficient. Additional or earlier laboratory testing to confirm the euthyroid state may be needed if a patient has gained or lost a significant amount of body weight, or if the patient has new medical conditions or new medications that might affect their requirement for LT4. Although the gold standard for assessing thyroid status has been considered to be the serum TSH, some studies suggest that thyroid hormone levels may be important too. As previously mentioned, a recent systematic review found that clinical parameters representing the cardiac, bone, and metabolism systems were more associated with FT4 levels than TSH levels, suggesting that further research is needed to understand the importance of monitoring and potentially adjusting therapy based on thyroid hormone levels as well as TSH levels.

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PART 6  Thyroid

Goals of Therapy

20

are to restore the patient to a euthyroid status by reversing the signs and symptoms of hypothyroidism and restoring the patient’s TSH and thyroid hormone levels to normal. The biochemical goals are readily achieved, although T3 levels, if assessed, may be in the lower part of the normal range, particularly in athyreotic patients.53 With respect to signs of hypothyroidism, although weight gain is associated with the hypothyroid state, reversal of hypothyroidism, although achieving a median TSH that was decreased from 18.3 mIU/L to 2.3 mIU/L, was not associated with any weight loss.54 Even treatment of more severe hypothyroidism, despite lowering the TSH from 102 mIU/L to 2.2 mIU/L, was associated only with a weight loss of 4.3 kg. This was not caused by a decrease in fat mass, but a decrease in the mass of the lean body compartment only.55 Although symptoms may improve following LT4 therapy, they may not reach the level of improvement anticipated if the control population is used as the comparison. For example, in a study in which quality of life was assessed using a thyroid specific instrument (ThyPRO), after 6 months of therapy, during which the median TSH was decreased from 8.1 mIU/L to 2.6 mIU/L, improvement was noted in 11 out of 13 subscales compared with baseline, but was only improved compared with the general population in seven out of 13 subscales.56 Additional goals are to avoid iatrogenic hypothyroidism or iatrogenic hyperthyroidism, which unfortunately occurs in up to 40% of patients being treated for hypothyroidism.57,58 Avoidance of long-­ term iatrogenic hypothyroidism or hyperthyroidism is desirable in all patients treated for hypothyroidism. However, this is particularly important in vulnerable populations such as patients who are pregnant, elderly, or have cardiac disease and other medical conditions. Thus, the ideal TSH value for a particular patient may depend on the patient’s age, coexisting medical conditions, and any benefits that may be associated with specific TSH values. A particular TSH target can be achieved by serial adjustment of a patient’s LT4 dose. Maintenance of that TSH should then be possible with continuation of that same dose if no other influences act to perturb the equilibrium. Despite such ease of adjustment, many patients with hypothyroidism have out-­of-­range TSH values. Of a population attending a health fair, 18% were receiving inadequate LT4 therapy, and 22% were receiving excessive LT4 therapy.58 Other studies show similar percentages of patients with abnormal TSH values.7 This is particularly a problem in those over 65 years of age, where iatrogenic thyrotoxicosis is especially common.57 This is of particular concern in patients with comorbidities, which primarily affect elderly patients. One risk of overtreatment with LT4 is adverse effects on the heart, which manifest as decreased cardiac reserve and impaired exercise capacity, and, based on data from populations with both exogenous and endogenous hyperthyroidism, an increased risk of atrial fibrillation. The other major adverse consequence is accelerated bone loss, which may be compounded by hypogonadism in the elderly. Undertreatment with LT4 is also associated with adverse outcomes. Whenever possible, therapy should be adjusted to avoidance iatrogenic hypothyroidism or hyperthyroidism. Serum TSH should be checked annually, and perhaps more frequently in those who have a history of dose instability. Alterations in weight, as well as new medical comorbidities, medications, or symptoms should also trigger consideration of whether repeat laboratory assessment is indicated. Additional Goals. Other goals of therapy should also be considered. In two studies funded by a pharmaceutical company, frequent LT4 dosage changes were associated with lost time and wages for patients, with reduced patient satisfaction. Based on these studies, it would seem to be important to ensure stability of treatment by minimizing influences that may lead to changing dose requirements.59,60

Serum TSH (mlU/L)

Standard-­of-­Care Goals. The goals of treating hypothyroidism 15

10

5

0 BB

WB HS LT4 timing regimen Fig. 74.6  The effect of the timing of levothyroxine (LT4) administration on serum thyroid-­stimulating hormone (TSH). (From Bach-­ Huynh T, Nayak B, Loh J, et al. Timing of levothyroxine administration affects serum thyrotropin concentration. J Clin Endocrinol Metabol. 2009;94:3905–3912.)

Optimization of Therapy. A consistent schedule for daily administration of LT4 seems desirable to facilitate adherence. Scheduling of Levothyroxine. More important than the particular time that is identified for taking LT4 is maintaining a regular schedule so that absorption varies as little as possible and so that doses are not omitted.7 Potential times for LT4 administration are 60 or 30 minutes before breakfast, with breakfast, and at bedtime. A wealth of studies have compared these regimens, and in general the schedules that are most associated with maintaining a TSH that is consistently within the normal range are 60 minutes before breakfast and at bedtime61 (Fig. 74.6). Taking LT4 with breakfast results in the most variation in serum TSH. A consistent schedule should be adopted that is convenient to the patient and the least disruptive to their daily routine. Regimens that involve the patient awakening early from sleep do not seem optimum. The use of a prefilled tablet container is encouraged, particularly for those individuals who are taking multiple medications. If an LT4 dose is omitted, the options include taking the dose are soon as the omission is noted or taking two doses the following day. Absorption of Levothyroxine. LT4 absorption occurs mostly in the jejunum and ileum. The maximum absorption of LT4 is approximately 75% to 80%, and this occurs when the patient consumes LT4 in a fasting state. The presence of an acidic environment in the stomach also seems to promote absorption.7 Any food that is coadministered with LT4 can potentially reduce its absorption. Soy products, fiber, and milk, for example, can reduce absorption. Beverages such as Italian espresso can also impair absorption. The manifestation of impaired absorption is an elevated TSH. Medications and supplements are also known to reduce absorption, with common culprits being calcium carbonate, iron, multivitamins, and proton pump inhibitors62-­64 (Table 74.3). Calcium carbonate and iron seem to impair LT4 absorption by adsorbing to LT4 in the stomach. Proton pump inhibitors may exert their effects by reducing stomach acidity. Vitamin C stands alone as the one product that increases LT4 absorption and may have this effect by virtue of increasing stomach acidity. A particular example where the reduced LT4 absorption could have adverse effects for the patient is that seen when LT4 is given concurrently with enteral tube feeds in a hospitalized patient.65 The impaired absorption associated with foods, beverages, and medications may be overcome by changing the time of the LT4 administration, or a higher dose of LT4 may be administered to compensate for the effect of the decreased absorption.

CHAPTER 74  Hypothyroidism

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TABLE 74.3  Medical Conditions, Foods, and Drugs Affecting Levothyroxine (LT4) Absorption Factor

Strength of Evidence

Result of Interaction (Clinical Significance) Recommendation

Disorder Celiac disease

Very good

↑↑ need for LT4 (clinically significant)

Lactose intolerance

Very good

↑↑ need for LT4 (clinically significant)

Atrophic gastritis and Helicobacter pylori Giardiasis

Very good

↑↑ need for LT4 (clinically significant)

Poor

↑↑ need for LT4 (clinically significant)

Bariatric surgery*

Controversial

↑/↓ need for LT4 (clinically significant)

Caution during treatment because of iatrogenic hyperthyroidism Caution during treatment because of iatrogenic hyperthyroidism Caution during treatment because of iatrogenic hyperthyroidism Caution during treatment because of iatrogenic hyperthyroidism Monitor thyroid hormones after surgery

Food Fiber Soybeans Coffee* Grapefruit Vitamin C†

Controversial Very good Very good Good Very good

↑ need for LT4 (clinically significant) ↑↑ need for LT4 (clinically significant) ↑↑ need for LT4 (clinically significant) ↑ need for LT4 (clinically significant) ↓↓↓ need for LT4 (clinically significant)

Avoid concomitant use Avoid concomitant use Avoid concomitant use Avoid concomitant use Can improve absorption

Drug Aluminum hydroxide Calcium salts Iron preparations

Very good Very good Very good

↑↑ need for LT4 (D) ↑↑ need for LT4 (D) ↑↑ need for LT4 (D)

Lanthanum Orlistat Polystyrene sulfonate Raloxifene

Very good Controversial Poor Good

↑↑ need for LT4 (D) ↑↑↑ need for LT4 (D) ↑↑↑ need for LT4 (D; moderate) ↑↑ need for LT4 (D; minor)

Bile acid sequestrants

Very good

↑↑↑ need for LT4 (D; moderate)

Sevelamer Simethicone Ciprofloxacin Rifampicin Sucralfate Chromium H2-­receptor antagonists Proton pump inhibitorsa

Very good Poor Very good Very good Controversial Very good Controversial Good

↑↑↑ need for LT4 (D; moderate) ↑↑ need for LT4 (D; moderate) ↑↑↑ need for LT4 (C; moderate) ↑/↓ need for LT4 (C; moderate) ↑↑↑ need for LT4 (C; moderate) ↑ need for LT4 (B; moderate) ↑ need for LT4 (B) ↑↑ need for LT4 (B; moderate)

Avoid concomitant use for >2 h Avoid concomitant use for at least 2 h Avoid concomitant use as much as possible Avoid concomitant use Avoid concomitant use for at least 2 h Avoid concomitant use Avoid concomitant use as much as possible Avoid concomitant use as much as possible Avoid concomitant use Avoid concomitant use Avoid concomitant use Monitor thyroid hormones Avoid concomitant use Monitor thyroid hormones Monitor thyroid hormones Monitor thyroid hormones

↑ = increase; ↓ = decrease. Interaction was refuted for soft gel capsules and liquid form of LT4. aPatients with impaired absorption. (From Skelin M, Lucijanić T, Amidžić Klarić D, et al. Factors affecting gastrointestinal absorption of levothyroxine: a review. Clin Ther. 2017;39:378– 403.)

In addition to decreased LT4 absorption being associated with the food and medications listed earlier, several gastrointestinal conditions may also be associated with a decrement in LT4 absorption. These include celiac disease, lactose intolerance, ulcerative colitis, and atrophic gastritis associated with Helicobacter pylori infection.62,66 Patients with these conditions may require greater than the predicted weight-­based LT4 dose before their treatment, but with resolution of the higher requirement if the condition is successfully treated. The balance of the competing effects of weight loss and malabsorption after bariatric surgery most commonly seems to result in patients requiring a reduced LT4 dose after surgery.67 Drugs Affecting Levothyroxine Dose. In addition to the drugs and conditions mentioned earlier that alter LT4 dose requirement by affecting, and usually reducing, absorption, a number of drugs may

also alter LT4 dose requirement through their effects on LT4 transport and metabolism (Table 74.4). The classic examples of drugs affecting LT4 transport that increase and reduce the requirement for LT4, respectively, are estrogen and testosterone.7,63 These influences occur because of the effects of these sex steroids to increase (estrogen) and decrease (testosterone) the concentration of thyroxine-­binding globulin. LT4 is metabolized by deiodination, glucuronidation, and sulfation, and many drugs act to increase its metabolism. The conjugation with glucuronates and sulfates occurs in the liver, and several drugs act as hepatic enzyme inducers and enhance LT4 metabolism. Examples of such drugs are phenobarbital, phenytoin, carbamazepine, rifampin, and nicardipine.7,68 Other drugs such as tyrosine kinase inhibitors may act by altering deiodinase activity. The increased TSH that occurs in previously euthyroid patients taking LT4 when drugs such as sunitinib and sorafenib are initiated appears to be caused by increased activity of

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TABLE 74.4  Examples of Medications Causing Altered Transport or Metabolism of

Levothyroxine (LT4)

Alteration in LT4 transport Increased TBG Decreased TBG Increased TBG or increased transport Decreased TBG or decreased transport

Alteration in LT4 Metabolism Increased hepatic metabolism via induction of P450

Medication Estrogen Capecitabine Androgens Estrogen, raloxifene, tamoxifen, heroin, methadone, 5-­fluorouracil, mitotane, clofibrate Androgens, anabolic steroids, glucocorticoids, nicotinic acid, salicylates, furosemide, heparin, NSAIDs, phenytoin, carbamezepine Medication Phenobarbital, rifampin, phenytoin, sertraline, carbamazepine

Decreased activation of T4 to T3 via inhibition of Beta-­blockers, steroids, PTU, radiographic contrast agents, type 2 deiodinase amiodarone Increased deactivation of T4 and T3 via acceleration Tyrosine kinase inhibitors (imatinib, sorafenib, motesanib, of type 3 deiodinase sunitinib)

Effect Increased LT4 requirement Increased LT4 requirement Decreased LT4 requirement May not cause thyroid dysfunction May not cause thyroid dysfunction

Effect Can increase LT4 requirement or may not cause thyroid dysfunction Can increase LT4 requirement or may not cause thyroid dysfunction Can increase LT4 requirement

TBG, thyroxine-binding globulin; NSAID, nonsteroidal antiinflammatory drugs; PTU, propylthiouracil.

the type 3 deiodinase, as evidenced by decreased T4 and T3 levels and increased reverse T3 levels.69 Levothyroxine Preparations. LT4 is available as tablet preparations and also as a liquid preparation, or as a liquid contained within a gel capsule. The various LT4 tablets include brand-­name and generic preparations. The tablet preparations are all similar in the content of their active ingredient (LT4 itself) but differ with respect to their inert ingredients in the form of excipients (mixers, fillers, and colorants). The product that is most affordable to a patient may vary according to the patient’s insurance company and pharmacy, and also may not stay constant over time. There are reports of significant changes in serum TSH when a patient’s LT4 is changed either from one brand name to another, or from a brand name to generic product, or from one generic to another.7 The altered TSH is thought to occur because the various products may have different absorption because of the different excipients. Because of their different absorption, each of these products has slightly different bioequivalence. Bioequivalence is a measure of absorption or bioavailability that the US Food and Drug Administration (FDA) uses to predict therapeutic efficacy. Bioavailability is determined by studying the pharmacokinetics of large oral doses of LT4 in volunteers who have normal endogenous thyroid function.7 Pharmacokinetic parameters including the maximum serum concentration (Cmax), time to Cmax, and area under the concentration-­time curve (AUC) are used to compare different products. If the two 90% confidence intervals from the natural logarithms of the AUC at 48 hours and the Cmax are both within the 80% to 125% range, the products are deemed bioequivalent by the FDA and can be substituted for each other. Measures of systemic exposure (AUC, Cmax) are used in this testing, rather than clinically relevant or biochemical endpoints such as serum TSH levels. As such bioequivalence methodology does not detect LT4 doses with approximately 12.5% difference from each other (e.g., 100-­mcg and 112-­mcg tablets), it is always advisable to recheck serum TSH in individuals who have been switched from one preparation to another to be sure that iatrogenic hyperthyroidism or hypothyroidism has not developed and that the serum TSH is still in the target range.7 Furthermore, where possible it is best to maintain a patient on the same “identifiable” product. The name of the manufacturer is on the label of the bottle, so patients can request LT4 made by the same manufacturer when obtaining refills.

Liquid LT4 preparations are available in the United States in which the LT4 is dissolved in glycerol and contained either within a gelatin capsule or in a solution. Liquid preparations are reported by the manufacturer to have 100% absorption from the gastrointestinal tract, compared with approximately 75% absorption in the fasting state for other LT4 preparations. The dissolution of the gelatin capsule preparation appears to be relatively unaffected by pH. Therefore, its absorption, in theory, might be less affected by the altered pH of the gastric environment associated with food consumption, atrophic gastritis, and use of proton pump inhibitors than other LT4 products. There is, in fact, a growing body of evidence that liquid preparations may have better absorption in situations where patients are receiving proton pump inhibitors70 or tube feeds71 or have other causes of malabsorption.72 It is also plausible that gelatin capsules may be better tolerated than tablets in individuals with allergies to the excipients contained within tablet preparations.

Levothyroxine Therapy in Specific Situations Pregnant Patients. During pregnancy it is recommended that patients being treated for hypothyroidism be frequently monitored so that their TSH values can be maintained in the lower half of the trimester-­specific reference range. When such reference ranges are not available, a TSH below 2.5 mIU/L is a reasonable goal.73 Women being treated for hypothyroidism typically require a 20% to 30% increase in their LT4 dose early in the first trimester of pregnancy. The magnitude of the increase is greater in thyroidectomized patients than in those with some residual thyroid function. Pediatric Population. Treatment of hypothyroidism in the pediatric population is discussed elsewhere. The management of hypothyroidism in children is similar to its management in adults. However, there are special considerations based on the requirement of normal thyroid function for neurocognitive development as well as growth and development. As a reflection of their unique physiology, newborns, children, and adolescents typically require higher LT4 doses than adults. As an individual advances from childhood into adulthood, LT4 replacement doses decrease, with newborns typically requiring 10 mcg/kg/day, 1-­year-­old children 4 to 6 mcg/kg/day, and adolescents 2 to 4 mcg/kg/day, with transition to the average adult dose of 1.6 mcg/ kg/day once endocrine maturation is complete.

CHAPTER 74  Hypothyroidism

Nonadherent Patients. If patients take a consistent dose of LT4, their serum TSH levels should remain within a fairly narrow range. If patients experience unexpected fluctuations in their serum TSH, or persistently elevated TSH concentrations despite the administration of large doses of LT4, factors affecting LT4 formulation, absorption, and metabolism should be investigated as potential causes. If, however, such factors do not appear to be responsible, variable adherence or nonadherence to LT4 therapy should be considered. Although one survey of patients prescribed LT4 found a self-­reported nonadherence rate of 22%, a study of a large insurance claims database indicated nonadherence rates of 40% to 52%. Using another database, adherent patients had lower overall healthcare costs and less diagnosis of other diseases than nonadherent patients. However, it should be noted that the latter two studies were funded by a pharmaceutical company. Because LT4 is generally a lifelong medication, it is important for patients to identify a medication schedule that facilitates adherence. Patients may be taking multiple other medications, including those that require specific administration conditions (e.g., bisphosphonates) or those that impair absorption. It may be necessary to choose a schedule of medication administration that is practical, even if absorption is affected, if this ensures that doses are not omitted. If a combination of a high TSH and a high-­normal or high T4 concentration is documented by laboratory testing, this pattern could be consistent with the syndrome of thyroid hormone resistance, a TSH-­secreting pituitary adenoma, or recent resumption of LT4 intake before a scheduled blood test. Intuitively, patient education regarding the benefits of euthyroidism and the risks of iatrogenic thyroid disease would seem to a logical approach to reducing nonadherence. However, a study providing education in the form of booklets mailed to patients’ homes did not affect serum TSH, which was used as a surrogate marker of adherence. If efforts to encourage regular daily consumption of LT4 are unsuccessful, options include observed therapy, including twice weekly or weekly therapy. Weekly therapy is associated with supratherapeutic concentrations of FT4 for about 24 hours and elevated TSH levels prior to the administration of the weekly dose, although FT3 levels remain within the normal range, and patients appear not to report side effects. Parenteral administration of LT4 is also possible. Central Hypothyroidism. When treating patients with hypothyroidism because of TSH deficiency, a parameter other than serum TSH is needed to guide LT4 replacement therapy. Based on a randomized trial comparing two doses of LT4, it has been recommended that FT4 levels be kept in the upper half of the normal range.7 Slightly lower FT4 levels, perhaps in the mid-­normal range, have been suggested for frail or older individuals. Patients with Thyroid Cancer. A subgroup of patients who are intentionally kept with TSH values below the normal range are those with intermediate or high-­risk differentiated thyroid cancer. Studies show either increased survival or increased relapse-­free survival in these risk categories with TSH suppression, but not in patients with low-­risk thyroid cancer, who can be maintained with a serum TSH in the lower half of the normal range. In higher-­risk patients, the benefits of TSH suppression and the risks, such as potential bone loss and adverse cardiac effects, have to be balanced against potential benefits. Older Patients and Patients With Medical Conditions. Patients older than 60 to 65 years of age, patients who have severe, longstanding hypothyroidism, or those with coronary artery disease need a gradual approach to LT4 replacement. In patients known to have ischemic heart disease, treatment should be initiated with lower doses of LT4 such as 25 mcg daily. In other patients at risk for coronary artery disease, but without documentation of such disease, a conservative starting dose of approximately 50 mcg per day may be advisable. This caution would apply to patients who are in older age groups or who have had longstanding severe hypothyroidism. Older individuals may

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have other chronic medical conditions that may require altered doses of LT4 to maintain a normal TSH. In addition, euthyroidism should be carefully maintained to avoid iatrogenic thyroid disease that may exacerbate the patient’s underlying medical condition. For unclear reasons, sick patients older than 65 years who are taking other medications for a variety of comorbidities in addition to LT4 require lower doses of LT4 to normalize their serum TSH than do healthy controls of a similar age who taking only LT4. The difference in LT4 dose requirement persisted even after accounting for body weight. As other examples of medical conditions, patients with glomerular disease may require higher LT4 doses at times when their urinary protein losses are greatest,74 and those with cirrhosis may also have an increased requirement for LT4, possibly because of increased levels of thyroxine-­binding globulin. Hospitalized Patients. When patients with hypothyroidism are hospitalized, their LT4 therapy should continue uninterrupted. If patients are unable to take their LT4 by mouth, it should be provided by other enteral routes such as via feeding tubes or the intravenous route, as appropriate. However, in a review of LT4 replacement therapy in an intensive care unit, it was found that 17% did not have their LT4 prescribed for more than 7 days, and 21% did not have their LT4 administered when enteral feeding was instituted.75 Patients with TSH levels either above or below the reference range had longer median hospital lengths of stay (22 days) than those with normal TSH values (15 days).75 Coexistent Adrenal Insufficiency. If a patient has both hypothyroidism and suspected or documented adrenal insufficiency, cortisol replacement should be started concurrently with LT4 to avoid restoration of normal renal function increasing cortisol disposal, or normalization of metabolic rate increasing the requirement for cortisol, and thus precipitating an adrenal crisis.7 Subclinical Hypothyroidism. SCH is the one of the most prevalent thyroid diseases in developed countries, with a reported overall prevalence rate of 4.3% in a study of 17,353 individuals in the United States.14 This prevalence was higher, at 9%, when individuals attending a health fair in the United States were studied.58 Other studies of the general population reported prevalence rates of 4.9% and 3% in women and men, respectively, in the Netherlands, 7.5% and 2.8% in women and men respectively, in the United Kingdom, and 11% and 7.3% in women and men, respectively, in China.13 However, different prevalence rates can be observed depending on the specific subpopulation studied. For example, when examining individuals over 60 to 65 years of age, the prevalences for women and men, respectively, were 7.1% and 2.7% in the United States, 11.6% and 2.9% in the United Kingdom, and 6.7% and 6.1% in Brazil.13 Each of the studies cited earlier, as well as other studies examining the prevalence of SCH, used a variety of different upper limits of normal for the TSH reference interval that was used to define SCH. Arguments have been made both in favor of lowering the upper end of the reference interval and in favor of maintaining it. The proponents of lowering the upper limit of the normal TSH range suggest that patients may have mild symptoms and abnormalities in lipids and cardiovascular function that can be improved with LT4 therapy. The concern would be labeling a larger population with having SCH, with treatment potentially being undertaken, without proven benefits of lowering TSH values that fall within this range. Indeed, it has been suggested that treatment decisions should not be guided so much by reference intervals, but by data showing benefit of treatment for particular degrees of TSH elevation. Moreover, reference intervals themselves may be better defined by the risk of disease rather than on population-­based ranges. There is an ongoing debate regarding the risks and benefits of treating SCH.33 Many reviews regarding SCH conclude that there are less adverse consequences of mild SCH, compared with greater degrees of

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PART 6  Thyroid

SCH, in most populations, and that this is particularly true in specific populations such as the elderly.76 This discussion has to be qualified with a consideration of whether specific populations can be defined as having SCH if a TSH reference interval that is derived from that particular population has not been used. For example, does a 75-­year-­old with a persistent TSH value of 5.5 mIU/L have SCH? This TSH value may well fall within the normal range if an age-­adjusted reference interval is employed. Does a 25-­year-­old African American presenting with a TSH of 4.9 mIU/L not have SCH, when the reference intervals specific to his/her race and age are considered? This TSH value may be above the 97.5th percentile when race-­adjusted reference ranges are employed.77 Formulae for adjusting the TSH reference interval for age, ethnicity, and sex within the US population have been devised.78 However, not all experts believe that there is a need for specific reference intervals for these subpopulations. Even when there is agreement about the existence of SCH, benefits of treating generally have not been shown, except possibly in specific age groups.79 On the other hand, there are some preliminary data that even serum TSH values that fall within the upper end of the TSH reference interval are associated with adverse outcomes.43 A metaanalysis studied the risk of developing adverse outcomes in individuals with serum TSH values in the upper end of the normal reference range compared with the lower end of the range. Cardiovascular, metabolic, and skeletal outcomes were examined. The odds ratio for developing adverse cardiovascular and metabolic outcomes was significant at 1.21 (95% CI: 1.15−1.27) and 1.37 (95% CI: 1.27−1.48), respectively, whereas the odds ratio for adverse skeletal outcomes was significantly reduced at 0.55 (95% CI: 0.41−0.72).43 Should this be confirmed, this might suggest that trials of treatment of SCH should enroll patients with even milder degrees of TSH elevation to better determine the risks and benefits of such treatment. A recent clinical practice guideline suggested that treatment of SCH should not be considered unless the TSH is above 20 mIU/L, except in certain subgroups.80 This conclusion was reached despite the fact that only two of the trials in their associated metanalysis included patients with TSH values above 10 mIU/L. An alternative approach is to carefully consider the clinical context when deciding whether treatment of SCH is of benefit.81 Two recent studies have addressed the effect of LT4 on hypothyroid symptoms in older patients with SCH. Those over 65 years of age were studied in the TRUST trial,82 and individuals over 80 years of age were studied in the IEMO80-­plus Thyroid Trial.83 Both of these trials enrolled patients with persistent TSH elevations of over 4.6 mIU/L. In the TRUST trial the mean TSH values at enrollment were 6.41 and 6.38 mIU/L, with declines to 3.63 and 5.48 mIU/L, in the LT4 group and the placebo group, respectively. In the study of those over 80 years (which included some individuals from the IEMO80-­plus Thyroid Trial and some from the TRUST trial), the TSH values at enrollment were 6.50 and 6.20 mIU/L, with reduction to 3.69 and 5.49 mIU/L, in the LT4 and placebo groups, respectively. Neither of these trials showed any difference in their primary outcomes of hypothyroid symptoms or tiredness at 1 year. There was no effect of LT4 on secondary outcomes either. One could speculate that these trials would potentially have had different results if the TSH reference interval used was age-­adjusted and resulted in a different definition of SCH, with a higher TSH value such as greater than 7 to 10 mIU/L being used for enrollment. Of note, a recent survey of clinicians found that 11% to 29% of those responding to the survey would treat a 80-­year-­old patient with tiredness and a TSH of 6.5 to 6.8 mIU/L with LT4.84 Myxedema Coma. Severe hypothyroidism can culminate in myxedema coma, a life-­threatening condition characterized by hypothermia, bradycardia, hypotension, altered mental status, and multisystem organ failure. Risk factors include advanced age, poor access to healthcare, and

other underlying major organ system diseases. Patients may present more frequently in the winter months, and most patients have severe and longstanding thyroid hormone deficiency. Treatment should include T4 (1.6–1.8 mcg/kg/day, with or without a 200-­to 500-­mcg loading dose). The rationale for the loading dose is to reoccupy the empty binding sites on thyroxine-­binding globulin, so that administered LT4 can contribute to raising FT4 levels. Some experts advocate coadministration of LT3 in divided doses to compensate for impaired conversion of T4 to T3. No controlled trials have been performed to evaluate the relative benefits and risks of these different approaches. Glucocorticoids should be administered in stress doses until the results of a cosyntropin stimulation test performed to check for evidence of concomitant adrenal insufficiency are available. Care should be taken to avoid exposure to potent sedative or analgesic agents that may exacerbate altered mental status. Hypothermia should be treated with external warming to reduce the risk of circulatory collapse. KEY POINTS:  Levothyroxine Therapy • Hypothyroidism generally requires life-long therapy with thyroid hormone replacement. • There are well established protocols for initiation of levothyroxine therapy • Levothyroxine therapy must be continually monitored as dose requirements may change with changes in a patient’s physiological and medical situation. • Both overtreatment and undertreatment of hypothyroidism can have adverse effects and monitoring is necessary to maintain euthyroidism. • Specific treatment goals may need to be considered for specific patient populations. • The majority of patients are believed to feel well while taking levothyroxine. • A subset of patients do not feel well while taking levothyroxine, despite the best attempts to optimize therapy.

Therapy Other Than Levothyroxine The standard of care therapy for hypothyroidism is LT4. The majority of patients, when they are treated to achieve a normal TSH, feel well while taking LT4. However, there are a subset of patients who do not feel fully restored to health and report reduced quality of life despite being biochemically euthyroid. This has led to studies to try and understand the underpinning of this reduced quality of life and also to investigate whether alternative therapies for hypothyroidism might yield greater levels of satisfaction.

Residual Symptoms While Taking Levothyroxine and Potential Underpinnings. Studies that have assessed well-­being using the thyroid symptom questionnaire identified dissatisfaction in both the patients treated for hypothyroidism and also in age-­and sex-­matched patients without hypothyroidism. Approximately 14% more patients with hypothyroidism were dissatisfied compared with the control group.85 The hypothyroid group did have more coexistent medical conditions, but the difference between the groups remained significant even after adjusting for these. Another study, in which subjects were recruited by letter and then subsequently took part in cognitive testing and completed questionnaires assessing well-­being, also showed lower scores in both these areas in the euthyroid patients taking LT4 compared with standard reference values.86 Several factors have been proposed as contributing to the decreased quality of life in patients who are apparently euthyroid. These factors include failure to optimize serum TSH, coexistent medical conditions, low serum T3 levels during therapy, the presence of deiodinase or thyroid hormone transporter polymorphisms, and the presence of TPO Abs.87 With respect to fine-­tuning the TSH value in treated patients,

CHAPTER 74  Hypothyroidism most studies do not show improvement in patients’ symptoms with lower TSH values. For example, a recent trial in which patients were randomly assigned to receive LT4 doses that resulted in three different ranges of TSH values showed that these different TSH targets had no effect on quality of life, mood, or cognition.88 Patients with hypothyroidism do appear to have more chronic medical conditions than other populations,89 but whether this accounts for their decreased quality of life is not clear. LT4 therapy is associated with steady serum levels of T3 but is also associated with a higher T4/T3 ratio than is seen in endogenous euthyroidism. Many studies have examined the T3 levels achieved during LT4 monotherapy. These studies have had different methodology, including with respect to the group used as a comparator. In a study using patients as their own controls, T3 levels were better maintained.53 However, most of these studies show that T3 levels are lower in a proportion of LT4-­treated patients7 and may even be below the T3 reference range in some studies.90 In a small study of thyroidectomized patients, lower T3 levels were associated with being either homozygous or heterozygous for the Thr92Ala polymorphism of the type 2 deiodinase.91 There are some limited data about the effect of type 2 deiodinase polymorphism status on either response to LT4 therapy or preference for combination therapy. One study performed a retrospective analysis of data from a study of combination therapy by Saravanan et al.92 Participants underwent genotyping for the presence of the mutation encoding the Thr92Ala polymorphism and their scores on the General Health Questionnaire, both while taking LT4 and while taking combined LT4/LT3, were analyzed according to their polymorphism status. Ala/Ala homozygotes had worse General Health Questionnaire scores while taking LT4.93 Moreover, they also had a better response to combination therapy based on their General Health Questionnaire scores.93 Different TSH values could not be implicated, as TSH values did not differ between patients with the same genotype receiving combination therapy or monotherapy with LT4. However, other studies have not shown any association between scores of quality of life and cognition in LT4-­treated patients according to their Thr92Ala status.94,95 One small study found that patients with both the Thr92Ala polymorphism and a polymorphism in one of the thyroid hormone transporters preferred combination therapy with both LT4 and LT3.96 There are data suggesting that the presence of TPO Abs, even in patients who have maintained endogenous euthyroidism, may be associated with more symptoms than are reported by euthyroid patients without thyroid autoimmunity. One study found more symptoms and lower quality of life in those with elevated titers of thyroid peroxidase antibodies, whereas another study reported more depression.

Combination Therapy With Levothyroxine and Liothyronine Synthetic Combination Therapy. A relative T3 deficiency in those being treated with traditional LT4 monotherapy in the setting of some patient dissatisfaction with this therapy may prompt consideration of combination therapy with both LT4 and liothyronine (LT3). If the intention were to replicate the molar ratio of T4:T3 achieved by endogenous thyroid functioning in humans, the prescribed ratio would be approximately 14:1 to 15:1.7 Thus, this would be best achieved using synthetic LT3, with adjustment of the LT3 dose by the prescribing physician. However, it is not well-­understood what parameters should be used to make adjustments, given the short half-­life of LT3. Fourteen randomized trials examining the issue of combination therapy for patients with hypothyroidism have been performed.7,97 Following an initial study in 1999, 12 additional studies were performed between 2002 and 2010, with a subsequent study in 2016. These studies had variable designs with respect to the outcomes studied, the use of crossover or parallel groups, blinding methodology, and the ratio of T4 to T3 employed. However, autoimmune hypothyroidism was the

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predominant diagnosis in most studies. With some exceptions (see later), these studies failed to demonstrate a benefit of LT3 combination therapy. Four metaanalyses or reviews that incorporated the trials completed at the time of the analysis also concluded that there is no advantage to combination therapy.7 However, these metaanalyses were made difficult by the heterogeneity of the trials with respect to causes of hypothyroidism, different dosing regimens, different outcome measures, different duration of treatment, and different TSH and T3 levels achieved in the combination therapy groups. Other design issues included nonvalidated outcome measures, carryover effects in some studies, overtreatment in some studies, and failure to study therapy in men and the elderly. Recent reviews and guidelines have stressed the need for larger, better-­designed studies of longer duration.7,98 Health-­related quality of life or mood was studied in 13 trials, with very heterogeneous results.7 There was superiority of combination therapy on multiple measures in two trials, one by Bunevicius et al.99 and one by Nygaard et al.100 These two trials included patients with thyroid cancer and low TSH values, and used a large single dose of LT3 (20 mcg), respectively. Superiority of combination therapy for a minority of measures was seen in two trials.92,101 In one of these trials the benefit was seen at 3 months, but was no longer seen at 12 months.92 Of note, the latter is the only combination therapy study of 12 months’ duration, with the next longest trials being 4 months and 6 months in duration. The remaining nine trials did not show any superiority of combination therapy with respect to improving quality of life. Neurocognitive functioning was studied in ten trials, again with heterogeneous results.7 Superiority of combination therapy on multiple measures was found in one trial.99 Superiority of combination therapy for a minority of measures was also found in one trial.102 The remaining eight trials did not show a superior effect of combination therapy on neurocognitive functioning. An example of a negative trial with respect to both quality of life and cognitive functioning is a 4-­month study by Clyde et al.103 In this study, LT4 alone was compared with combination therapy by substituting 50 mcg of LT4 with 7.5 mcg of LT3. This study demonstrated no beneficial changes in body weight, serum lipid levels, hypothyroid symptoms as measured by a health-­related quality of life questionnaire, and standard measures of cognitive performance. Patient preference has been studied in five blinded crossover design trials and two blinded, parallel design trials of combination therapy.7 With respect to the crossover design trials, the combination therapy was preferred in four trials, which, when combined, total 128 patients.99,100,102,104 Another trial of 101 patients did not demonstrate a preference for combination therapy.105 Of the two parallel design trials, there was a preference for combination therapy in one trial of 130 patients106 and no preference for combination therapy in another trial of 573 patients.92 The former trial was characterized by some degree of overtreatment, and preference for combination therapy was associated with weight loss. The latter trial was the largest and longest-­duration trial of combination therapy conducted thus far. Natural Combination Therapy. There is considerable patient interest in “natural” combination therapy in the form of DTE. One small trial of DTE randomized patients to either LT4 or DTE and then switched them to the other therapy after 16 weeks, for a further 16 weeks of therapy.107 During the DTE treatment arm, patients had significantly higher serum levels of T3 and lower levels of FT4. Multiple different parameters of quality of life were assessed during the trial, and these parameters did not differ between the two groups. However, 49% of patients preferred the DTE, compared with 19% preferring LT4 and 33% having no preference.107 Preference for DTE was found to be associated with weight loss. A recent online survey of patients with hypothyroidism found that those responding to the survey had a higher level of satisfaction with DTE than those receiving other

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PART 6  Thyroid

therapies.108 Reported satisfaction level on a scale of 1 to 10 was 7 (interquartile range 4, 8) for DTE, compared with 6 (3, 8) for synthetic combination therapy with LT4 and LT3 and 5 (3, 7) for LT4 alone. These data need to be evaluated with the self-­selected nature of the respondents being considered.

Monotherapy With Liothyronine. Monotherapy with LT3 has rarely been used, in part because of the need to take LT3 multiple times a day to maintain a reasonably stable TSH. Preference for LT3 alone compared with LT4 alone has not been studied in a controlled fashion.

Reduction of Thyroid Peroxidase Antibodies. Whether TPO Abs themselves affect patient quality of life, including in the setting of a normal TSH, has been investigated. In a recent randomized trial, patients with continued symptoms while receiving LT4, who also had significantly elevated TPO Ab titers of greater than 1000 IU/mL (median 2232 IU/mL), were randomized to thyroidectomy or continued medical management. In those who underwent thyroidectomy, TPO Abs declined to a median of 152 IU/mL. Health-­related quality of life and fatigue improved in the thyroidectomized patients and was sustained at 12 to 18 months.109 There was no sham-­thyroidectomized group. An earlier nonrandomized study of thyroidectomy for hypothyroid symptoms did not identify a similar benefit for those without TPO Abs.110 Other therapies tested as a means of reducing TPO Abs, such as specific diets, have not been sufficiently studied.

KEY POINTS:  Therapy Other Than Levothyroxine • If patients have unresolved symptoms with levothyroxine therapy, potential causes of these symptoms should be fully investigated. • If no cause of patient dissatisfaction is identified, a trial of personalized combination therapy with addition of liothyronine can be contemplated, and can be continued as long as patient benefit and safety is maintained. • Trials of combination therapy with levothyroxine and liothyronine have not shown patient benefit, but these trials were largely underpowered, of short duration and used once daily liothyronine dosing. • Future combination therapy trials that address some of the weaknesses of prior trials, and also utilize a sustained release liothyronine preparation, may show different results. • A future goal is to prevent the cascade of events that leads to autoimmune destruction of thyroid tissue.

SUMMARY AND AREAS OF FUTURE RESEARCH Despite the significant advances that have been made in our understanding of thyroid hormone therapy, some of these insights have not been easy to translate into practical applications. Additionally, some of the challenges that continue to face physicians treating patients with hypothyroidism concern basic fundamentals. With respect to the former issue, although we recognize that LT4 does not recapitulate all aspects of normal thyroid physiology, we still do not have a “physiologic” replacement therapy. As an example of the latter issue, a significant number of patients with hypothyroidism are either undertreated or overtreated. This is a complex problem, as out-­of-­range TSH values may be attributed to multiple factors as diverse as confounding medications, interfering conditions, or intrinsic patient variables. The patient variables may be as basic as adhering to prescribed therapy. Such problems, with multiple etiologies, may require creative and sustained efforts in order for them to be addressed. These challenges are also combined with exciting opportunities for understanding the nuances of fully reversing the consequences of hypothyroidism. Advances in our understanding of genetic diversity in

the function of deiodinases, and possibly thyroid hormone transporters, may ultimately enable us to provide therapy tailored to individual patients. Future prospects include understanding the genetic underpinning of thyroid hormone delivery to tissues, developing more physiologic, sustained release or combination thyroid hormone therapies incorporating T3, and facilitating achievement of adequate thyroid hormone levels in all tissues. Although so far only shown in animal models, another hope for the future is the regeneration of functional thyroid follicles from embryonic or pluripotent stem cells. Such cells, once differentiated into thyroid follicular cells, have been shown to be capable of expressing thyroid-­specific genes, responding to TSH stimulation, actively transporting iodine, forming three-­dimensional follicles, and expressing thyroglobulin. Should such advances be extended to humans, this would not only provide the opportunity for a better understanding of thyroid physiology and disease but may also pave the way for regenerating the full hormonal profile of a normally functioning thyroid gland and thereby restoring euthyroidism in its entirety.

REFERENCES 1. Slater S. The discovery of thyroid replacement therapy. Part 1: In the beginning. J R Soc Med. 2011;104:15–18. 2. Bauduer F, Tankersley KB. Evidence of an ancient (2000 years ago) goiter attributed to iodine deficiency in North America. Med Hypotheses. 2018;118:6–8. 3. Mehra MR, Campbell HR. The Mona Lisa decrypted: allure of an imperfect reality. Mayo Clin Proc. 2018;93:1325–1327. 4. Slater S. The discovery of thyroid replacement therapy. Part 2: the critical 19th century. J R Soc Med. 2011;104:59–63. 5. Slater S. The discovery of thyroid replacement therapy. Part 3: A complete transformation. J R Soc Med. 2011;104:100–106. 6. McAninch EA, Bianco AC. The history and future of treatment of hypothyroidism. Ann Intern Med. 2016;164:50–56. 7. Jonklaas J, Bianco AC, Bauer AJ, et al. Guidelines for the treatment of hypothyroidism: prepared by the American Thyroid Association Task Force on thyroid hormone replacement. Thyroid. 2014;24:1670–1751. 8. Hoermann R, Midgley JEM, Larisch R, et al. Recent advances in thyroid hormone regulation: toward a new paradigm for optimal diagnosis and treatment. Front Endocrinol (Lausanne). 2017;8:364. 9. van der Spek AH, Fliers E, Boelen A. The classic pathways of thyroid hormone metabolism. Mol Cell Endocrinol. 2017;458:29–38. 10. Groeneweg S, van Geest FS, Peeters RP, et al. Thyroid hormone transporters. Endocr Rev. 2020;41:bnz008. 11. Mendoza A, Hollenberg AN. New insights into thyroid hormone action. Pharmacol Ther. 2017;173:135–145. 12. Astapova I. Role of co-­regulators in metabolic and transcriptional actions of thyroid hormone. J Mol Endocrinol. 2016;56:73–97. 13. Taylor PN, Albrecht D, Scholz A, et al. Global epidemiology of hyperthyroidism and hypothyroidism. Nat Rev Endocrinol. 2018;14:301–316. 14. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab. 2002;87:489–499. 15. Chaker L, Bianco AC, Jonklaas J, et al. Hypothyroidism. Lancet. 2017;390:1550–1562. 16. Canaris GJ, Steiner JF, Ridgway EC. Do traditional symptoms of hypothyroidism correlate with biochemical disease? J Gen Intern Med. 1997;12:544–550. 17. Ott J, Promberger R, Kober F, et al. Hashimoto’s thyroiditis affects symptom load and quality of life unrelated to hypothyroidism: a prospective case-­control study in women undergoing thyroidectomy for benign goiter. Thyroid. 2011;21:161–167. 18. Kim J. Images in clinical medicine. Myxedema. N Engl J Med. 2015;372:764. 19. Taguchi T. Brittle nails and hair loss in hypothyroidism. N Engl J Med. 2018;379:1363.

CHAPTER 74  Hypothyroidism 20. Caturegli P, De Remigis A, Rose NR. Hashimoto thyroiditis: clinical and diagnostic criteria. Autoimmun Rev. 2014;13:391–397. 21. Bhattacharya S, Goyal A, Kaur P, et al. Anticancer drug-­induced thyroid dysfunction. Eur Endocrinol. 2020;16:32–39. 22. Li Z, Qiu Y, Fei Y, et al. Prevalence of and risk factors for hypothyroidism after hemithyroidectomy: a systematic review and meta-­analysis. Endocrine. 2020;70:243–255. 23. Zimmermann MB, Boelaert K. Iodine deficiency and thyroid disorders. Lancet Diabetes Endocrinol. 2015;3:286–295. 24. Duntas LH. The catalytic role of iodine excess in loss of homeostasis in autoimmune thyroiditis. Curr Opin Endocrinol Diabetes Obes. 2018;25:347–352. 25. Leung AM, Braverman LE. Iodine-­induced thyroid dysfunction. Curr Opin Endocrinol Diabetes Obes. 2012;19:414–419. 26. Lee SY, Rhee CM, Leung AM, et al. A review: Radiographic iodinated contrast media-­induced thyroid dysfunction. J Clin Endocrinol Metab. 2015;100:376–383. 27. Calil-­Silveira J, Serrano-­Nascimento C, Kopp PA, et al. Iodide excess regulates its own efflux: a possible involvement of pendrin. Am J Physiol Cell Physiol. 2016;310:C576–C582. 28. Wassner AJ. Unraveling the genetics of congenital hypothyroidism: challenges and opportunities. J Clin Endocrinol Metab. 2020;105:dgaa454. 29. Persani L, Cangiano B, Bonomi M. The diagnosis and management of central hypothyroidism in 2018. Endocr Connect. 2019;8:R44–R54. 30. Rurale G, Di Cicco E, Dentice M, et al. Thyroid hormone hyposensitivity: from genotype to phenotype and back. Front Endocrinol (Lausanne). 2019;10:912. 31. Luongo C, Trivisano L, Alfano F, et al. Type 3 deiodinase and consumptive hypothyroidism: a common mechanism for a rare disease. Front Endocrinol (Lausanne). 2013;4:115. 32. Baloch Z, Carayon P, Conte-­Devolx B, et al. Laboratory medicine practice guidelines. Laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13:3–126. 33. Jonklaas J, Razvi S. Reference intervals in the diagnosis of thyroid dysfunction: treating patients not numbers. Lancet Diabetes Endocrinol. 2019;7:473–483. 34. Estrada JM, Soldin D, Buckey TM, et al. Thyrotropin isoforms: implications for thyrotropin analysis and clinical practice. Thyroid. 2014;24:411–423. 35. Favresse J, Burlacu MC, Maiter D, et al. Interferences with thyroid function immunoassays: clinical implications and detection algorithm. Endocr Rev. 2018;39:830–850. 36. Over R, Mannan S, Nsouli-­Maktabi H, et al. Age and the thyrotropin response to hypothyroxinemia. J Clin Endocrinol Metab. 2010;95: 3675–3683. 37. van Deventer HE, Soldin SJ. The expanding role of tandem mass spectrometry in optimizing diagnosis and treatment of thyroid disease. Adv Clin Chem. 2013;61:127–152. 38. Fitzgerald SP, Bean NG, Falhammar H, et al. Clinical parameters are more likely to be associated with thyroid hormone levels than with thyrotropin levels: a systematic review and meta-­analysis. Thyroid. 2020;30:1695–1709. 39. Beck-­Peccoz P, Rodari G, Giavoli C, et al. Central hypothyroidism -­a neglected thyroid disorder. Nat Rev Endocrinol. 2017;13:588–598. 40. Barth JH, Spencer JD, Goodall SR, et al. Reference intervals for thyroid hormones on Advia Centaur derived from three reference populations and a review of the literature. Ann Clin Biochem. 2016;53:385–389. 41. Strich D, Karavani G, Levin S, et al. Normal limits for serum thyrotropin vary greatly depending on method. Clin Endocrinol (Oxf). 2016;85:110–115. 42. LeFevre ML, US Preventative Task Force. Screening for thyroid dysfunction: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med. 2015;162:641–650. 43. Taylor PN, Razvi S, Pearce SH, et al. Clinical review: A review of the clinical consequences of variation in thyroid function within the reference range. J Clin Endocrinol Metab. 2013;98:3562–3571. 44. Thayakaran R, Adderley NJ, Sainsbury C, et al. Thyroid replacement therapy, thyroid stimulating hormone concentrations, and long term health outcomes in patients with hypothyroidism: longitudinal study. BMJ. 2019;366:l4892.

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45. Cappola AR, Cooper DS. Screening and treating subclinical thyroid disease: getting past the impasse. Ann Intern Med. 2015;162:664–665. 46. Pollock MA, Sturrock A, Marshall K, et al. Thyroxine treatment in patients with symptoms of hypothyroidism but thyroid function tests within the reference range: randomised double blind placebo controlled crossover trial. BMJ. 2001;323:891–895. 47. Roos A, Linn-­Rasker SP, van Domburg RT, et al. The starting dose of levothyroxine in primary hypothyroidism treatment: a prospective, randomized, double-­blind trial. Arch Intern Med. 2005;165(15):1714–1720. 48. Kabadi UM. Optimal daily levothyroxine dose in primary hypothyroidism. Its relation to pretreatment thyroid hormone indexes. Arch Intern Med. 1989;149:2209–2212. 49. Kabadi UM, Jackson T. Serum thyrotropin in primary hypothyroidism. A possible predictor of optimal daily levothyroxine dose in primary hypothyroidism. Arch Intern Med. 1995;155:1046–1048. 50. Mistry D, Atkin S, Atkinson H, et al. Predicting thyroxine requirements following total thyroidectomy. Clin Endocrinol (Oxf). 2011;74:384–387. 51. Santini F, Pinchera A, Marsili A, et al. Lean body mass is a major determinant of levothyroxine dosage in the treatment of thyroid diseases. J Clin Endocrinol Metab. 2005;90:124–127. 52. Younis IR, Ahmed MA, Burman KD, et al. Stable isotope pharmacokinetic studies provide insight into effects of age, sex, and weight on levothyroxine metabolism. Thyroid. 2018;28:41–49. 53. Jonklaas J, Davidson B, Bhagat S, et al. Triiodothyronine levels in athyreotic individuals during levothyroxine therapy. JAMA. 2008;299:769– 777. 54. Lee SY, Braverman LE, Pearce EN. Changes in body weight after treatment of primary hypothyroidism with levothyroxine. Endocr Pract. 2014;20:1122–1128. 55. Karmisholt J, Andersen S, Laurberg P. Weight loss after therapy of hypothyroidism is mainly caused by excretion of excess body water associated with myxoedema. J Clin Endocrinol Metab. 2011;96:E99–E103. 56. Winther KH, Cramon P, Watt T, et al. Disease-­specific as well as generic quality of life is widely impacted in autoimmune hypothyroidism and improves during the first six months of levothyroxine therapy. PLoS One. 2016;11:e0156925. 57. Somwaru LL, Arnold AM, Joshi N, et al. High frequency of and factors associated with thyroid hormone over-­replacement and under-­ replacement in men and women aged 65 and over. J Clin Endocrinol Metab. 2009;94:1342–1345. 58. Canaris GJ, Manowitz NR, Mayor G, et al. The Colorado thyroid disease prevalence study. Arch Intern Med. 2000;160:526–534. 59. Ernst FR, Barr P, Elmor R, et al. The economic impact of levothyroxine dose adjustments: the CONTROL HE study. Clin Drug Investig. 2017;37:71–83. 60. McMillan M, Sandulli W, Engelken D, et al. Levothyroxine dose changes and hypothyroid patient satisfaction-­results of the CONTROL TS study. Annals Thyroid Res. 2017;3:109–114. 61. Bach-­Huynh T, Nayak B, Loh J, et al. Timing of levothyroxine administration affects serum thyrotropin concentration. J Clin Endocrinol Metab. 2009;94:3905–3912. 62. Skelin M, Lucijanić T, Amidžić Klarić D, et al. Factors affecting gastrointestinal absorption of levothyroxine: a review. Clin Ther. 2017;39:378– 403. 63. Irving SA, Vadiveloo T, Leese GP. Drugs that interact with levothyroxine: an observational study from the Thyroid Epidemiology, Audit and Research Study (TEARS). Clin Endocrinol (Oxf). 2015;82:136–141. 64. Burch HB. Drug effects on the thyroid. N Engl J Med. 2019;381:749–761. 65. Dickerson RN, Maish GO, Minard G, et al. Clinical relevancy of the levothyroxine-­continuous enteral nutrition interaction. Nutr Clin Pract. 2010;25:646–652. 66. Virili C, Stramazzo I, Santaguida MG, et al. Ulcerative colitis as a novel cause of increased need for levothyroxine. Front Endocrinol (Lausanne). 2019;10:233. 67. Gadiraju S, Lee CJ, Cooper DS. Levothyroxine dosing following bariatric surgery. Obes Surg. 2016;26:2538–2542. 68. Ross DS, Burch HB, Cooper DS, et al. 2016 American Thyroid Association guidelines for diagnosis and management of hyperthyroidism and other causes of thyrotoxicosis. Thyroid. 2016;26:1343–1421.

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69. Torino F, Barnabei A, Paragliola R, et al. Thyroid dysfunction as an unintended side effect of anticancer drugs. Thyroid. 2013;23:1345–1366. 70. Vita R, Saraceno G, Trimarchi F, et al. Switching levothyroxine from the tablet to the oral solution formulation corrects the impaired absorption of levothyroxine induced by proton-­pump inhibitors. J Clin Endocrinol Metab. 2014;99:4481–4486. 71. Pirola I, Daffini L, Gandossi E, et al. Comparison between liquid and tablet levothyroxine formulations in patients treated through enteral feeding tube. J Endocrinol Invest. 2014;37:583–587. 72. Pirola I, Formenti AM, Gandossi E, et al. Oral liquid L-­thyroxine (L-­t4) may be better absorbed compared to L-­T4 tablets following bariatric surgery. Obes Surg. 2013;23:1493–1496. 73. Alexander EK, Pearce EN, Brent GA, et al. 2017 Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the postpartum. Thyroid. 2017;27:315–389. 74. Benvenga S, Vita R, Di Bari F, et al. Do not forget nephrotic syndrome as a cause of increased requirement of levothyroxine replacement therapy. Eur Thyroid J. 2015;4:138–142. 75. Barrett NA, Jones A, Whiteley C, et al. Management of long-­term hypothyroidism: a potential marker of quality of medicines reconciliation in the intensive care unit. Int J Pharm Pract. 2012;20:303–306. 76. Baumgartner C, Blum MR, Rodondi N. Subclinical hypothyroidism: summary of evidence in 2014. Swiss Med Wkly. 2014;144:w14058. 77. Boucai L, Surks MI. Reference limits of serum TSH and free T4 are significantly influenced by race and age in an urban outpatient medical practice. Clin Endocrinol (Oxf). 2009;70:788–793. 78. Boucai L, Hollowell JG, Surks MI. An approach for development of age-­, gender-­, and ethnicity-­specific thyrotropin reference limits. Thyroid. 2011;21:5–11. 79. Razvi S, Weaver JU, Butler TJ, et al. Levothyroxine treatment of subclinical hypothyroidism, fatal and nonfatal cardiovascular events, and mortality. Arch Intern Med. 2012;172:811–817. 80. Bekkering GE, Agoritsas T, Lytvyn L, et al. Thyroid hormones treatment for subclinical hypothyroidism: a clinical practice guideline. BMJ. 2019;365:l2006. 81. Sawka AM, Cappola AR, Peeters RP, et al. Patient context and thyrotropin levels are important when considering treatment of subclinical hypothyroidism. Thyroid. 2019;29:1359–1363. 82. Stott DJ, Rodondi N, Bauer DC, et al. Thyroid hormone therapy for older adults with subclinical hypothyroidism. N Engl J Med. 2017;377:e20. 83. Mooijaart SP, Du Puy RS, Stott DJ, et al. Association between levothyroxine treatment and thyroid-­related symptoms among adults aged 80 years and older with subclinical hypothyroidism. JAMA. 2019;322:1977–1986. 84. Razvi S, Arnott B, Teare D, et al. Multinational survey of treatment practices of clinicians managing subclinical hypothyroidism in older people in 2019. Eur Thyroid J. 2021;10:330–338. 85. Saravanan P, Chau WF, Roberts N, et al. Psychological well-­being in patients on ‘adequate’ doses of l-­thyroxine: results of a large, controlled community-­based questionnaire study. Clin Endocrinol (Oxf). 2002;57:577–585. 86. Wekking EM, Appelhof BC, Fliers E, et al. Cognitive functioning and well-­being in euthyroid patients on thyroxine replacement therapy for primary hypothyroidism. Eur J Endocrinol. 2005;153:747–753. 87. Jonklaas J. Persistent hypothyroid symptoms in a patient with a normal thyroid stimulating hormone level. Curr Opin Endocrinol Diabetes Obes. 2017;24:356–363. 88. Samuels MH, Kolobova I, Niederhausen M, et al. Effects of altering levothyroxine (L-­T4) doses on quality of life, mood, and cognition in L-­T4 treated subjects. J Clin Endocrinol Metab. 2018;103:1997–2008. 89. Peterson SJ, McAninch EA, Bianco AC. Is a normal TSH synonymous with “euthyroidism” in levothyroxine monotherapy? J Clin Endocrinol Metab. 2016;101:4964–4973. 90. Gullo D, Latina A, Frasca F, et al. Levothyroxine monotherapy cannot guarantee euthyroidism in all athyreotic patients. PLoS One. 2011;6:e22552. 91. Castagna MG, Dentice M, Cantara S, et al. DIO2 Thr92Ala reduces deiodinase-­2 activity and serum-­T3 levels in thyroid-­deficient patients. J Clin Endocrinol Metab. 2017;102:1623–1630.

92. Saravanan P, Simmons DJ, Greenwood R, et al. Partial substitution of thyroxine (T4) with tri-­iodothyronine in patients on T4 replacement therapy: results of a large community-­based randomized controlled trial. J Clin Endocrinol Metab. 2005;90:805–812. 93. Panicker V, Saravanan P, Vaidya B, et al. Common variation in the DIO2 gene predicts baseline psychological well-­being and response to combination thyroxine plus triiodothyronine therapy in hypothyroid patients. J Clin Endocrinol Metab. 2009;94:1623–1629. 94. Wouters HJ, van Loon HC, van der Klauw MM, et al. No effect of the Thr92Ala polymorphism of deiodinase-­2 on thyroid hormone parameters, health-­related quality of life, and cognitive functioning in a large population-­based cohort study. Thyroid. 2017;27:147–155. 95. Young Cho Y, Jeong Kim H, Won Jang H, et al. The relationship of 19 functional polymorphisms in iodothyronine deiodinase and psychological well-­being in hypothyroid patients. Endocrine. 2017;57:115–124. 96. Carlé A, Faber J, Steffensen R, et al. Hypothyroid patients encoding combined MCT10 and DIO2 gene polymorphisms may prefer L-­T3 + L-­T4 combination treatment -­data using a blind, randomized, clinical study. Eur Thyroid J. 2017;6:143–151. 97. DiStefano J, Jonklaas J. Predicting optimal combination LT4 + LT3 therapy for hypothyroidism based on residual thyroid function. Front Endocrinol (Lausanne). 2019;10:746. 98. Jonklaas J, Cappola AR, Celi FS. Editorial: combination therapy for hypothyroidism: the journey from bench to bedside. Front Endocrinol (Lausanne). 2020;11:422. 99. Bunevicius R, Kazanavicius G, Zalinkevicius R, et al. Effects of thyroxine as compared with thyroxine plus triiodothyronine in patients with hypothyroidism. N Engl J Med. 1999;340:424–429. 100. Nygaard B, Jensen EW, Kvetny J, et al. Effect of combination therapy with thyroxine (T4) and 3,5,3´-­triiodothyronine versus T4 monotherapy in patients with hypothyroidism, a double-­blind, randomised cross-­over study. Eur J Endocrinol. 2009;161:895–902. 101. Valizadeh M, Seyyed-­Majidi MR, Hajibeigloo H, et al. Efficacy of combined levothyroxine and liothyronine as compared with levothyroxine monotherapy in primary hypothyroidism: a randomized controlled trial. Endocr Res. 2009;34:80–89. 102. Escobar-­Morreale HF, Botella-­Carretero JI, Gómez-­Bueno M, et al. Thyroid hormone replacement therapy in primary hypothyroidism: a randomized trial comparing L-­thyroxine plus liothyronine with L-­thyroxine alone. Ann Intern Med. 2005;142:412–424. 103. Clyde PW, Harari AE, Getka EJ, et al. Combined levothyroxine plus liothyronine compared with levothyroxine alone in primary hypothyroidism: a randomized controlled trial. JAMA. 2003;290:2952–2958. 104. Bunevicius R, Jakuboniene N, Jakubonien N, et al. Thyroxine vs thyroxine plus triiodothyronine in treatment of hypothyroidism after thyroidectomy for Graves’ disease. Endocrine. 2002;18:129–133. 105. Walsh JP, Shiels L, Lim EM, et al. Combined thyroxine/liothyronine treatment does not improve well-­being, quality of life, or cognitive function compared to thyroxine alone: a randomized controlled trial in patients with primary hypothyroidism. J Clin Endocrinol Metab. 2003;88:4543–4550. 106. Appelhof BC, Fliers E, Wekking EM, et al. Combined therapy with levothyroxine and liothyronine in two ratios, compared with levothyroxine monotherapy in primary hypothyroidism: a double-­blind, randomized, controlled clinical trial. J Clin Endocrinol Metab. 2005;90:2666– 2674. 107. Hoang TD, Olsen CH, Mai VQ, et al. Desiccated thyroid extract compared with levothyroxine in the treatment of hypothyroidism: a randomized, double-­blind, crossover study. J Clin Endocrinol Metab. 2013;98:1982–1990. 108. Peterson SJ, Cappola AR, Castro MR, et al. An online survey of hypothyroid patients demonstrates prominent dissatisfaction. Thyroid. 2018;28:707–721. 109. Guldvog I, Reitsma LC, Johnsen L, et al. Thyroidectomy versus medical management for euthyroid patients with hashimoto disease and persisting symptoms: a randomized trial. Ann Intern Med. 2019;170:453–464. 110. Promberger R, Hermann M, Pallikunnel SJ, et al. Quality of life after thyroid surgery in women with benign euthyroid goiter: influencing factors including Hashimoto’s thyroiditis. Am J Surg. 2014;207:974–979.

75 Thyroid and Pregnancy Tim I.M. Korevaar and Elizabeth N. Pearce

OUTLINE Thyroid Disease and Fertility, 1251 Epidemiology, 1251 Physiology, 1251 Physiology During Assisted Reproduction Technology, 1252 Clinical Outcomes and Management, 1252 Maternal and Fetal Physiology in Pregnancy, 1254 Thyroid Function Tests in Pregnancy, 1254 Hyperthyroidism in Pregnancy, 1254 Diagnosis, 1255 Treatment and Monitoring, 1255 Lactation, 1257 Hypothyroidism and Hypothyroxinemia In Pregnancy, 1257

Overt Hypothyroidism, 1257 Subclinical Hypothyroidism, 1257 Isolated Hypothyroxinemia, 1258 Thyroid Autoimmunity in Pregnancy, 1258 Associated Obstetric and Fetal Outcomes, 1259 Potential Mechanisms for Associations, 1259 Implications for Therapy and Monitoring, 1259 Screening for Thyroid Dysfunction in Pregnancy, 1260 Thyroid Nodules and Cancer During Pregnancy, 1260 Thyroid Disorders in the Postpartum Period, 1261 Postpartum Thyroiditis, 1261 Considerations in Lactating Women, 1262 Summary and Future Directions, 1262

 

THYROID DISEASE AND FERTILITY Adequate thyroid hormone availability is important for female reproduction. Thyroid hormone regulates key processes in female reproduction, both indirectly and directly. Various studies have shown that thyroid disease, including subclinical thyroid dysfunction and thyroid autoimmunity, is associated with infertility and suboptimal fertility treatment outcomes. However, the majority of these studies have been limited by their retrospective nature, small sample size, or selected population (typically women undergoing assisted reproduction). The overrepresentation of studies in subfertile women necessitates generalization and extrapolation of results for decision-­making in fertile women. Furthermore, clinical decision-­making strategies are also based on our understanding of thyroid physiology and pathophysiology, as well as efforts aimed at restoring homeostasis. Diagnosis and interpretation of thyroid function test abnormalities should not only focus on the preconception period, but also anticipate effects on gestation and fetal development.

Epidemiology Thyroid disorders are relatively common among women of reproductive age because of the sex and age distributions of Hashimoto thyroiditis and Graves hyperthyroidism, as well as the increased detection of subclinical disease entities because of preconception screening. Data on the true prevalence of thyroid disease and thyroid autoimmunity in women of reproductive age are scarce because the majority of studies focus only on subclinical disease or thyroid autoimmunity in populations of subfertile or infertile (used synonymously hereafter) women. Furthermore, instead of normal nonpregnancy reference ranges, many studies have used an upper limit for thyroid-­stimulating hormone (TSH) of 2.50 mIU/L, which is an incorrect cutoff that leads to considerable overdiagnosis and overtreatment in up to 20% of otherwise

healthy women. Of women referred to a fertility center or with a previous miscarriage, almost 5% will have a thyroid function test abnormality. Overt hypothyroidism or overt hyperthyroidism occur only in approximately 0.2% to 0.3%, whereas subclinical hypothyroidism occurs in approximately 2.4% and subclinical hyperthyroidism in approximately 1.3% of women. Furthermore, among women referred to a fertility center and/or with a previous miscarriage, 9.5% to 12.3% are thyroid peroxidase antibody (TPOAb)–positive, which is probably slightly higher than in otherwise healthy pregnant women (mean 7.5%; range 4.7%–15.0%).1-­3 Similar to the general population, risk factors for thyroid disease and autoimmunity in women of reproductive age include ethnicity (e.g., a higher risk of Hashimoto thyroiditis in Whites but a higher risk of Graves hyperthyroidism in Black and Asian women), increased age, the presence of other autoimmune disorders, smoking (Graves hyperthyroidism), and insufficient or excessive iodine intakes.

Physiology Thyroid hormone can affect female reproduction either directly, through regulation of key cellular processes, or indirectly, through interference with other hormonal axes. Thyroid hormone receptors are abundantly present throughout the human female reproductive tract, including in granulosa cells and oocytes.4-­8 Preclinical studies show that thyroid hormone regulates the stimulatory effects of follicle-­ stimulating hormone (FSH) and luteinizing hormone (LH) on granulosa cells, follicular growth, and oocyte development.9,10 These effects are mediated via thyroid hormone regulation of 3,β-­hydroxysteroid dehydrogenase (the enzyme that facilitates the final step in progesterone formation), aromatase (the enzyme that facilitates the final step in estradiol formation), and LH/human chorionic gonadotropin (hCG) receptor expression. Indirect effects of thyroid hormone on female reproduction can be extrapolated from clinical observations

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PART 6  Thyroid a 1.8-­to 4-­fold higher risk of miscarriage15 and slightly higher risks of other adverse pregnancy outcomes such as preterm birth and preeclampsia. Overt hypothyroidism requires thyroid hormone replacement with a preconception or pre-assisted reproduction technology (ART) target TSH of less than 2.5 mIU/L. Women should be counseled to contact their healthcare provider before starting ART or at the time of a positive pregnancy test so that their thyroid function can be checked and the levothyroxine dosage can be increased by roughly 30% as soon as they are pregnant. Any thyroid hormone formulations containing liothyronine should be avoided preconception and during pregnancy, because triiodothyronine (T3) is unlikely to reach the fetal brain, and it can decrease maternal serum thyroxine (T4) concentrations. For women with hyperthyroidism, especially Graves disease, a permanent preconception treatment modality is recommended either before ART or pregnancy (see later). In euthyroid TPOAb-­positive women, clinical follow-­up throughout COH and gestation is advised, owing to the high risk of thyroid function deterioration to the point of a treatment indication during pregnancy or during COH (Fig. 75.2). Because of a lack of high-­quality data, the decision about whether or not to start levothyroxine treatment for TPOAb-­negative subclinical hypothyroidism should be tailored to each specific patient and should take into account data from studies in women undergoing infertility treatment (see later), as well as other risk factors for adverse outcomes such as a prior history of miscarriage or preterm birth, age, body mass index (BMI), and comorbidities (Fig. 75.3). The majority of studies thus far have focused on the associations of preconception subclinical hypothyroidism and thyroid autoimmunity (mainly TPOAb positivity) in infertile women with early in vitro fertilization (IVF) outcomes, miscarriage, and live birth rates. In these heterogeneous study populations, it remains difficult to establish whether subclinical hypothyroidism is a risk factor for adverse outcomes in women undergoing ART.16 A metaanalysis of three small randomized controlled trials (RCTs) has indicated that levothyroxine treatment in women with subclinical hypothyroidism may increase the number of fertilized oocytes, and reduce the relative risk of miscarriages by 31%.17 Data regarding thyroid autoimmunity and the risk of adverse ART outcomes are more consistent. TPOAb positivity is a more specific risk factor for adverse fertility or pregnancy outcomes than thyroglobulin antibody (TgAb) positivity. Although TPOAb positivity is

of menstrual and ovulatory disturbances in both hypo-­and hyperthyroidism. Hypothyroidism leads to a blunted FSH and LH response to gonadotropin-­releasing hormone (GnRH), hyperprolactinemia, and lower sex hormone–binding globulin (SHBG) and estradiol concentrations, the clinical consequences of which can be reversed by reestablishing euthyroidism.11 On the other hand, hyperthyroidism is associated with a greater LH and FSH response to GnRH, higher SHBG and estradiol concentrations, and high testosterone and androstenedione concentrations.11 Although it is likely that similar physiological changes occur in subclinical thyroid disease, studies remain scarce.

Physiology During Assisted Reproduction Technology Thyroid function test results remain stable throughout a normal menstrual cycle but can change during controlled ovarian hyperstimulation (COH; as employed for assisted reproduction). The rapid increase in estradiol during COH increases thyroxine-­binding globulin (TBG) concentrations and type 3 deiodinase (D3) gene transcription.12 Therefore, an increase in thyroid hormone production is required to ensure adequate thyroid hormone availability (Fig. 75.1). hCG administration may slightly increase free thyroxine (FT4) concentrations (see Fig. 75.1). The increased demand for thyroid hormone during COH is also demonstrated by the changes in TSH and FT4 concentrations in women treated with a fixed dose of levothyroxine. Furthermore, this is supported by data from TPOAb-­positive women, who likely have a reduced thyroid functional capacity, as TPOAb-­positive women typically exhibit a considerable and sustained increase in TSH following COH (Fig. 75.2).

Clinical Outcomes and Management Data on the association of preconception thyroid function with fertility or pregnancy outcomes in apparently healthy women are sparse, of low quality, and conflicting. For example, a large population-­based study13 performed in China showed that women with a high-­normal or increased preconception TSH had a slightly higher risk of abortion and preterm birth, but this was not replicated in a cohort of healthy Australian women.14 There is a clear indication to treat preconception overt thyroid disease from the view of fertility, conception, and pregnancy. Women with adequately treated preconception hypothyroidism have a similar risk of adverse pregnancy outcomes as euthyroid women. However, suboptimal levothyroxine treatment in women with overt hypothyroidism during early pregnancy is associated with Estradiol

TBG FT4

TSH Before COH

4-7 days after stimulation

At time of hCG stimulation

1 week after hCG stimulation

2 weeks after embryo transfer

2 weeks pregnant

Thyroxine degradation by D3* * Depiction of expected increase in thyroxine degradation by D3 based on estradiol stimulated DIO3 gene transcription

Fig. 75.1  Thyroid physiology throughout controlled ovarian hyperstimulation. COH, Controlled ovarian hyperstimulation; hCG, human chorionic gonadotropin; D3, type 3 deiodinase; TSH, thyroid-­stimulating hormone; TBG, thyroxine-­binding globulin; FT4, free thyroxine.

CHAPTER 75  Thyroid and Pregnancy

1253

A) TSH concentrations TBG Estradiol TPOAb positive Treated with fixed LT4 dose

TSH(TPOAb negative/non-treated) Before COH

4-7 days after stimulation

At time of hCG stimulation

1 week after hCG stimulation

2 weeks after embryo transfer

2 weeks pregnant

B) FT4 concentrations TBG Estradiol FT4

TPOAb positive FT4(TPOAb negative/non-treated)

Before COH

Treated with fixed LT4 dose

At time of 1 week after 2 weeks after 2 weeks 4-7 days hCG stimulation embryo transfer pregnant after stimulation hCG stimulation Fig. 75.2  Thyroid function throughout controlled ovarian hyperstimulation (COH) in thyroid peroxidase antibody (TPOAb)-­positive women and women treated with levothyroxine. hCG, Human chorionic gonadotropin; TBG, thyroxine-­binding globulin; FT4, free thyroxine; LT4, levothyroxine.

Female subfertility? check

TSH (with reflex FT4 if abnormal ) and TPOAbs

TSH increased TSH normal FT4 normal

FT4 low

TSH < 0.4

TPO antibodies neg

pos

No intervention or follow-up

Check TFTs during COH/ART and pregnancy

TPO antibodies neg

Start LT4 1.3-1.8 ug/kg/day Aim for TSH 10 mU/L consider checking macro TSH or heterophilic antibodies, if negative there is an indication for treatment Consider treatment if for example: TSH >7 mU/L, undergoing ART, or if previous gestational thyroid disease, other risk factors for hypothyroidism related adverse pregnancy outcomes.

.

Fig. 75.3  Treatment decision flowchart for thyroid function testing in women with subfertility. TSH, Thyroid-­stimulating hormone; FT4, free thyroxine; TPO, thyroid peroxidase; TPOAb, thyroid peroxidase antibody; LT4, levothyroxine; COH, controlled ovarian hyperstimulation; ART, assisted reproductive technology; TRAb, thyroid-­stimulating hormone receptor antibody; TFT, thyroid function tests.

not associated with early IVF outcomes such as oocyte quality/yield or implantation rate, observational studies show that TPOAb positivity is associated with an approximately 44% higher relative risk of miscarriage and, consequently, with a 35% lower relative chance of live birth.18 Two randomized trials have indicated that, for euthyroid

TPOAb-­positive patients with a prior miscarriage or undergoing fertility treatment, levothyroxine treatment has no beneficial effects. One randomized trial compared 226 euthyroid TPOAb-­ positive women who received levothyroxine (25–50 μg/day, depending on their TSH concentration) to an equally sized control group and found

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similar rates of miscarriage (10.6% vs. 10.3%) and live births (32.3% vs. 31.7%).3 Interestingly, subanalyses indicated a beneficial effect of levothyroxine treatment on the risk of miscarriage specifically in cases of a female cause of infertility, as opposed to a male or idiopathic cause of infertility (levothyroxine 7.3% vs. control 16.1%), although the study was not powered to show statistical significance for those analyses.3 Another randomized trial compared 476 euthyroid TPOAb-­positive women who received a fixed TSH dosage of 50 μg/ day to an equally sized control group and found similar rates of miscarriage (28.2% vs. 29.6%) and live births (37.4% vs. 37.9%).19 These studies provide good evidence for a lack of benefit from levothyroxine treatment for euthyroid TPOAb-­positive women, even if the TSH is above 2.5 mIU/L. Nonetheless, TPOAb positivity is an indication for follow-­up thyroid function tests during COH, pregnancy, and postpartum. TPOAb positivity with a preconception TSH above the reference range is considered to be an indication to start levothyroxine treatment, particularly when other risk factors for adverse ART or pregnancy outcomes are present, including a prior history of miscarriage or failed ART and age greater than 35 years (Fig. 75.3). The main argument for this is that these women are highly unlikely to normalize their thyroid function during COH or pregnancy and will thus eventually have a treatment indication. If levothyroxine therapy is elected, treatment should start with a low dose (25–50 μg/day), with subsequent dose titration to target a serum TSH of less than 2.5 mIU/L. KEY POINTS  • Thyroid dysfunction and thyroid autoimmunity are common among women referred for fertility treatment. Overt thyroid dysfunction should be screened for and treated preconception in women with subfertility. Decisions about the treatment of subclinical hypothyroidism in the preconception setting must be individualized.

MATERNAL AND FETAL PHYSIOLOGY IN PREGNANCY The maternal supply of thyroid hormone to the fetus, an increase in TBG concentrations under the influence of high maternal estrogen, an increase in renal iodine clearance, and the degradation of thyroid hormone by placental D3 together necessitate an increase in thyroid hormone production to ensure adequate availability for mother and fetus.11 This increased demand for thyroid hormone production is normally met through additional thyroid stimulation by high concentrations of the pregnancy hormone hCG, a weak agonist of the TSH receptor (Fig. 75.4).20 These pregnancy-­specific changes and increased demand may expose preexisting mild thyroid dysfunction or cause new hypothyroidism. Thyroid hormone is crucial for normal fetal brain development because it regulates the migration, proliferation, and differentiation of fetal neuronal cells, as well as synaptogenesis and myelination. In human fetuses, early neurogenesis begins at approximately 5 weeks postconception, and thyroid hormone receptors have been detected in the fetal brain at as early as 8 weeks’ gestation. Because the fetal thyroid gland is not functionally mature before week 18 to 20 of pregnancy, the fetus largely depends on the supply of maternal T4 during the early stages of intrauterine brain development. Later in pregnancy, maternal T4 continues to contribute to fetal thyroid hormone availability (Fig. 75.4). Thyroid autoimmunity, generally reflected by TPOAb positivity, is the most important risk factor for thyroid dysfunction during pregnancy. Recent studies show that the thyroidal response to hCG stimulation is severely impaired in TPOAb-­positive

women, leading to a relative thyroid hormone shortfall during early pregnancy.21 KEY POINTS  • Maternal thyroid hormone synthesis normally increases by 50% in pregnancy. Maternal thyroid hormone is critical for fetal brain development during early gestation, before the onset of fetal thyroid function.

THYROID FUNCTION TESTS IN PREGNANCY The major changes in thyroid physiology necessitate the use of pregnancy-­specific reference ranges to diagnose thyroid disease during gestation. Although many centers previously used fixed upper limits for TSH of 2.5 mIU/L in the first trimester and 3.0 mIU/L in the second or third trimester, it has been shown that these cutoffs are too low and cause considerable overdiagnosis. Ideally, a stepwise approach should be used to define the optimal reference range for each specific institution. Lab-­specific, gestational age-­specific TSH and FT4 reference ranges are the gold standard. A lab-­specific reference range for TSH and FT4 can be calculated as the 2.5th to 97.5th percentiles for a group of at least 400 women who are free of thyroid function-interfering factors (major disease, severe iodine deficiency, TPOAb positivity, thyroid [interfering] drug use, higher physiological hCG [e.g., twin pregnancies and/or IVF treatment]).22 If it is not possible to obtain lab-­specific reference ranges, reference ranges from other centers using the same assay and with a similar patient population can be adopted. Finally, if proper reference ranges cannot be obtained via either of the previous steps, a fixed upper limit for TSH of 4.0 mIU/L may be used.23 FT4 is required to distinguish between overt and subclinical thyroid disease. However, it is not possible to identify an evidence-­based fixed lower limit for FT4, because published reference ranges differ widely and are highly assay-­specific. Therefore, the lower range for FT4 is best defined using a laboratory-­specific or “adopted” reference range approach. Commonly used FT4 immunoassays have been reported to be inaccurate because of a shift in thyroid hormone-binding proteins during pregnancy.24 This shift affects FT4 measurements during the late second and third trimester and is therefore less likely to affect thyroid function tests in the first half of pregnancy. Furthermore, because between-­assay correlation for FT4 concentrations is very high,25-­27 commercial assays can be used to adequately identify women with abnormal FT4 if pregnancy-­specific and assay-­specific reference ranges are used, as shown in metaanalyses.28,29 Total T4 is a suboptimal marker for thyroid function during pregnancy, as it represents a very crude estimate of the fraction of available thyroid hormone, is highly dependent on changes in TBG, and there is no association with adverse outcomes. For the same reason, T3 concentrations are not used for defining abnormal thyroid function during pregnancy, and the general quality of free T3 assays limits their use both outside of and during pregnancy. KEY POINTS  • Thyroid function shifts over the course of gestation. Assay- and gestational age-specific reference ranges for TSH and free T4 levels should be used when available.

HYPERTHYROIDISM IN PREGNANCY In otherwise healthy women, a biochemical diagnosis of overt hyperthyroidism can be made in 0.7% to 0.9%, and another 1.0% to 2.1% of women can be diagnosed with subclinical hyperthyroidism.30 However, from a clinical perspective, two major subtypes of overt

CHAPTER 75  Thyroid and Pregnancy

hCG

1255

Estrogen Total T4 Thyroxine binding globulin FT4

TSH

week 5

week 10

Birth

Conception

Thyroid antibodies

week 20 Fetal thyroid hormone production

Fetal dependency on maternal thyroxine

T4 degradation by placental type 3 deiodinase Iodine clearance

Fig. 75.4  Thyroid physiology throughout pregnancy. TSH, Thyroid-­stimulating hormone; hCG, human chorionic gonadotropin; FT4, free thyroxine.

hyperthyroidism should be distinguished. First, the majority of women with gestational hyperthyroidism will present with a physiological form of transient biochemical hyperthyroidism (also referred to as gestational transient thyrotoxicosis). This is caused by high hCG concentrations, which typically peak between 8 to 12 weeks of pregnancy and are higher in IVF and twin pregnancies (Fig. 75.4). Most of these women have no or mild (transient) thyrotoxic symptoms and rarely require symptomatic treatment (i.e., propranolol). Trophoblastic diseases, partial and complete hydatidiform moles, and choriocarcinoma are other rare causes of hCG-­mediated (but pathological) hyperthyroidism in pregnancy. Non-hCG-­ mediated hyperthyroidism, the second major type of overt hyperthyroidism in pregnancy, is caused by Graves disease or, less commonly, autonomous thyroid hormone production (toxic adenoma or multinodular toxic goiter). Non-hCG-­ mediated hyperthyroidism during pregnancy is rare and can be further categorized as preexisting Graves disease (estimated prevalence 0.5%), new-­onset Graves disease (0.05%), and autonomous thyroid hormone production (0.1%).31,32 When untreated, the vast majority of women with Graves disease will present with overt biochemical abnormalities and thyrotoxic symptoms. Overt hyperthyroidism from Graves disease is associated with a high risk of adverse outcomes such as preeclampsia, preterm birth, low birth weight, and maternal heart failure.31,33 The clinical presentation of women with hyperthyroidism because of a toxic nodule or toxic multinodular goiter is more heterogeneous. In some cases, the additional hCG stimulation of pregnancy causes only transient hyperthyroidism, while preconception hyperthyroidism because of nodular disease may be more severe and in some cases necessitate treatment with antithyroid drugs, surgery, or radioablation therapy.

Diagnosis Although some clinical clues, including the personal and family past medical history, preconception onset of symptoms, goiter, or ophthalmopathy, can raise suspicion for Graves hyperthyroidism, biochemical evaluation is the key to diagnosis (Fig. 75.5). Typically, Graves hyperthyroidism in pregnancy presents with a FT4 concentration greater than 1.5 times the upper limit of normal and relatively high T3 concentrations but, most importantly, markers of autoimmunity in the form of positive serum thyroid-­stimulating immunoglobulin or TSH receptor antibody (TRAb). Because new-­onset Graves disease during pregnancy is very rare, some physicians prefer to perform a targeted approach to TRAb testing, while others prefer a universal testing approach in women with hyperthyroidism during pregnancy. In rare cases of a suspected nonpalpable toxic (T3-­producing) adenoma, thyroid ultrasound may be useful, although there is a high baseline risk of thyroid incidentaloma of up to 33%.34 Thyroid ultrasound cannot be used to distinguish physiological, hCG-­mediated hyperthyroidism from Graves hyperthyroidism.

Treatment and Monitoring Gestational thyrotoxicosis and subclinical hyperthyroidism typically do not require treatment. A conservative approach, for example monitoring thyroid function every 4 to 6 weeks until FT4 concentrations decrease, suffices for the vast majority of cases. Many women with gestational thyrotoxicosis have severe morning sickness or hyperemesis gravidarum and may need supportive care with antiemetics or intravenous hydration. For toxic adenomas and Graves disease, biochemical control of hyperthyroidism is essential to prevent adverse maternal, fetal, and neonatal complications. Maternal

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Fig. 75.5  Treatment decision flowchart for hyperthyroidism during pregnancy. TSH, Thyroid-­stimulating hormone; FT4, free thyroxine; TRAb, thyroid-­stimulating hormone receptor antibody; MMI, methimazole; ULN, upper limit of normal; TFT, thyroid function tests; Hx, history.

FT4 concentrations should be used to monitor treatment, because TSH can remain suppressed and (F)T3 concentrations do not adequately reflect fetal thyroid hormone availability. The treatment target should be an FT4 concentration in the upper third of the reference range, because the fetal thyroid is more sensitive to antithyroid drugs (ATDs), and lower maternal FT4 concentrations are associated with a higher risk of fetal hypothyroidism (upper third: 10% vs. 36% for lower two-­thirds).43 When hyperthyroidism is diagnosed preconception, various treatment modalities can be used to establish biochemical control. Of note, there is an important role for prepregnancy counseling and shared decision-­making, because toxic adenomas and the vast majority of Graves disease complicating pregnancy will predate conception. One option that can be considered is radioactive iodine, which is contraindicated during pregnancy and lactation. When considered preconception, women should be counseled to avoid conception for 6 months post-radioactive iodine therapy because of risk of teratogenicity from the radiation and the need to establish euthyroidism on levothyroxine. TRAb titers typically increase for approximately 1 year following radioactive iodine administration,35 and women with high baseline TRAb titers should be counseled that this may increase the risk for fetal and neonatal complications. Another option is thyroidectomy, which results in relatively swift biochemical control and normalization of serum TRAb concentrations but confers a risk of surgical complications and requires lifelong thyroid hormone therapy. Finally, treatment with ATDs is effective in establishing biochemical control and normalization of serum TRAb concentrations but, when used in the first trimester, is associated with an additional 3% to 5% absolute risk increase in fetal birth defects. That is in addition to the well-­known, but very rare, side effects of hepatic failure for propylthiouracil (PTU) or agranulocytosis for both ATDs.36-­38 PTU is the preferred drug during organogenesis in the first trimester, because large population studies indicate that the use of PTU during early

pregnancy is associated with a slightly lower risk of birth defects (absolute risk increase: 3% vs. 5% for methimazole [MMI]) but also less severe fetal anomalies as compared with MMI.36,37,38 To limit the potential adverse outcomes associated with ATDs, several pregnancy-­specific ATD strategies can be applied. First, in women of childbearing age with hyperthyroidism, the options for definitive treatment and the patient’s preference should be discussed preconception. Second, if the patient prefers ATDs or is already pregnant while on ATDs, so-­called block and replace therapy (a high dose of ATD with levothyroxine titration) should be avoided, and therapy should consist of ATD monotherapy with titration to euthyroidism with the lowest possible dose of PTU. Women using MMI preconception who are likely to require continuing therapy during pregnancy (i.e., those with more severe disease) should be switched to PTU preconception. It remains unknown whether switching from MMI to PTU in the first trimester of pregnancy reduces the risk of fetal birth defects. Switching strategies increases the risk of biochemical uncontrolled disease, and the single study that compared women who switched from PTU to MMI in the first trimester with those who continued MMI therapy found no difference in the risk of fetal birth defects.38 Owing to immune tolerance and the subsequent alterations in maternal immune response during pregnancy, the dose of ATDs should typically be reduced from the second half of pregnancy onwards, guided by maternal FT4 levels, to avoid overtreatment and fetal hypothyroidism.39-­40 Third, in women who are biochemically controlled with a stable low dose of MMI (5–10 mg) or PTU (100–200 mg) before pregnancy, the chance of relapse is lower than the overall 30% to 50% risk, and relapse would most likely occur after a few weeks to months.41 Therefore, stopping ATD treatment immediately at the time of a positive pregnancy test and monitoring thyroid function every 2 weeks can be considered to prevent the risks related to ATD exposure.23 In women with a history of active Graves disease, measurement of TRAbs should be performed in early pregnancy (Fig. 75.5).

CHAPTER 75  Thyroid and Pregnancy If biochemical control is not established before the second half of pregnancy, or if TRAbs are increased more than three times the upper limit of normal, additional maternal and fetal monitoring is required. Additional monitoring to determine the risk of hyperthyroidism (from maternal TRAb) or hypothyroidism (from ATD) in the fetus can start from midpregnancy (i.e., at 28, 32, and 36 weeks) via assessment of the fetal heart rate (normal 70% - 90%

> 1 cm: Biopsy + Endocrinology referral < 1 cm: Endocrinology referral

Intermediate suspicion

10% - 20%

> 1 cm: Biopsy + Endocrinology referral < 1 cm: U/S follow-up in 1 - 2 years

Low suspicion

5% - 10%

> 1.5 cm: Endocrinology referral < 1.5 cm: U/S follow-up in 1 - 2 years

Very low suspicion

< 3%

Clinical follow-up and U/S in 2 years

Table 6, figure 2 in reference 76

Fig. 77.5, cont’d  B, Pathway for the workup of thyroid nodules.

thyroid hormone (i.e., 75 μg/day T4 for 2 weeks followed by 150 μg/day for 2 weeks), after which uptake in all nonautonomous tissue will be suppressed, and thyroid autonomy is unmasked. Measurement of thyroid autoantibodies is not routinely performed in TA and TMNG. However, in iodine-­deficient areas, distinction between Graves disease and TMNG can be difficult if extrathyroidal

manifestations of the former are absent and diagnostic findings are “atypical,” e.g., presence of thyroid nodules (∼27%–34% by ultrasonography) in suspected Graves disease patients and patchy rather than diffuse uptake on scintigraphy. In these conditions, determination of TSHR antibodies is helpful to establish the correct diagnosis.83 Urinary iodine excretion can be measured in case

CHAPTER 77  Euthyroid and Hyperthyroid Nodules and Goiter

TABLE 77.1  Clinical Signs and Symptoms

of Multinodular Goiter

• S lowly growing nodular anterior neck mass • Tracheal deviation or compression, upper airway obstruction, dyspnea, dysphonia • Occasional cough and dysphagia, globus sensation • Sudden pain or enlargement secondary to hemorrhage • Superior vena cava obstruction syndrome • Pemberton’s sign: obstruction of the thoracic inlet by extending the arms over the head • Gradually developing hyperthyroidism • Iodide-­induced thyrotoxicosis • Recurrent nerve palsy (rare) • Phrenic nerve palsy (rare) • Horner syndrome (rare) • Enlargement during pregnancy

TABLE 77.2  Diagnosis of Multinodular

Goiter

• M  ultinodularity on examination • Asymmetry, tracheal deviation • Thyroid-­stimulating hormone normal or decreased, free thyroxine and free triiodothyronine normal or increased, thyroglobulin elevated • Calcitonin normal • Thyroid antibodies negative in approximately 90% • Scintigraphy with hot and cold areas • Ultrasound finding of nodularity (nonhomogeneity); cysts and calcifications are common • Computed tomography, magnetic resonance, positron emission tomography/computed tomography imaging demonstrating a nonhomogeneous mass • Lung function testing may demonstrate impaired inspiratory capacity • Benign cytology by fine-­needle aspiration of dominant nodules

of suspected iodine contamination. CT and magnetic resonance imaging techniques are not routinely indicated for the diagnosis of thyroid autonomy, but may be used for presurgical planning in patients with large and partly intrathoracic goiters.

Scintigraphy. The American Thyroid Association (ATA) guidelines recommend that a radionuclide thyroid scan should be performed if the serum TSH is subnormal.66 However, the American Association of Clinical Endocrinologists/American College of Endocrinology/ Associazione Medici Endocrinologi (AACE/ACE/AME) thyroid nodule guidelines recommend performing thyroid scintigraphy only in the presence of a thyroid nodule or MNG in combination with a suppressed TSH level, or when ectopic thyroid tissue or a retrosternal goiter is suspected. In iodine-­deficient regions, thyroid scintigraphy is recommended to exclude autonomy of a thyroid nodule or MNG even when the TSH level is low-­normal.73 The latter is supported by a metaanalysis of eight studies with 2761 hot thyroid nodules that reported a pooled prevalence of hot nodules with normal TSH levels of 50%.84 Moreover, thyroid nodules are not only classified as either “cold,” or “hot,” but may also show “normal” uptake on scintigraphy. In Europe, approximately 50% to 85% of all nodules are “cold,” up to 40% are scintigraphically indifferent, and approximately 10% are “hot.”85,86 Moreover, there is some geographic variation that is influenced by iodine intake, as well as the clinical setting. Smaller thyroid nodules

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(95% negative predictive value based on local data may replace diagnostic lobectomy for Bethesda System for Reporting Thyroid Cytopathology atypia of undetermined significance/follicular lesion of undetermined significance or follicular neoplasm/suspicious for follicular neoplasm cytology by appropriate surgery (lobectomy for mutations with low malignancy risk mutations or total thyroidectomy for mutations with high malignancy risk mutations) or follow-­up.

lobectomy, or by identifying patients with malignant lesions or a high likelihood for malignancy for total thyroidectomy as definitive surgical treatment (Fig. 77.6). Currently available molecular FNAC tests are based on testing for somatic mutations, gene expression evaluation, and microRNA (miRNA)-­based classifiers.102-107 The ThyroSeq test (version 3) uses targeted next-­generation sequencing analysis of 112 cancer-­related genes to identify point mutations, gene fusions, copy number alterations, or abnormal gene expression. Validation in 257 cytologically indeterminate nodules with surgical pathology reported a sensitivity of 94% and a specificity of 82%, with a negative predictive value of 97% and a positive predictive value of 66%. The test may eliminate the need for diagnostic surgery in up to 61% of patients with indeterminate nodules.103 The Afirma Genomic Sequencing Classifier uses RNA sequencing and consists of 12 classifiers composed of 10,196 genes. It has a reported specificity of 68% and a positive predictive value of 47% with a sensitivity of 91% and a negative predictive value of 96%.103 It is expected to reduce the frequency of diagnostic surgeries.108 Thus, the ThyroSeq and Afirma assays reported positive and negative predictive values that make them suitable for use in both rule-­in and rule-­out testing. The validation studies for both tests reported sufficient sensitivities and specificities to be used as rule-­in or rule-­out tests for indeterminate FNACs. However, the validation study cohort for the Afirma assay was not representative of the populations in whom the assay has been used, thus calling into question its reported diagnostic performance, including its negative predictive value.109 In the case of availability of similar postmarketing studies for the ThyroSeq test, a similar problem would likely also surface for the ThyroSeq test, because the validation study was mostly performed in tertiary referral centers. These findings emphasize once again the requirement for local evaluation of test performance as part of a diagnostic pathway. Currently, the further remaining problems are a lack of independent validation studies, a lack of long-­term outcome studies for ThyroSeq and Afirma benign tests, and the high cost that currently limits their application outside the United States. Although longer follow-­up is needed, recent postvalidation and clinical experience studies have demonstrated improved performance of both tests for

1289

indeterminate FNAC with Hürthle cell-dominant cytology,110 in spite of the predominance of widespread chromosomal losses and mitochondrial DNA alterations in Hürthle cell carcinomas.111,112 ThyroPrint, a gene expression classifier based on interrogation of only 10 miRNAs, has also displayed good performance (sensitivity 96%, specificity 87%, and positive and negative predictive values of 78% and 98%, respectively).113 Promising results have also been reported for a combination assay using miRNA classification (ThyraMIR) and next-­ generation sequencing mutation analysis (ThyGeNEXT) (positive predictive value 74%, negative predictive value 94%).104 However, the assay has yet to be subjected to independent validation. ThyroSPEC is a cost-­efficient matrix-­assisted laser desorption/ionization–time of flight mass spectrometry-­based mutation detection panel that detects the most prevalent 117 point mutations and 23 gene fusions in thyroid cancer.107 All molecular diagnostic test except the ThyroSPEC molecular test require additional liquid FNAC material generated by dedicated FNAC passes in preserving solutions, whereas ThyroSPEC analyzes the residual liquid material or the FNAC material of a representative FNAC glass slide smear after routine FNAC.107 A systematic review of studies examining RAS mutations reported a range from 0% to 48% for the prevalence of RAS mutation in benign lesions, 0% to 68% for RET/PTC rearrangements, and 0% to 55% for PAX8/PPARG rearrangements in benign lesions.114 Therefore, the presence of these biomarkers and the tremendous variation in reports of their prevalence in benign lesions suggests the need for caution when including these markers in diagnostic decisions. Further characterization of the importance of these markers, as well as newly discovered markers of thyroid malignancy, is required in order to avoid overtreatment of patients with benign thyroid tumors. However, the interpretation of these results is further complicated by the large interobserver for differentiating adenomatous nodules, thyroid adenomas, and minimally invasive FTCs.84 This high rate of histopathological discordance hampers translational studies assessing the value of molecular markers that aim to improve presurgical diagnosis, because the histological reference for these studies can be ambiguous or discrepant. Therefore, it is likely that the current histology-­based classification of thyroid tumors1 will ultimately be complemented by molecular/genetic characteristics for a more accurate classification, akin to other endocrine tumors or other cancers. The cost-­effectiveness of molecular FNAC tests varies dramatically depending on the healthcare setting (for example, in a multidisciplinary tertiary care setting vs. a primary care setting), the cost of the test, the cost of a lobectomy, the risk of malignancy for the respective FNAC diagnosis, and the sensitivity, specificity, and negative and positive predictive values and accuracy and impact on clinical decision-­making, and the quality of evidence reported for the respective molecular test. Consequently, the cost-­effectiveness of molecular FNAC tests needs to be evaluated for each specific interdisciplinary diagnostic pathway setting, and end users need to perform independent studies on the basis of regional costs and population demographics and ultrasound and FNAC performance.115 This was recently demonstrated by the first large patient cohort study before and after ThyroSeq implementation with 773 consecutive patients. It reported a doubling of AUS/FLUS and FN/SFN cytologies and an increase of the overall cost of care for patients with thyroid nodules.116 This study did not include any information on ultrasound malignancy risk stratification and emphasizes again the importance of local outcome evaluations taking into account all building blocks of the diagnostic pathway for the characterization of a nodule.

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The purpose of thyroid surgery is to cure thyrotoxicosis by removing all autonomously functioning thyroid tissue and other macroscopically visible nodular thyroid tissue. The extent of surgery is determined by preoperative ultrasound and, importantly, intraoperative morphologic inspection. For TA, hemithyroidectomy is usually adequate. In the case of TMNG, a subtotal, near-­total, or total thyroidectomy is indicated. The advantages of surgery (removal of all nodular tissue, rapid and permanent resolution of thyrotoxicosis, and definite histologic diagnosis) have to be weighed against general (risk of anesthesia) and thyroid-­specific side effects, the latter of which are largely dependent on the surgeon’s training and expertise (vocal cord paralysis ∼1%, permanent hypoparathyroidism 80–100 mL), and especially with suspicion of malignancy, thyroid surgery is recommended. Different protocols have been suggested for therapy in benign thyroid disease. Some investigators prefer to administer a standard dose, e.g., 10 or 20 mCi (370–740 MBq) per thyroid gland, while others apply a certain activity per gram of thyroid tissue. Different algorithms exist for dose calculation, e.g., in Germany, dosage is mostly calculated according to the Marinelli formula, which takes into account the maximum radioiodine uptake. Advantages of radioiodine are its simplicity and, in many countries, its outpatient-­based applicability. Disadvantages are the “time to euthyroidism” period (6 weeks to >3 months) during which drug therapy has to be continued and thyroid function monitored at 3-­to 6-­week intervals (Table 77. 3). Radioiodine treatment is contraindicated in pregnancy, and contraception is recommended for at least 6 months after receiving therapy. Side effects of radioiodine treatment include transient local pain and tenderness. Significant exacerbation of thyrotoxicosis due to destructive thyroiditis is uncommon. In general, most studies have not shown an increased risk of thyroid cancer, leukemia, or other malignancies, reproductive abnormalities, or congenital defects in the offspring, in adults treated with radioiodine.119 However, a more recent study has suggested a modest positive association with the risk of death from solid carcinomas, including breast cancer, in patients treated with radioiodine for hyperthyroidism,120 and the safety in children remains controversial.121 Hypothyroidism after therapy for TMNG and TA usually develops insidiously. The prevalence of hypothyroidism depends on the extent of TSH suppression prior to therapy and the protocol applied, and, importantly, increases with the duration of follow-­up. In a retrospective study of 346 patients with hyperfunctioning thyroid nodules, the occurrence of hypothyroidism was 7.6% at 1 year, 28% at 5 years, 46% at 10 years, and 60% at 20 years of follow-­up.122 These data emphasize the requirement of long-­term monitoring of thyroid function in all patients receiving therapy. Radioiodine treatment is considered safe and appropriate in nearly all types of hyperthyroidism, especially in older adult patients.123,124 Generally, radioiodine therapy is thought to carry a lower rate of complications and a lower cost than surgery. This fact has led a number of centers to offer radioiodine therapy as the first choice of therapy in the majority of patients. In contrast to surgery, which cures nearly all patients and normalizes hyperthyroidism within a few days,125 only 50% become euthyroid within 3 months of therapy, given that antithyroid drugs are not administered.126 Twenty to forty percent need additional therapy, and even up to five treatment sessions will not cure all patients. In contrast, persistence of hyperthyroidism after surgery is rare.125 A summary of the advantages and disadvantages of the different treatments for TA and TMNG is shown in Table 77.4. For patients who refuse or are not suitable for surgery or radioiodine therapy, radiofrequency or laser ablation of the hot nodule is increasingly accepted as an alternative treatment option.127

Radioiodine Therapy

Follow-­Up

Radioiodine therapy is widely used for treatment of thyroid autonomy and is highly effective in terms of eradicating thyrotoxicosis

The long-­term management of TA and TMNG patients is aimed at the detection and adequate treatment of thyroid dysfunction, prevention,

KEY POINTS  • Thyroid nodule and thyroid cancer diagnostics should be organized as a pathway. Deficiencies for one of the diagnostic steps or expertise cannot be compensated for by (over)emphasizing any other step or expertise. • As part of an integrated approach of careful clinical, ultrasound, and fine-­ needle aspiration cytology assessment with local outcome data, molecular testing may be able to improve diagnostic outcomes for thyroid nodules.

TREATMENT OF AUTONOMOUS THYROID NODULES Antithyroid drugs, usually in combination with beta-­blocking drugs, are the first-­line treatment in all patients with overt thyrotoxicosis. Depending on the type of antithyroid drug, an initial dosage of 10 to 30 mg/day of methimazole, 20 to 60 mg/day of carbimazole, or 150 to 300 mg/day of propylthiouracil is recommended. Higher doses are associated with more frequent adverse effects (3%–12%) and will only result in marginally faster resolution of thyrotoxicosis. Furthermore, a trial of low-­dose drug therapy (5–10 mg methimazole per day) may be justified in selected patients with symptomatic subclinical thyrotoxicosis. While the purpose of antithyroid drug therapy is to render the patient euthyroid, there is, contrary to Graves disease, practically no spontaneous resolution of thyroid autonomy. This implies that, once thyroid autonomy becomes clinically manifest, one needs to either consider definitive treatment or choose long-­term therapy with a thionamide in selected patients. However, benefits and risks of such “long-­term” drug therapy have to be weighed against the potential advantages and limitations of definitive treatment.83 Several ablative treatment options are available for TA and TMNG: thyroid surgery, radioiodine therapy, and thermal ablation techniques.

Surgery

CHAPTER 77  Euthyroid and Hyperthyroid Nodules and Goiter

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TABLE 77.3  Treatment Options for Patients with Toxic Multinodular Goiter Advantages

Disadvantages

Comments

Surgery

Significant goiter reduction Rapid achievement of euthyroidism Allows pathologic examination

Standard therapy for a large goiter or when rapid decompression is required Treatment of choice if radioiodine is not feasible

Antithyroid drugs

Easiest treatment option Mainly used at preparation for radiodiodine or surgery

Not all patients eligible Surgical mortality and morbidity a little higher than in nontoxic goiter Persistence or recurrence of hyperthyroidism Hypothyroidism* Lifelong treatment needed Very little chance of remission Adverse effects in approximately 5% Continuous goiter growth

Radioiodine

Effective in rendering patients euthyroid and in reducing thyroid volume Most often outpatient Few subjective side effects

Thermal ablation -­ Radiofrequency -­ Laser ablation

Alternative options to surgical treatment or radioiodine

Only gradual reversal of hyperthyroidism Gradual reduction of the goiter Small risk for acute goiter enlargement Risk for thyroiditis: 3% Risk for transition into Graves disease: 5% Five-­year risk for hypothyroidism: 15% Small risk for radiation-­induced ophthalmopathy Treatment needs to be repeated in some In experienced hands, serious side effects are very rare

Major indications are before thyroid surgery and before and after radioiodine, particularly in older adult patients and those with concurrent health problems Long-­term treatment recommended only when surgery or radioiodine cannot be used Standard therapy unless the goiter is very large (>100 mL). High-­dose radioiodine (inpatient treatment) may be an option in those with a very large goiter not fit for or who decline surgery

Mostly used for euthyroid nodules

*The percentage of patients affected depends on the extent of surgery.

TABLE 77.4  Treatment Options for Patients with Nontoxic Multinodular Goiter Treatment

Advantages

Disadvantages

Comments

Surgery

Significant goiter reduction Rapid decompression of vital structures Allows pathologic examination

Standard therapy for large goiters or when rapid decompression is required Total thyroidectomy should be considered to avoid goiter recurrence

Thyroxine

Outpatient Low cost May help prevent new nodule formation or goiter growth

Radioiodine

Halving of thyroid volume within 1 year Improves inspiratory capacity in long term Most often outpatient Can be repeated successfully Few subjective side effects

Thermal ablation -­ Radiofrequency -­ Laser ablation

Alternative options to surgical treatment or observation alone for solid nodules, purely or predominantly cystic nodules that recur after aspiration and ethanol ablation, and those that have a residual solid component after such therapy

Not all patients eligible Postoperative hemorrhage (1%) Recurrent laryngeal nerve injury (1%–2%) Hypoparathyroidism (0.5%–5%) Hypothyroidism* and goiter recurrence* Postoperative tracheomalacia (rare) Risk rates are slightly increased in cases of large goiter, intrathoracic extension, or reoperation Low efficacy Mainly impact on the perinodular volume Treatment, if lifelong and aimed at TSH suppression, could induce side effects caused by subclinical hyperthyroidism and inadvertent effects on bone and the heart Gradual reduction of the goiter Decreasing effect with increasing size Small risk for acute goiter enlargement Risk for thyroiditis: 3% Risk for transition into Graves disease: 5% One-­year risk for hypothyroidism: 15%–20% Small risk for radiation-­induced ophthalmopathy Treatment needs to be repeated in some Risk for radiation-­induced malignancy unsettled In experienced hands, serious side effects are very rare.

*The percentage of patients affected depends on the extent of surgery. TSH, Thyroid-­stimulating hormone; rhTSH, recombinant human thyroid-­stimulating hormone.

Its role on the wane due to possible adverse effects and low efficacy in reducing size Not recommended by the authors Low-­dose treatment may help prevent growth Has replaced surgery as the standard therapy in some European countries. Should be contemplated instead of reoperation and in those who decline or are not fit for surgery, also in case of a very large goiter (high-­dose radioiodine) For data in relation to rhTSH-­stimulated therapy, please see the text Only 20% and 38% show significant regrowth in the long term after radiofrequency ablation or laser ablation, respectively

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and detection of novel nodular thyroid disease, and, in the case of surgery, detection and treatment of postsurgical hypoparathyroidism. With radioiodine therapy, long-­term follow-­up for the development of hypothyroidism is mandatory.

of the thyroid nodules, LT4 treatment may not be appropriate in many patients.132 Based on the aforementioned considerations, LT4 treatment has been abandoned by many endocrinologists for this indication.13

TREATMENT OF EUTHYROID NODULES (SEE TABLE 77.3)

Surgery

Nodular thyroid disease is very common, but most of these goiters do not cause significant symptoms and are best left untreated. Treatment may be indicated in the case of the following: 1. Large goiter or progressive growth of the entire gland or individual nodules 2. Signs of cervical compression 3. Overt or subclinical hyperthyroidism 4. Marked neck disfigurement or cosmetic complaints 5. Suspicion of thyroid cancer In a number of clinical situations, a discrepancy is seen between clinical findings and patient complaints. In this context, the decision whether to treat can be more difficult. There is no ideal treatment for goiter, and practice patterns differ quite widely. This is, for example, reflected by the fact that one third of clinicians would refrain from treating a patient with moderate discomfort due to a multinodular nontoxic goiter of 50 to 80 g, where malignancy has been ruled out.13 Comparative studies of available options are sparse. Thus, treatment is not only a matter of goiter reduction. Patient satisfaction, the risks for hypothyroidism and goiter recurrence, and fear of overlooking a thyroid cancer, age, and comorbidities are all important factors that should be taken into account. It follows that the optimal treatment of toxic and nontoxic multinodular goiter is controversial, and the therapeutic approach must be individualized.

Iodine Supplementation Some clinicians, particularly in Europe, use iodine supplementation for treatment of goiter.13 In nodular goiter, iodine is no better than levothyroxine (LT4) suppression therapy for goiter reduction in comparative trials. In addition, the major hindrance for the use of iodine consists in the fact that a sudden increase of iodine intake may induce thyrotoxicosis in individuals with underlying thyroid autonomy.

Thyroid Hormone Treatment Thyroid hormone therapy for suppression of pituitary TSH secretion has been widely used in nontoxic multinodular goiter. Although LT4 suppressive therapy is effective in reducing the volume of diffuse nontoxic goiters by up to 30%, there are few controlled studies in nontoxic multinodular goiter, in which sonography has been employed for objective size monitoring. In one study, 58% of patients had a significant (>13%) decrease in thyroid volume, but regrowth was seen after discontinuation of therapy.128 In a randomized trial, Wesche and coworkers129 found a median reduction of goiter volume in the radioiodine treated group of 38% and 44% after 1 and 2 years, while the corresponding values in the LT4-­treated group were 7% and 1%, respectively, and nonsignificant. In a German study, LT4 in combination with elementary iodine supplementation resulted in a goiter volume reduction of only 7.9%, and a nodule volume reduction of 17.3% within 1 year, as compared with placebo.130 For volume reduction, the LT4 dose is often adjusted to reach a partly suppressed serum TSH level.130 The consequence is subclinical hyperthyroidism, which may adversely affect the skeleton and the cardiovascular system.131 Because lifelong therapy is probably needed to avoid goiter recurrence, and the natural history of the disease is progression toward hyperthyroidism due to autonomous function

For a comprehensive overview of thyroid surgery, see Chapter 82. The goal of surgery is removal of all thyroid tissue with a nodular appearance, usually by a unilateral hemithyroidectomy and subtotal resection of the contralateral lobe. A bilateral subtotal resection cannot be recommended. Only extremely rarely is a thoracic approach necessary. Further resection is not usually recommended if the final pathologic evaluation incidentally reveals a unilateral cancer less than 1 cm in size. This rather frequent finding accounts for most cancers found in surgical series, the majority of which are of little, if any, clinical significance.133 Macroscopically normal perinodular tissue often harbors microscopic growth foci, which explains the relatively high risk for recurrence in these patients.134 In the case of a toxic nodular goiter, thyroid function is more rapidly normalized after surgery than after antithyroid drug therapy. Surgery leads to rapid decompression resulting in improved respiratory function, if affected presurgically.135 Not all patients are surgical candidates, but among those undergoing surgery, the surgical mortality rate is less than 1% in experienced centers. Disadvantages include the general risks and side effects of a surgical procedure. Specific risks include transient (6%) or permanent (2%) vocal cord paralysis, transient (6%) or permanent (5%) hypoparathyroidism, and postoperative bleeding (1%).135 Others have found lower figures.136 Complications are related to increasing goiter size and extent of the resection.137 Novel techniques may reduce the operation time, postoperative pain, and length of hospital stay. Postoperative tracheomalacia, necessitating intubation, can occur after the removal of large goiters but is a rare complication. A matter of concern is the apparently high prevalence (7%–17%) of thyroid carcinomas in substernal goiters,138 but this seemingly high frequency is probably influenced by selection bias.139 The long-­term risk for hypothyroidism after subtotal resection of multinodular goiters is insufficiently described, but is approximately 10% to 20%, as reported for toxic multinodular goiters, and related to the extent of the resection. Recurrence of the nontoxic multinodular goiter is seen in 15% to 40% of patients with long-­term follow-­up.13 Postoperative use of LT4 is preferred by many clinicians to avoid recurrence; but based on results from randomized trials, use of LT4 is generally not recommended, and neither is iodine.13,132 A reoperation for the recurrent goiter results in a 3-­fold to 10-­fold increase in risk for permanent vocal cord paralysis or hypoparathyroidism.136,137 Goiter recurrence can be completely avoided if a total thyroidectomy is carried out initially, an approach that does not have a higher complication compared with subtotal thyroidectomy if the intervention is performed by high-­volume surgeons.140

Radioiodine Therapy Besides being able to cure hyperthyroidism, radioiodine therapy can also be used for volume reduction. During the last 25 years, radioiodine therapy for symptomatic multinodular nontoxic goiter has therefore been introduced in a number of mainly European centers as a nonsurgical alternative to LT4 therapy. Thyroid volume reduction is of the same magnitude in toxic and nontoxic multinodular goiters, that is, approximately 40% after 1 year126,129,141,142 and 50% to 60% after 2 years, without further reduction.141,143 Sixty percent of this decrease is seen within 3 months of therapy.141,142 In addition to a relief of compressive symptoms, radioiodine therapy also results in decreased tracheal

CHAPTER 77  Euthyroid and Hyperthyroid Nodules and Goiter compression, which can improve pulmonary function, particularly the inspiratory component.142,144 If used for very large goiters, normalization of thyroid volume, as seen in diffuse toxic and nontoxic goiters, is rarely achieved, but symptoms are in most cases considerably improved, and patient satisfaction is high.144 If a secondary increase in thyroid volume is seen, this should raise the suspicion of malignancy. Generally, therapy doses of 100 μCi (3.7 MBq) per gram of thyroid tissue corrected for 100% 24-­hour therapy uptake have been given.144,145 However, it has been questioned whether such dose adjustment is worthwhile,123,145 and fixed doses are given in a number of centers if nuclear medicine regulations allow to do so.123 The treatment can be repeated if further goiter reduction in a euthyroid patient is required.141 Radiation thyroiditis is seen in 3% within the first months of radioiodine therapy124 and is easily treated with salicylates or corticosteroids. Another possible complication consists in “Graves-­like autoimmune hyperthyroidism,” which is seen in up to 5%. Rare cases of radioiodine therapy-induced Graves ophthalmopathy have also been reported. The presence of anti-­TPO antibodies prior to radioiodine therapy is associated with an increased risk for this complication, which is most likely triggered by therapy related release of thyroid antigens and the appearance of TSHR antibodies typically 3 to 6 months after radioiodine therapy. This phenomenon can also be seen after surgical manipulation of the thyroid or after subacute thyroiditis. The condition is often self-­limiting, but may necessitate therapy. Although goiter enlargement caused by radioiodine therapy may be seen initially after treatment, there is usually no significant acute thyroid enlargement.124,142,146 The risk for permanent hypothyroidism after therapy in multinodular goiters ranges from 14% to 58% within 5 to 8 years.124 It occurs more commonly in patients with a small goiter and when anti-­TPO antibodies are present. Radioiodine therapy, given for Graves disease for decades, is not thought to be followed by any clinically significant increased risk for cancer deaths,119,124,147 although one study questions this view (see earlier).148 Most patients treated are older than 50 years of age and are less susceptible to radiation-­induced malignancy than young individuals. Data regarding radioiodine therapy in multinodular goiter are sparse, and in the case of nontoxic goiter, nonexistent. In a study by Ron and coworkers,147 1089 patients were treated for a toxic nodular goiter, and these individuals had a 31% increase in overall cancer mortality, nearly exclusively attributable to thyroid malignancy. However, a similar pattern was seen in patients not treated with radioiodine. Hence, the detection of a thyroid cancer in a nodular goiter after radioiodine therapy raises the question of whether malignancy in a nodule had been overlooked prior to radioiodine therapy. In some European countries, radioiodine therapy has replaced surgery as the treatment of choice in most patients.13,124 However, the optimal treatment modality remains to be established, ideally through comparative randomized trials, including data on effect, side effects, cost, and patient satisfaction. The efficacy of radioiodine therapy in multinodular goiter is hampered by the irregular therapy uptake in the gland, and the relative goiter reduction is inversely correlated with the initial goiter size.124,142 A high dietary iodine intake diminishes efficacy. Recombinant human TSH (rhTSH) has the potential of increasing the 24-­hour radioiodine therapy uptake more than 4-­fold, and the effect is inversely correlated to the initial thyroid radioiodine therapy uptake.124,149-152 Moreover, pretreatment with rhTSH causes a more homogeneous distribution of radioiodine therapy within the nodular gland.153 These properties of rhTSH are ideal in the context of therapy of multinodular goiter. Several studies, including two randomized double-­blind trials,124 have shown that rhTSH, in doses from 0.1 mg to 0.9 mg and administered 24 hours before radioiodine therapy, improves goiter reduction by

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35% to 55% within 1 year, compared with radioiodine therapy without stimulation. RhTSH-­augmented radioiodine therapy also results in reduced tracheal compression and an enhancement of the inspiratory reserve, as shown in a randomized trial.154 Long-­term follow-­up studies have documented that the enhanced goiter volume reduction obtained with rhTSH-­augmented therapy improves the reduction in goiter-­related symptoms and reduces the need for additional therapy.124 The administration of rhTSH may also permit the successful use of lower activities of radioiodine to achieve goiter reduction.117,155 Indeed, in a randomized double-­blind study,155 it was demonstrated that an equivalent goiter reduction after radioiodine therapy can be obtained by a much lower thyroid dose if rhTSH prestimulation is used.155,156 Although rhTSH stimulation has several attractive aspects for volume reduction in selected patients, it is important to emphasize that it forms an “off-­label” treatment for multinodular goiter. RhTSH-­augmented radioiodine therapy is generally well tolerated, particularly when using a dose of 0.1 mg or lower. A drawback of rhTSH-­augmented radioiodine therapy is the fact that, in parallel with the improved goiter reduction, a higher frequency of hypothyroidism is encountered.124 If rhTSH stimulation is used in order to lower the amount of the radioactive dose delivered to the thyroid gland, the risk for permanent hypothyroidism is comparable to that seen after radioiodine therapy alone.155

Nonsurgical Thermal Ablation of Thyroid Nodules Thermal ablation has shown efficacy in the treatment of a spectrum of benign thyroid nodules in multiple studies. In benign nodules that cause pressure symptoms and/or cosmetic concerns, when balancing efficacy, side effects, and cost, both laser ablation and radiofrequency ablation are alternative options to surgical treatment or observation for solid nodules, purely or predominantly cystic nodules that recur after aspiration and ethanol ablation, and those that have a residual solid component after such therapy.127,157 Only 20% and 38% show significant regrowth in the long term after radiofrequency ablation or laser ablation, respectively. In experienced hands, serious side effects are very rare. Thermal ablation for properly selected patients is widely used in some countries, but currently still more rarely used in others. Prior to thermal ablation, FNAC is warranted to characterize the nature of the nodule. Size and location, as well as operator experience, influence outcomes. Lesions that are located in the posterior and medial part of the thyroid may not be amenable to thermal ablation because of the proximity to the recurrent laryngeal nerve and the esophagus (“danger triangle”). Proper identification of patients who are most likely benefit from these localized therapies is key. There is no reason for any therapeutic intervention (thermal ablation or surgery) for sonographically visible nodules per se without local symptoms or cosmetic reasons. Percutaneous ethanol injection therapy (PEIT) has been used for two decades in solitary hot, toxic, and even cold thyroid nodules.158 The most convincing effect is seen in solitary thyroid cysts.127 In centers with access to thermal ablation techniques, PEIT now generally plays a secondary role. KEY POINTS  • Surgery remains the primary therapy in symptomatic patients with benign nontoxic goiter, and should be as complete as possible to avoid recurrence. • Radioiodine can be used as a nonsurgical therapy option in benign nontoxic nodular goiter. Efficacy can be augmented by recombinant human thyroid-­ stimulating hormone prestimulation of iodine uptake. • Thermal ablation has repeatedly shown efficacy in the treatment of a spectrum of benign thyroid nodules.

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24. Krohn K, Emmrich P, Ott N, et al. Increased thyroid epithelial cell proliferation in toxic thyroid nodules. Thyroid. 1999;9:241–246. 25. Deleu S, Allory Y, Radulescu A, et al. Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology. Thyroid. 2000;10:131–140. 26. Studer H, Peter HJ, Gerber H. Natural heterogeneity of thyroid cells: the basis for understanding thyroid function and nodular goiter growth. Endocrine Rev. 1989;10:125–135. 27. Krohn K, Reske A, Ackermann F, et al. Ras mutations are rare in solitary cold and toxic thyroid nodules. Clin Endocrinol. 2001;55:241–248. 28. Dohan O, De LV, Paroder V, et al. The sodium/iodide symporter (NIS): characterization, regulation, and medical significance. Endocrine Rev. 2003;24:48–77. 29. Krohn K, Paschke R. BRAF mutations are not an alternative explanation for the molecular etiology of ras-­mutation negative cold thyroid nodules. Thyroid. 2004;14:359–361. 30. Kimura ET, Nikiforova MN, Zhu Z, et al. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-­RAS-­BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 2003;63:1454–1457. 31. Krohn K, Stricker I, Emmrich P, et al. Cold thyroid nodules show a marked increase in proliferation markers. Thyroid. 2003;13:569–575. 32. Knudsen N, Bulow I, Jorgensen T, et al. Comparative study of thyroid function and types of thyroid dysfunction in two areas in Denmark with slightly different iodine status. Eur J Endocrinol. 2000;143:485–491. 33. Xing M. Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer. 2013;13:184–199. 34. Sponziello M, Lavarone E, Pegolo E, et al. Molecular differences between human thyroid follicular adenoma and carcinoma revealed by analysis of a murine model of thyroid cancer. Endocrinology. 2013;154:3043–3053. 35. Nikiforova MN, Biddinger PW, Caudill CM, et al. PAX8-­PPARgamma rearrangement in thyroid tumors: RT-­PCR and immunohistochemical analyses. Am J Surg Pathol. 2002;26:1016–1023. 36. Ye L, Zhou X, Huang F, et al. The genetic landscape of benign thyroid nodules revealed by whole exome and transcriptome sequencing. Nat Commun. 2017;8:15533. 37. Yoo SK, Lee S, Kim SJ, et al. Comprehensive analysis of the transcriptional and mutational landscape of follicular and papillary thyroid cancers. PLoS Genet. 2016;12:e1006239. 38. Thomas JL, Leclere J, Hartemann P, et al. Familial hyperthyroidism without evidence of autoimmunity. Acta Endocrinol-­Cop. 1982;100:512–518. 39. Parma J, Duprez L, Van Sande J, et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature. 1993;365:649–651. 40. Duprez L, Parma J, Van Sande J, et al. Germline mutations in the thyrotropin receptor gene cause non-­autoimmune autosomal dominant hyperthyroidism. Nat Genet. 1994;7:396–401. 41. Kopp P, Vansande J, Parma J, et al. Brief report -­congenital hyperthyroidism caused by a mutation in the thyrotropin-­receptor gene. N Engl J Med. 1995;332:150–154. 42. Sunthornthepvarakul T, Gottschalk ME, Hayashi Y, et al. Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-­receptor gene. N Engl J Med. 1995;332:155–160. 43. Hébrant A, van Staveren WCG, Maenhaut C, et al. Genetic hyperthyroidism: hyperthyroidism due to activating TSHR mutations. Euro J Endocrinol. 2011;164:1–9. 44. Stephenson A, Lau L, Eszlinger M, et al. The thyrotropin receptor mutation database update. Thyroid. 2020;30:931–935. 45. Paschke R, Niedziela M, Vaidya B, et al. 2012 European thyroid association guidelines for the management of familial and persistent sporadic non-­autoimmune hyperthyroidism caused by thyroid-­ stimulating hormone receptor germline mutations. Eur Thyroid J. 2012;1:142–147. 46. Burgi H. Iodine excess. Best Pract Res Clin Endocrinol Metab. 2010;24:107–115. 47. Lee SY, Rhee CM, Leung AM, et al. A review: radiographic iodinated contrast media-­induced thyroid dysfunction. J Clin Endocrinol Metab. 2015;100:376–383.

CHAPTER 77  Euthyroid and Hyperthyroid Nodules and Goiter

48. Wolff J, Chaikoff IL, Goldberg RC, et al. The temporary nature of the inhibitory action of excess iodine on organic iodine synthesis in the normal thyroid. Endocrinology. 1949;45:504–513. 49. Bartalena L, Bogazzi F, Chiovato L, et al. 2018 European Thyroid Association (ETA) guidelines for the management of amiodarone-­associated thyroid dysfunction. Eur Thyroid J. 2018;7:55–66. 50. Rhee CM, Bhan I, Alexander EK, et al. Association between iodinated contrast media exposure and incident hyperthyroidism and hypothyroidism. Archiv Internal Med. 2012;172:153–159. 51. Bervini S, Trelle S, Kopp PA, et al. Prevalence of iodine-­induced hyperthyroidism after administration of iodinated contrast during radiographic procedures: a systematic review and meta-­analysis of the literature. Thyroid. 2021;31:1020–1029. 52. Reinwein D, Benker G, Konig MP, et al. The different types of hyperthyroidism in Europe. Results of a prospective survey of 924 patients. J Endocrinol Invest. 1988;11:193–200. 53. Vejbjerg P, Knudsen N, Perrild H, et al. Effect of a mandatory iodization program on thyroid gland volume based on individuals’ age, gender, and preceding severity of dietary iodine deficiency: a prospective, population-­ based study. J Clin Endocrinol Metabol. 2007;92:1397–1401. 54. Dirikoc A, Polat SB, Kandemir Z, et al. Comparison of ultrasonography features and malignancy rate of toxic and nontoxic autonomous nodules: a preliminary study. Ann Nucl Med. 2015;29:883–889. 55. Jaeschke H, Undeutsch H, Patyra K, et al. Hyperthyroidism and papillary thyroid carcinoma in thyrotropin receptor D633H mutant mice. Thyroid. 2018;28:1372–1386. 56. Sandrock D, Olbricht T, Emrich D, et al. Long-­term follow-­up in patients with autonomous thyroid adenoma. Acta Endocrinol (Copenh). 1993;128:51–55. 57. Hamburger JI. Evolution of toxicity in solitary nontoxic autonomously functioning thyroid nodules. J Clin Endocrinol Metabol. 1980;50:5. 58. Sandrock D, Olbricht T, Emrich D, et al. Long-­term follow-­up in patients with autonomous thyroid adenoma. Acta Endocrinologica. 1993;128:51–55. 59. Laurberg P, Pedersen KM, Vestergaard H, et al. High incidence of multinodular toxic goitre in the elderly population in a low iodine intake area vs. high incidence of Graves’ disease in the young in a high iodine intake area: comparative surveys of thyrotoxicosis epidemiology in East-­Jutland Denmark and Iceland. J Internal Med. 1991;229:415–420. 60. Durante C, Grani G, Lamartina L, et al. The diagnosis and management of thyroid nodules: a review. J Am Med Assoc. 2018;319:914–924. 61. Kwong N, Medici M, Angell TE, et al. The influence of patient age on thyroid nodule formation, multinodularity, and thyroid cancer risk. J Clin Endocrinol Metab. 2015;100:4434–4440. 62. Volzke H, Ludemann J, Robinson DM, et al. The prevalence of undiagnosed thyroid disorders in a previously iodine-­deficient area. Thyroid. 2003;13:803–810. 63. Guth S, Theune U, Aberle J, et al. Very high prevalence of thyroid nodules detected by high frequency (13 MHz) ultrasound examination. Eur J Clin Invest. 2009;39:699–706. 64. Russ G, Bonnema SJ, Erdogan MF, et al. European Thyroid Association guidelines for ultrasound malignancy risk stratification of thyroid nodules in adults: the EU-­TIRADS. Eur Thyroid J. 2017;6:225–237. 65. Sharma SD, Jacques T, Smith S, et al. Diagnosis of incidental thyroid nodules on 18F-­fluorodeoxyglucose positron emission tomography imaging: are these significant? J Laryngol Otol. 2015;129:53–56. 66. Haugen BR, Alexander EK, Bible KC, et al. 2015 American thyroid association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American thyroid association guidelines Task Force on thyroid nodules and differentiated thyroid cancer. Thyroid. 2016;26:1–133. 67. Tessler FN, Middleton WD, Grant EG, et al. ACR Thyroid Imaging, Reporting And Data System (TI-­RADS): white paper of the ACR TI-­RADS committee. J Am Coll Radiol. 2017;14:587–595. 68. Nakamura H, Hirokawa M, Ota H, et al. Is an increase in thyroid nodule volume a risk factor for malignancy? Thyroid. 2015;25:804–811. 69. US Preventive Services Task Force; Bibbins-Domingo K, Grossman DC, Curry SJ, et al. Screening for thyroid cancer: US preventive Services Task Force recommendation statement. JAMA. 2017;317:1882–1887.

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70. Ahn HS, Kim HJ, Welch HG. Korea’s thyroid-­cancer “epidemic”— screening and overdiagnosis. N Engl J Med. 2014;371:1765–1767. 71. Vaccarella S, Franceschi S, Bray F, et al. Worldwide thyroid-­cancer epidemic? the increasing impact of overdiagnosis. N Engl J Med. 2016;375:614–617. 72. Morris LG, Sikora AG, Tosteson TD, et al. The increasing incidence of thyroid cancer: the influence of access to care. Thyroid. 2013;23:885–891. 73. Gharib H, Papini E, Garber JR, et al. American Association of Clinical Endocrinologists, American College of Endocrinology, and Associazione Medici Endocrinologi medical guidelines for clinical practice for the diagnosis and management of thyroid nodules. Endocr Pract. 2016;22:622– 639. 74. Paschke R, Cantara S, Crescenzi A, et al. European Thyroid Association guidelines regarding thyroid nodule molecular fine-­needle aspiration cytology diagnostics. EurThyroid J. 2017;6:115–129. 75. Van den Bruel A, Francart J, Dubois C, et al. Regional variation in thyroid cancer incidence in Belgium is associated with variation in thyroid imaging and thyroid disease management. J Clin Endocrinol Metab. 2013;98:4063–4071. 76. Persichetti A, Di Stasio E, Guglielmi R, et al. Predictive value of malignancy of thyroid nodule ultrasound classification systems: a prospective study. J Clin Endocrinol Metab. 2018;103:1359–1368. 77. Cibas ES, Ali SZ. The 2017 Bethesda System for reporting thyroid cytopathology. Thyroid. 2017;27:1341–1346. 78. Eszlinger M, Ullmann M, Ruschenburg I, et al. Low malignancy rates in fine-­needle aspiration cytologies in a primary care setting in Germany. Thyroid. 2017;27:1385–1392. 79. Basaria S, Salvatori R. Images in clinical medicine. Pemberton’s sign. N Engl J Med. 2004;350. 80. Trivalle C, Doucet J, Chassagne P, et al. Differences in the signs and symptoms of hyperthyroidism in older and younger patients. J Am Geriatr Soc. 1996;44:50–53. 81. Costante G, Durante C, Francis Z, et al. Determination of calcitonin level in C-­cell disease: clinical interest and potential pitfalls. Nat Clin Pract Endocrinol Metab. 2009;5:35–44. 82. Nygaard B, Hegedus L, Nielsen KG, et al. Long-­term effect of radioactive iodine on thyroid function and size in patients with solitary autonomously functioning toxic thyroid nodules. Clin Endocrinol (Oxf). 1999;50:197–202. 83. Ross DS, Burch HB, Cooper DS, et al. 2016 American Thyroid Association guidelines for diagnosis and management of hyperthyroidism and other causes of thyrotoxicosis. Thyroid. 2016;26:1343–1421. 84. Franc B, de la Salmoniere P, Lange F, et al. Interobserver and intraobserver reproducibility in the histopathology of follicular thyroid carcinoma. Hum Pathol. 2003;34:1092–1100. 85. Belfiore A, La Rosa GL, La Porta GA, et al. Cancer risk in patients with cold thyroid nodules: relevance of iodine intake, sex, age, and multinodularity. Am J Med. 1992;93:363–369. 86. Knudsen N, Perrild H, Christiansen E, et al. Thyroid structure and size and two-­year follow-­up of solitary cold thyroid nodules in an unselected population with borderline iodine deficiency. Euro J Endocrinol. 2000;142:224–230. 87. Symonds CJ, Seal P, Ghaznavi S, et al. Thyroid nodule ultrasound reports in routine clinical practice provide insufficient information to estimate risk of malignancy. Endocrine. 2018;61:303–307. 88. Seifert P, Gorges R, Zimny M, et al. Interobserver agreement and efficacy of consensus reading in Kwak-­, EU-­, and ACR-­thyroid imaging recording and data systems and ATA guidelines for the ultrasound risk stratification of thyroid nodules. Endocrine. 2020;67:143–154. 89. Pantano AL, Maddaloni E, Briganti SI, et al. Differences between ATA, AACE/ACE/AME and ACR TI-­RADS ultrasound classifications performance in identifying cytological high-­risk thyroid nodules. Eur J Endocrinol. 2018;178:595–603. 90. Grani G, Lamartina L, Ascoli V, et al. Reducing the number of unnecessary thyroid biopsies while improving diagnostic accuracy: toward the “right” TIRADS. J Clin Endocrinol Metab. 2019;104:95–102. 91. Tessler FN. Thyroid nodules and real estate: location matters. Thyroid. 2020;30:349–350.

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92. Garber JR, Papini E, Frasoldati A, et al. American Association of Clinical Endocrinology And Associazione Medici Endocrinologi Thyroid Nodule Algorithmic Tool. Endocr Pract. 2021;27:649–660. 93. Bongiovanni M, Spitale A, Faquin WC, et al. The Bethesda system for reporting thyroid cytopathology: a meta-analysis. Acta Cytol. 2012;56: 333–339. 94. Nikiforov YE. Ramifications of new terminology for encapsulated follicular variant of papillary thyroid carcinoma-­reply. JAMA Oncol. 2016;2:1098–1099. 95. Wienhold R, Scholz M, Adler JR, et al. The management of thyroid nodules: a retrospective analysis of health insurance data. Dtsch Arztebl Int. 2013;110:827–834. 96. Trimboli P, Treglia G, Guidobaldi L, et al. Detection rate of FNA cytology in medullary thyroid carcinoma: a meta-­analysis. Clin Endocrinol (Oxf). 2015;82:280–285. 97. Essig Jr GF, Porter K, Schneider D, et al. Fine needle aspiration and medullary thyroid carcinoma: the risk of inadequate preoperative evaluation and initial surgery when relying upon FNAB cytology alone. Endocr Pract. 2013;19:920–927. 98. d’Herbomez M, Caron P, Bauters C, et al. Reference range of serum calcitonin levels in humans: influence of calcitonin assays, sex, age, and cigarette smoking. Eur J Endocrinol. 2007;157:749–755. 99. Wells Jr SA, Asa SL, Dralle H, et al. Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid. 2015;25:567–610. 100. Allen L, Al Afif A, Rigby MH, et al. The role of repeat fine needle aspiration in managing indeterminate thyroid nodules. J Otolaryngol Head Neck Surg. 2019;48:16. 101. Stewart R, Leang YJ, Bhatt CR, et al. Quantifying the differences in surgical management of patients with definitive and indeterminate thyroid nodule cytology. Eur J Surg Oncol. 2020;46:252–257. 102. Steward DL, Carty SE, Sippel RS. Performance of a multigene genomic classifier in thyroid nodules with indeterminate cytology: a prospective blinded multicenter study. JAMA Oncol. 2019;1:204–212. 103. Patel KN, Angell TE, Babiarz J, et al. Performance of a genomic sequencing classifier for the preoperative diagnosis of cytologically indeterminate thyroid nodules. JAMA Surg. 2018;153:817–824. 104. Labourier E, Shifrin A, Busseniers AE, et al. Molecular testing for miRNA, mRNA and DNA on fine needle aspiration improves the preoperative diagnosis of thyroid nodules with indeterminate cytology. J Clin Endocrinol Metab. 2015;100:2743–2750. 105. Gonzalez HE, Martínez JR, Vargas-­Salas S, et al. A 10-­gene classifier for indeterminate thyroid nodules: development and multicenter accuracy study. Thyroid. 2017;27:1058–1067. 106. Eszlinger M, Bohme K, Ullmann M, et al. Evaluation of a two-­year routine application of molecular testing of thyroid fine-­needle aspirations using a seven-­gene panel in a primary referral setting in Germany. Thyroid. 2017;27:402–411. 107. Stewardson P, Khalil M, Ghaznavi S, et al. Prospective Evaluation of the ThyroSPECTM Mutation Panel for the Diagnosis of Indeterminate Thyroid fine Needle Aspiration Cytologies (FNAC) in the Southern Alberta Healthcare Region. Chicago: 89th annual meeting of the American Thyroid Association; 2019. 108. Angell TE, Heller HT, Cibas ES, et al. Independent comparison of the Afirma genomic sequencing classifier and gene expression classifier for cytologically indeterminate thyroid nodules. Thyroid. 2019;29:650–656. 109. Valderrabano P, Hallanger-­Johnson JE, Thapa R, et al. Comparison of postmarketing findings vs the initial clinical validation findings of a thyroid nodule gene expression classifier: a systematic review and meta-­ analysis. JAMA Otolaryngol Head Neck Surg. 2019;145:783–792. 110. Endo M, Nabhan F, Angell TE, et al. Letter to the editor: Use of molecular diagnostic tests in thyroid nodules with hurthle cell-­dominant cytology. Thyroid. 2020;30:1390–1392. 111. Ganly I, Makarov V, Deraje S, et al. Integrated genomic analysis of Hurthle cell cancer reveals oncogenic drivers, recurrent mitochondrial mutations, and unique chromosomal landscapes. Cancer Cell. 2018;34:256–270.e5.

112. Gopal RK, Kubler K, Calvo SE, et al. Widespread chromosomal losses and mitochondrial DNA alterations as genetic drivers in Hurthle cell carcinoma. Cancer Cell. 2018;34:242–255.e5. 113. Zafereo M, McIver B, Vargas-­Salas S, et al. A thyroid genetic classifier correctly predicts benign nodules with indeterminate cytology: two independent, multicenter, prospective validation trials. Thyroid. 2020;30:704–712. 114. Najafian A, Noureldine S, Azar F, et al. RAS mutations, and RET/ PTC and PAX8/PPAR-­gamma chromosomal rearrangements are also prevalent in benign thyroid lesions: implications thereof and a systematic review. Thyroid. 2017;27:39–48. 115. Eszlinger M, Lau L, Ghaznavi S, et al. Molecular profiling of thyroid nodule fine-­needle aspiration cytology. Nat Rev Endocrinol. 2017;13:415–424. 116. Fazeli SR, Zehr B, Amraei R, et al. ThyroSeq v2 testing: impact on cytologic diagnosis, management, and cost of care in patients with thyroid nodule. Thyroid. 2020;30:1528–1534. 117. Nieuwlaat WA, Huysmans DA, van den Bosch HC, et al. Pretreatment with a single, low dose of recombinant human thyrotropin allows dose reduction of radioiodine therapy in patients with nodular goiter. J Clin Endocrinol Metab. 2003;88:3121–3129. 118. Bonnema SJ, Bennedbaek FN, Gram J, et al. Resumption of methimazole after 131I therapy of hyperthyroid diseases: effect on thyroid function and volume evaluated by a randomized clinical trial. Euro J Endocrinol. 2003;149:485–492. 119. Franklyn JA, Maisonneuve P, Sheppard M, et al. Cancer incidence and mortality after radioiodine treatment for hyperthyroidism: a population-­ based cohort study. Lancet. 1999;353:2111–2115. 120. Kitahara CM, Berrington de Gonzalez A, Bouville A, et al. Association of radioactive iodine treatment with cancer mortality in patients with hyperthyroidism. JAMA Intern Med. 2019;179:1034–1042. 121. American Thyroid Association Taskforce On Radioiodine Safety; Sisson JC, Freitas J, McDougall IR, et al. Radiation safety in the treatment of patients with thyroid diseases by radioiodine 131I : practice recommendations of the American Thyroid Association. Thyroid. 2011;21:335–346. 122. Ceccarelli C, Bencivelli W, Vitti P, et al. Outcome of radioiodine-131 therapy in hyperfunctioning thyroid nodules: a 20 years’ retrospective study. Clin Endocrinol (Oxf). 2005;62:331–335. 123. Weetman AP. Radioiodine treatment for benign thyroid diseases. Clin Endocrinol. 2007;66:757–764. 124. Bonnema SJ, Hegedus L. Radioiodine therapy in benign thyroid diseases: effects, side effects, and factors affecting therapeutic outcome. Endocr Rev. 2012;33:920–980. 125. Porterfield Jr JR, Thompson GB, Farley DR, et al. Evidence-­based management of toxic multinodular goiter (Plummer’s disease). World J Surg. 2008;32:1278–1284. 126. Nygaard B, Hegedus L, Ulriksen P, et al. Radioiodine therapy for multinodular toxic goiter. Archiv Internal Med. 1999;159:1364–1368. 127. Papini E, Monpeyssen H, Frasoldati A, et al. 2020 European Thyroid Association clinical practice guideline for the use of image-­guided ablation in benign thyroid nodules. Eur Thyroid J. 2020;9:172–185. 128. Berghout A, Wiersinga WM, Drexhage HA, et al. Comparison of placebo with L-­thyroxine alone or with carbimazole for treatment of sporadic non-­toxic goitre. Lancet. 1990;336:193–197. 129. Wesche MF, Tiel V, Lips P, et al. A randomized trial comparing levothyroxine with radioactive iodine in the treatment of sporadic nontoxic goiter. J Clin Endocrinol Metabol. 2001;86:998–1005. 130. Grussendorf M, Reiners C, Paschke R, et al. Reduction of thyroid nodule volume by levothyroxine and iodine alone and in combination: a randomized, placebo-­controlled trial. J Clin Endocrinol Metabol. 2011;96:2786–2795. 131. Surks MI, Ortiz E, Daniels GH, et al. Subclinical thyroid disease: scientific review and guidelines for diagnosis and management. J Am Med Assoc. 2004;291:228–238. 132. Fast S, Bonnema SJ, Hegedus L. The majority of Danish nontoxic goitre patients are ineligible for Levothyroxine suppressive therapy. Clin Endocrinol. 2008;69:653–658. 133. Ito Y, Uruno T, Nakano K, et al. An observation trial without surgical treatment in patients with papillary microcarcinoma of the thyroid. Thyroid. 2003;13:381–387.

CHAPTER 77  Euthyroid and Hyperthyroid Nodules and Goiter 134. Hegedus L, Nygaard B, Hansen JM. Is routine thyroxine treatment to hinder postoperative recurrence of nontoxic goiter justified? J Clin Endocrinol Metabol. 1999;84:756–760. 135. Erickson D, Gharib H, Li H, et al. Treatment of patients with toxic multinodular goiter. Thyroid. 1998;8:277–282. 136. al-­Suliman NN, Ryttov NF, Qvist N, et al. Experience in a specialist thyroid surgery unit: a demographic study, surgical complications, and outcome. Euro J Surg. 1997;163:13–20. 137. Thomusch O, Machens A, Sekulla C, et al. Multivariate analysis of risk factors for postoperative complications in benign goiter surgery: prospective multicenter study in Germany. World J Surg. 2000;24:1335– 1341. 138. Torre G, Borgonovo G, Amato A, et al. Surgical management of substernal goiter: analysis of 237 patients. Am Surg. 1995;61:826–831. 139. Hegedus L, Bonnema SJ. Approach to management of the patient with primary or secondary intrathoracic goiter. J Clin Endocrinol Metab. 2010;95:5155–5162. 140. Pappalardo G, Guadalaxara A, Frattaroli FM, et al. Total compared with subtotal thyroidectomy in benign nodular disease: personal series and review of published reports. Eur J Surg. 1998;164:501–506. 141. Nygaard B, Hegedus L, Gervil M, et al. Radioiodine treatment of multinodular non-­toxic goitre. BMJ. 1993;307:828–832. 142. Bonnema SJ, Bertelsen H, Mortensen J, et al. The feasibility of high dose iodine 131 treatment as an alternative to surgery in patients with a very large goiter: effect on thyroid function and size and pulmonary function. J Clin Endocrinol Metab. 1999;84:3636–3641. 143. Parle JV, Maisonneuve P, Sheppard MC, et al. Prediction of all-­cause and cardiovascular mortality in elderly people from one low serum thyrotropin result: a 10-­year cohort study. Lancet. 2001;358:861–865. 144. Huysmans DA, Hermus AR, Corstens FH, et al. Large, compressive goiters treated with radioiodine. Ann Intern Med. 1994;121:757–762. 145. Jarlov AE, Hegedus L, Kristensen LO, et al. Is calculation of the dose in radioiodine therapy of hyperthyroidism worth while? Clin Endocrinol (Oxf). 1995;43:325–329. 146. Nygaard B, Faber J, Hegedus L. Acute changes in thyroid volume and function following 131I therapy of multinodular goitre. Clin Endocrinol (Oxf). 1994;41:715–718. 147. Ron E, Doody MM, Becker DV, et al. Cancer mortality following treatment for adult hyperthyroidism. Cooperative thyrotoxicosis therapy follow-­up study group. J Am Med Assoc. 1998;280:347–355.

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148. Metso S, Jaatinen P, Huhtala H, et al. Increased cardiovascular and cancer mortality after radioiodine treatment for hyperthyroidism. J Clin Endocrinol Metab. 2007;92:2190–2196. 149. Huysmans DA, Nieuwlaat WA, Erdtsieck RJ, et al. Administration of a single low dose of recombinant human thyrotropin significantly enhances thyroid radioiodide uptake in nontoxic nodular goiter. J Clin Endocrinol Metab. 2000;85:3592–3596. 150. Nielsen VE, Bonnema SJ, Boel-­Jorgensen H, et al. Recombinant human thyrotropin markedly changes the 131I kinetics during 131I therapy of patients with nodular goiter: an evaluation by a randomized double-­ blinded trial. J Clin Endocrinol Metab. 2005;90:79–83. 151. Fast S, Nielsen VE, Grupe P, et al. Optimizing 131I uptake after rhTSH stimulation in patients with nontoxic multinodular goiter: evidence from a prospective, randomized, double-­blind study. J Nucl Med. 2009;50:732– 737. 152. Albino CC, Mesa Jr CO, Olandoski M, et al. Recombinant human thyrotropin as adjuvant in the treatment of multinodular goiters with radioiodine. J Clin Endocrinol Metab. 2005;90:2775–2780. 153. Nieuwlaat WA, Hermus AR, Sivro-­Prndelj F, et al. Pretreatment with recombinant human TSH changes the regional distribution of radioiodine on thyroid scintigrams of nodular goiters. J Clin Endocrinol Metab. 2001;86:5330–5336. 154. Bonnema SJ, Nielsen VE, Boel-­Jorgensen H, et al. Recombinant human thyrotropin-­stimulated radioiodine therapy of large nodular goiters facilitates tracheal decompression and improves inspiration. J Clin Endocrinol Metab. 2008;93:3981–3984. 155. Nielsen VE, Bonnema SJ, Hegedus L. Effects of 0.9 mg recombinant human thyrotropin on thyroid size and function in normal subjects: a randomized, double-­blind, cross-­over trial. J Clin Endocrinol Metab. 2004;89:2242–2247. 156. Fast S, Hegedus L, Grupe P, et al. Recombinant human thyrotropin-­ stimulated radioiodine therapy of nodular goiter allows major reduction of the radiation burden with retained efficacy. J Clin Endocrinol Metab. 2010;95:3719–3725. 157. Hegedus L, Miyauchi A, Tuttle RM. Nonsurgical thermal ablation of thyroid nodules: not if, but why, when, and how? Thyroid. 2020;30:1691– 1694. 158. Hegedus L. Thyroid ultrasound. Endocrinol Metabol Clinic North Am. 2001;30:339–360, viii–ix.

78 Differentiated Thyroid Cancer – Streamlining Diagnosis and Optimizing Management Amanda La Greca and Bryan R. Haugen

OUTLINE Epidemiology, 1298 Pathology, 1298 Papillary Thyroid Carcinoma Variants, 1299 Follicular Thyroid Carcinoma, 1300 Hurthle Cell (Oncocytic) Tumors, 1301 Poorly Differentiated Thyroid Carcinoma, 1301 Causes, 1301 Somatic Gene Alterations, 1301 Germline Genetic Factors, 1303 Ionizing Radiation, 1304 Diagnosis, Clinical Features, And Course, 1304 Staging, Risk Of Recurrence, And Response To Therapy, 1306

Response to Therapy, 1306 Prognostic Factors, 1308 Age and Gender, 1308 Histopathological Factors, 1308 Size of the Primary Tumor and Extrathyroidal Extension, 1308 Extrathyroidal Extension, 1308 Lymph Node Metastases, 1308 Therapy And Monitoring, 1308 Radioiodine Therapy, 1309 Levothyroxine Therapy, 1309 Monitoring for Disease Recurrence, 1310 Treatment of Metastatic Disease, 1310

 

EPIDEMIOLOGY Thyroid cancer is the fifth most common cancer in women, contributing 6.3% of the total number of new cases diagnosed in 2018. In the United States, the estimated incidence of new cases of thyroid carcinoma for 2020 was 52,890, representing 2.9% of all new cancer cases. The rate of death from thyroid cancer has been stable at 0.4%, suggesting that the increased incidence is primarily the result of diagnosis of subclinical, indolent tumors. The median age at diagnosis is approximately 45 to 50 years old, and differentiated thyroid carcinoma (DTC) is diagnosed two to four times more frequently in women than in men.1 The incidence of thyroid cancer was relatively stable until the early 1990s, after which it increased substantially.2 Since 1975, the incidence of thyroid cancer has nearly tripled, from 4.9 to 14.3 per 100,000 individuals (absolute increase, 9.4 per 100,000). Almost the entire increase was attributable to papillary thyroid cancer (PTC), from 3.4 to 12.5 per 100,000 (absolute increase, 9.1 per 100,000),2 suggesting that the increase is the result of diagnosis of subclinical, indolent tumors. The size distribution of detected thyroid cancer has shifted toward smaller lesions. In 1988 to 1989, 25% of detected thyroid cancers were 1 cm or smaller. In the most recent data (2008–2009), 39% were 1 cm or smaller.2 Autopsy series have shown that up to 6% of thyroid glands in autopsied adults in the United States, and over 20% in Japan, also harbor microscopically detectable foci of thyroid carcinoma, probably of no clinically significance.3,4 Compared with other malignancies, thyroid cancer has a high rate of cure with very high long-­term survival rates, such as 98.3% 5-­year relative survival.1 For PTC, the most frequent thyroid malignancy, the 5-­year relative survival rate for localized PTC is nearly 100%, for regional PTC is 99%, for patients who present with distant is metastasis is 76%, and for all Surveillance, Epidemiology, and End Results (SEER) stages combined is near 100%.5

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KEY POINTS  • The incidence of thyroid cancer has nearly tripled, from 4.9 to 14.3 per 100,000 individuals. Almost the entire increase was attributable to small papillary thyroid cancer. The rate of death from thyroid cancer has been stable at 0.4%, suggesting that the increased incidence is primarily the result of diagnosis of subclinical, indolent tumors. • The median age at diagnosis of thyroid cancer is approximately 45 to 50 years old, and differentiated thyroid cancer is diagnosed two to four times more frequently in women than in men. • Compared with other malignancies, thyroid cancer has a high rate of cure with very high long-­term survival rates, such as 98.3% 5-­year relative ­survival.

PATHOLOGY Papillary and follicular carcinomas are the two most common subtypes of thyroid cancer, referred to as DTC. The fourth edition World Health Organization (WHO) Classification of Tumors of Endocrine Organs was published in 2017, in which the new thyroid tumor classification was included.6 Several modifications to follicular cell tumors were made (Table 78.1). The major revisions were: i) the introduction of borderline tumors (uncertain malignant potential [UMP] and noninvasive follicular thyroid neoplasm with papillary-­like nuclear features [NIFTP] and hyalinizing trabecular tumors) in the thyroid tumor classification; ii) PTC comprising 15 variants, and a new (hobnail) histologic variant being included; iii) follicular thyroid carcinoma (FTC) being subdivided into three prognostic groups: minimally invasive (capsule invasion only), encapsulated angioinvasive, and widely invasive; iv) Hurthle/oncocytic cell tumors being separated from follicular adenoma (FA)/FTC in an independent chapter, as it has a different genetic profile from those of the other types of thyroid cancer; v) the diagnostic criteria of poorly differentiated carcinoma being more precisely defined

CHAPTER 78  Differentiated Thyroid Cancer – Streamlining Diagnosis And Optimizing Management and adopting the Turin consensus criteria; vi) emphasizing the prognostic value of genetic markers, such as BRAFV600E and telomerase reverse transcriptase (TERT) promoter mutation, in thyroid carcinoma of follicular cell origin; and vii) emphasizing the examination of the entire capsule to identify or exclude invasiveness in encapsulated thyroid tumors.7

Papillary Thyroid Carcinoma Variants Fifteen variants of PTC were listed in the most recent WHO classification of thyroid tumors (Table 78.2).6

Conventional/Classic Type. PTC is a malignant epithelial tumor demonstrating evidence of follicular cell differentiation and a set of distinct nuclear features (nuclear folding, increased mitoses, and TABLE 78.1  Modified from WHO

Classification of thyroid tumors (4th edition – 2017) 1. Papillary thyroid carcinoma (PTC) variants: - Conventional / classic variant - Papillary microcarcinoma - Encapsulated PTC – applicable to fully encapsulated classic PTC - Diffuse sclerosing variant - Tall cell variant - Columnar cell variant - Cribriform-morular variant - Hobnail variant – new entity PTC - PTC with fibromatosis/fascitiis-like stroma - Solid/trabecular variant - Oncocytic variant - Spindle cell variant - Clear cell variant - Warthin like variant (note that variants diffuse sclerosing, tall cell, columnar cell, hobnail and solid/trabecular are so-called aggressive variants). 2. Follicular thyroid carcinoma (FTC) groups: - Minimally invasive FTC – capsular invasion only - Angioinvasive FTC - Widely invasive FTC – grossly invasive 3. Hürthle cell (oncocytic) tumors as a separate entity: Include Hürthle cell adenoma and Hürthle cell carcinoma, previously classified as oncocytic variant of follicular adenoma and FTC. The term "Hürthle" is favored over "oncocytic" 4. Poorly differentiated thyroid carcinoma: Turin criteria were adopted

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hyperchromasia of the nucleus; the cell nuclei have a ground-­glass or “cat’s eye” appearance and intracellular inclusions are common) (Fig. 78.2). PTC usually does not have a true capsule (invades surrounding normal tissue, invades blood and lymph channels). Papillae, invasion, or cytological features of PTC are required according to the 2017 WHO classification (fourth edition).6 After publication of the 2017 WHO classification, the identification of definite invasion, as well as the demonstration of well-­ developed nuclear features and true papillae, have become essential components in the diagnosis of PTC.6 These criteria have become stricter to reduce overdiagnosis of indolent tumors.2 Papillary cancer is often multicentric and bilateral.8,9 Lymphatic spread within the thyroid is the likely reason for the high frequency of multifocality of the tumor. Vascular invasion and distant metastases (most often to the lung and bone) are rare and account for 2% to 5% of cases.

Papillary Microcarcinomas. Papillary microcarcinomas (PMCs) are tumors less than or equal to 1 cm in diameter. In some series, PMC comprise more than half of the surgically treated patients.2,10 They may have features of a classic papillary carcinoma, or they may appear as unencapsulated sclerotic nodules of a few millimeters, infiltrating the surrounding thyroid. Most of these small tumors are not biologically significant, considering the much lower incidence of clinically recognized cancer. Less than 1% of patients with PMC have more aggressive disease identified by the presence of distant metastases at presentation.

Encapsulated Papillary Thyroid Cancer––Fully Encapsulated Classic Papillary Thyroid Cancer. This variant is classic PTC completely surrounded by a fibrous capsule that may be intact or only focally infiltrated by the carcinoma.6 The prognosis is excellent, with close to a 100% survival rate, even though regional lymph node metastases may be present.11

Follicular Variant Papillary Thyroid Cancer. Follicular variant papillary thyroid cancer (FVPTC) has the basic histologic structure of follicular cancers, but the characteristic nuclei of papillary cancers. These constitute the “follicular variant” of papillary cancer, and behave more or less as do other papillary cancers (low-­grade tumors).12 The variant was subclassified into encapsulated and nonencapsulated (infiltrative) variants. The encapsulated FVPTCs were further divided into invasive and noninvasive. The noninvasive encapsulated follicular variant of PTC was reclassified from carcinoma to noninvasive follicular thyroid neoplasm with papillary-­like nuclear features (NIFTP).13 Those tumors with definite invasion remain in the category of invasive encapsulated FVPTC. A new definitions of FVPTC, NIFTP, and well-­differentiated tumor (WDT)-­UMP were incorporated into the fourth edition of the WHO classification.

TABLE 78.2  Inheritance, Gene Involved, and Risks of Familiar Cancer Syndromes for Developing Thyroid Cancer Histological Type

Gene Mutation

Location

Incidence of Thyroid Cancer

Pathological Variant of PTC

PTC

APC tumor ­suppressor gene

5q21

2%–12%

Cribiform-­morular classical variant

PTEN tumor ­suppressor gene

10q23.2

>10%

Carney complex

FTC, PTC, C-­cell hyperplasia FTC, PTC

PRKAR1-­x

Werner syndrome

FTC, PTC, ATC

WRN gene

2p16 17q22-­24 Spl1-­pi 2

60% 4% 18%

Disease FAP and Gardner syndrome Cowden syndrome

PTC, Papillary thyroid cancer; FAP, familial adenomatous polyposis; APC, adenomatous polyposis; FTC, follicular thyroid cancer; PTEN, phosphate and tensin; PRKAR1-­x, protein kinase A regulatory subunit type 1-­alpha; ATC, anaplastic thyroid cancer; WRN, Werner. From Son EJ, Nosé V. Familial follicular cell-­derived thyroid carcinoma. Front Endocrinol (Lausanne). 2012;3:61.

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PART 6  Thyroid

Capsular/vascular invasion Present Present

Questionable/incomplete

Follicular variant PTC Well-differentiated carcinoma, NOS

PTC type nuclear features

Well-differentiated tumor of uncertain malignant potential (WDT-UMP)

Questionable/ incomplete Absent

Follicular carcinoma

Follicular tumor of uncertain malignant (FT-UMP)

Absent Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)

Follicular adenoma

Fig. 78.1  Red indicates malignant tumors, green for borderline tumors, and light blue for benign follicular adenoma.

Noninvasive variants of FVPTC and WDT-­UMP were reclassified as NIFTP. Questionable/incomplete capsular invasion of FVPTC was merged into WDT-­UMP (Fig. 78.1).14

than those for patients with PTC. Disease-­specific mortality rates at 5 years were also higher for TCVPTC, at 6.4% compared with 2.3% for patients with PTC (P < 0.001).24

Diffuse Sclerosing Variant Papillary Thyroid Cancer. This vari-

Columnar Cell Variant. Columnar cell variant is a rare subtype of

ant is rare and is estimated to account for up to 6% of all PTCs.15 The typical histopathological characteristics include classic nuclear features of PTC, diffuse stromal fibrosis, dense lymphoid infiltration, squamous metaplasia, and abundant psammoma bodies.16 Diffuse sclerosing variant papillary thyroid cancer (DSVPTC) is found most often in children and young adults. Tumors are often multifocal and have more extrathyroidal extension (ETE) compared with classical PTC. Extensive lymphocytic infiltration of the thyroid gland is often found, and lymph node metastasis is present in up to 100% of cases. Disease recurrence is also more common in patients with DSVPTC than in those with PTC.17 In a large multicenter study, the risk of recurrence was significantly higher in DSVPTC patients than for all other PTC patients (hazard ratio [HR] 8.5 [5.2; 13.9], P < 0.0001), but not when compared with high-­risk PTC (HR 1.1 [0.6; 2.2], P = 0.5). Overall, the cancer-­related death rate was lower in non-­DSVPTC patients than in DSVPTC patients (1.8% vs. 10%, P = 0.05).18

PTC accounting for 0.15% to 0.2% of all PTCs.25 It has tall-­cell morphology without the typical nuclear cell features of PTC.26 The prognosis is poor when it exhibits ETE, but if the tumor is encapsulated, it has indolent clinical behavior.27

Tall Cell Variant Papillary Thyroid Carcinoma. These tumors have a papillary pattern (similar nuclear features of PTC), and the cells are tall and columnar in shape, with a granular, eosinophilic cytoplasm.19 According to the WHO classification, tall cell variant papillary thyroid carcinoma (TCVPTC) is a PTC composed of cancer cells that are two to three times taller than they are wide. The most recent WHO classification also defined that tall cells must account for more than 30% of all tumor cells for a diagnosis of the tall-­cell variant (revised from the previous definition of more than 50% by the 2004 WHO classification).6 A review of the literature by Silver at al. found that the incidence of TCVPTC ranged between 5% and 11% of PTCs.20 Several studies have shown that TCVPTC tumors had more aggressive features with advanced tumor stage at diagnosis, including higher incidence of ETE, vascular invasion, larger primary tumor size, lymph node metastases, and older patient age.21-­22 Also, the prevalence of BRAF and TERT mutations in TCVPTC is significantly higher than that in classical PTC. In a study performed by Dettmer et al., 7% of TCVPTCs had a TERT promoter mutation, whereas 61% demonstrated a BRAF mutation.23 Patients had poorer prognosis, with higher overall mortality at 10 years (up to 22% for patients with TCVPTC, compared with 8.6% for PTC). A large population-­based study by Kazaure et al. found that rates of distant metastases in patients with TCVPTC were 2.6% higher

Cribiform-­Morular Variant. This variant can be sporadic, but more than half of cases occur in patients with familial adenomatous polyposis (FAP) syndrome and APC gene alteration.28 The American Thyroid Association (ATA) guidelines recommend that pathologists specifically note this variant, which could lead clinicians to consider genetic counseling referral.29 This variant usually develops multiple encapsulated nodules in the thyroid gland, and in general it has a better prognosis, with lower frequency of lymph node metastases, recurrence rates, and distant metastases.30 Hobnail Variant. This variant is a new entity first introduced in 2017 to the WHO classification of thyroid tumors.6 It is a rare subtype, with a frequency of 0.15% to 0.2% of all PTCs. It is a moderately differentiated PTC with aggressive behavior and poor prognosis.31 This variant consists of complex papillary structures covered by follicular cells that contain eosinophilic voluminous cytoplasm and large apical nuclei (“hobnails”). Hobnail variant PTC was defined with a cutoff of greater than 30%, as some hobnail features could be seen in classic PTC and DSVPTC.32

Solid/Trabecular Variant. This variant is diagnosed when all or nearly all of the tumor has a solid, trabecular or insular growth pattern with nuclear features of PTC.6 This variant was frequently seen in pediatric patients following the Chernobyl nuclear accident,33 and it was referred to as a nonaggressive variant in children. However, adults with this same variant have a slightly higher risk of recurrence and cause-­ specific mortality (10% at 10 years).34

FOLLICULAR THYROID CARCINOMA Follicular carcinoma is typically a solitary, encapsulated nodule in the thyroid. The distinction between follicular adenoma and follicular carcinoma depends on the presence of capsular/vascular invasion. The diagnosis of malignancy is based on the demonstration of unequivocal vascular invasion and/or capsular invasion and the lack of typical nuclear features of PTC. Examination of the full capsule of the tumor is

CHAPTER 78  Differentiated Thyroid Cancer – Streamlining Diagnosis And Optimizing Management necessary to exclude the presence of invasion, therefore FTC cannot be distinguished from benign adenoma on fine-­needle aspiration cytology only (Fig. 78.5). The major driver mutation is in the RAS gene (RAS-­like tumors).35 The 2017 WHO classification subdivided FTCs, depending on the degree of invasiveness, into three groups: (i) minimally invasive FTC (capsular invasion only), which represent more than 50% of cases, (ii) angioinvasive FTC, and (iii) widely invasive FTC (grossly invasive).6 This classification highlights the importance of vascular classification in risk stratification of thyroid carcinomas.36 FTC invades blood vessels but rarely invades lymphatics. Metastases are spread hematogeneously to the lungs, bones, and, less commonly, the brain and liver.37

HURTHLE CELL (ONCOCYTIC) TUMORS The 2017 WHO classification has described these tumors as a separate entity, given their unique biological and clinical features and different genetic profile from FTC.38 This entity includes Hurthle cell adenoma and Hurthle cell carcinoma (HCC). These tumors are composed of cells derived from the follicular epithelium and characterized by large size with abundant granular, eosinophilic cytoplasm resulting from the large number of mitochondria inside the cells. The cells have large nuclei and prominent nucleoli. Hurthle cells can be found in papillary carcinomas and in other benign conditions such as Hashimoto thyroiditis, benign nodules, and hyperthyroidism. A diagnosis of HCC is based on the presence of capsular and vascular invasion, similar to FTC.39

POORLY DIFFERENTIATED THYROID CARCINOMA In the new WHO classification, poorly differentiated thyroid carcinoma (PDTC) was more precisely defined with the Turin consensus criteria: (i) a diagnosis of carcinoma of follicular cell origin by conventional criteria (presence of invasion and/or metastasis); (ii) the presence of solid/trabecular/insular growth over more than 50% of the tumor area; (iii) the absence of PTC-­N (nuclear features); and (iv) at least one of the following three features: convoluted nuclei (dedifferentiated nuclear feature of PTC), mitotic activity more than three per 10 high-­power fields, or tumor necrosis.40 PDTC occupies an intermediate position between differentiated (PTC and FTC) and anaplastic thyroid cancer, both histologically and clinically. Patients usually present with advanced stage at diagnosis, low radioactive iodine avidity, and frequent metastatic spread.41 KEY POINTS  • The fourth edition of the World Health Organization (WHO) Classification of Tumors of Endocrine Organs was published in 2017 and listed 15 variants of papillary thyroid carcinoma (PTC). • PTC is often multicentric and bilateral. Lymphatic spread is common, while vascular invasion and distant metastases are rare. • The noninvasive encapsulated follicular variant of PTC was reclassified from carcinoma to noninvasive follicular thyroid neoplasm with papillary-­ like nuclear features, and is now considered a precancerous lesion. • The prevalence of BRAF and TERT mutations in tall cell variant PTC is significantly higher than that in classical PTC, conferring poorer prognosis with higher overall mortality. • The distinction between follicular adenoma and follicular carcinoma depends on the presence of capsular/vascular invasion. Examination of the full capsule of the tumor is necessary to exclude the presence of invasion; therefore, follicular thyroid carcinoma (FTC) cannot be distinguished from benign adenoma on fine-­ needle aspiration cytology only. The major driver mutation is in the RAS gene (RAS-­like tumors). FTC invades blood vessels but rarely invades lymphatics. • The 2017 WHO classification has described these tumors as a separate entity, given their unique biological and clinical features and different genetic profile from FTC.

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CAUSES Somatic Gene Alterations The main genes that play a role in cancer are oncogenes and tumor suppressor genes. Protooncogenes are genes that normally help cells grow when growth is needed. When a protooncogene mutates, whether by rearrangements, insertions/deletions, point mutations, or copy number alterations, it becomes an active oncogene and confers a growth advantage to the cell, eventually leading to malignant transformation. Another possible cause is the loss of a tumor suppressor gene. In this case, the malignant transformation occurs when these genes are inactivated. Thyroid carcinoma is a genetically simple neoplasm with a low number of genetic alterations. It is one of better-­characterized malignancies where more than 90% of cases have a driver mutation or gene fusion identified.42 In well-­differentiated thyroid cancer, driver gene mutations are generally mutually exclusive (one per tumor). Less welldifferentiated thyroid cancers acquire additional genetic alterations as late events.43 The most common initiating alterations in thyroid cancer are BRAF and RAS point mutations and RET/PTC, NTRK, and PAX8/ PPARg chromosomal rearrangements, while TP53 inactivation, TERT promoter mutations, and alterations in the PI3 kinase (PI3K) pathway are later events. A comprehensive investigation of the genomic landscape of PTCs, the most common thyroid malignancy, was reported by The Cancer Genome Atlas Network (TCGA Network). Investigators of this group interrogated nearly 500 PTCs comprising mostly well-­differentiated, low-­to intermediate-­risk cases.44 These well-­differentiated tumors were found to have a low frequency of somatic alterations, with the majority harboring mutually exclusive activating mutations in BRAF (60%) and RAS-­family genes (13%), as well as fusion oncoproteins, primarily involving receptor tyrosine kinases (RTKs) such as RET (6%), NTRK1 or NTRK3 (2%), and ALK (1%). Distinct signaling and transcriptomic consequences were observed between BRAFV600E-­like tumors, which showed higher mitogen-­activated protein kinase (MAPK) transcriptional output and lower expression of genes involved in iodine metabolism, and RAS-­like tumors, which had lower MAPK signaling and comparatively preserved expression of iodine-­related genes. BRAF-­like tumors demonstrate a papillary growth pattern (conventional and tall cell variants), and have BRAFV600E mutations and rearrangement of the BRAF, RET, and MET genes. RAS-­like tumors exhibit a follicular growth pattern with a distinct tumor capsule in more than 80% of cases, and have mutations in the RAS, EIF1AX, and PTEN genes, and rearrangement of the PPARG, FGFR2, and THADA genes. TERT promoter mutations at low frequency (10–20 ng/mL on LT4 therapy) with negative RAI imaging, as diagnostic of metastatic disease and also as a prognostic tool. Patients with a negative 131I-­WBS and positive FDG-­PET have a more aggressive and less differentiated disease, with worse prognosis.109 CT scan of chest without intravenous (IV) contrast (imaging pulmonary parenchyma) or with IV contrast (to include the mediastinum)

TABLE 78.5  Ongoing Risk Stratification––

Modified from 2015 American Thyroid Association Guidelines

Biochemical Structural Excellent Indeterminate Incomplete Incomplete TSH target Serum Tg Neck US Stim Tg WBS Cross-­ sectional imaging

0.5–2 Yearly 5 years No No No

0.1–0.5 Yearly 1–3 years Maybe Maybe No

10 ng/mL) and negative neck and chest imaging.29 In Table 78.5, we summarize the use of ongoing risk stratification, modified from 2015 ATA guidelines.29

Treatment of Metastatic Disease

Distant Metastases. Distant metastases are observed in approximately 10% of DTC patients, and half of them are detected at presentation. They are usually located in the lungs (50%), bones (25%), or lungs and bones (20%), or at other sites (5%). Distant metastases at diagnosis confer an increase in morbidity and mortality in patients with PTC or FTC, but individual prognosis depends on several factors. In a study of patients with lung metastases from DTC who were observed for 40 years, young age at diagnosis and 131I uptake by metastases were the most important factors positively affecting survival time. RAI could also lead to longer survival time or complete recovery.110 Another retrospective study showed that age of 45 years or more, symptoms, site other than lung only or bone only, and no RAI for the metastasis were predictors of poor outcome, with 13%, 11%, 16%, and 12% 10-­year disease-­specific survival, respectively.112 Improved survival in patients with distant metastases is also associated with responsiveness to directed therapy (external beam radiation therapy, thermal ablation, surgery, etc.) and/or RAI.113

Surgery. After primary surgery, recurrences in the neck may develop in the thyroid bed and in surrounding soft tissues or in the regional lymph nodes. Any clinically detectable local biopsy-­proven persistent or recurrent disease should be treated by surgery if possible, although reoperations involving central neck dissection are difficult and increase the risk of complications to the parathyroid glands and recurrent laryngeal nerve. Recurrent disease in the lateral cervical nodes is easier to treat surgically, because the operative field has not been dissected previously. The preferred surgical procedure is a modified radical neck

CHAPTER 78  Differentiated Thyroid Cancer – Streamlining Diagnosis And Optimizing Management dissection.114 Most studies suggest that surgery results in a high clearance rate of structural disease in over 80% of patients. Lung metastases that do not concentrate radioactive iodine could be treated surgically in very selected cases, such as a single macronodular lesion or more than one in the same lobe. Bone metastasis surgery is generally palliative, as required in pathologic fractures or to ameliorate neurologic symptoms resulting from spinal cord compression by vertebral metastases.

Radioactive Iodine. RAI could be used for regional nodal metastases discovered on diagnostic WBS, and could be employed in patients with low-­volume disease or in combination with surgery. Significant variation exists in regard to RAI use, irrespective of the degree of disease or risk of recurrence. Micronodular diffuse lung metastases shown by WBS in the absence of radiographic changes have the greatest chance of remission when treated with RAI.115 This is important in children, who often have a diffuse pattern of metastatic pulmonary spread and do exceptionally well with RAI. Adult patients should be treated with RAI repeatedly every 6 to 12 months as long as disease continues to concentrate RAI and respond clinically; a cumulative administered activity of 600 mCi 131I is a limit in many patients. Children should be treated at longer intervals, 24 to 36 months if possible, and lower cumulative administered activities. In adult patients, the treatment dose is usually 100 to 200 mCi (or 100–150 mCi for patients ≥70 years old). Lower doses (approximately 1 mCi/kg body weight) should be used in children with lung metastases, particularly of the diffuse type, to avoid the risk for radiation-­induced pulmonary fibrosis.116 Radioiodine-­avid lung macronodules may benefit from RAI, but the ability to achieve and excellent response is very low. RAI of iodine-­ avid bone metastases has been associated with improved survival and should be employed, although RAI is rarely curative.117 The treatment dose can be given empirically (100–200 mCi) or determined by dosimetry. Radioactive-­iodine refractory thyroid cancer is defined in four ways: (i) the malignant metastatic tissue does not ever concentrate RAI (no uptake outside the thyroid bed at the first therapeutic WBS), (ii) the tumor tissue loses the ability to concentrate RAI after previous evidence of RAI-­avid disease (in the absence of stable iodine contamination), (iii) RAI is concentrated in some lesions but not in others; and (iv) metastatic disease progresses despite significant concentration of RAI.118 When a patient with DTC is classified as refractory to RAI, there is generally no indication for further RAI treatment. Directed Therapy. Several local treatment modalities to induce tumor control, other than surgery, may be used to treat distant metastatic disease in the brain, lung, liver, and bone lesions from thyroid carcinoma. The most commonly used techniques include surgery, thermal ablation (radiofrequency ablation or cryotherapy), stereotactic body radiation therapy, or intensity-­modulated radiotherapy. The main principle of these techniques is to selectively treat the lesion, especially when patients are symptomatic or at risk for complications, be minimally invasive, and be well tolerated with few side effects. Systemic Therapy. In 80% of PTCs, constitutive activation of the MAPK signaling pathway is the initiating event in the carcinogenesis of PTC. This includes RET/PTC rearrangement and point mutations in RAS and BRAF, with no overlap between these mutations in primary tumors. The PI3K pathway may also be activated in a few papillary and follicular cancers. Acquisition of additional mutations

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and gene amplifications that activate this pathway may be a common event in PDTCs. Angiogenesis is another potential molecular target for therapy. Various vascular endothelial growth factors (VEGF) and VEGF receptors (VEGFR-­1 [FLT1] and VEGFR-­2 [KDR]), as well as receptors for the fibroblast growth factor and for the platelet-­derived growth factor, are often overexpressed in the vascular endothelium of thyroid cancer tissues, and they also trigger the MAPK signaling pathway. Molecules that block kinase activity at distal steps in the MAPK pathway have been identified as drugs for refractory thyroid cancer. These tyrosine kinase inhibitors are multikinase inhibitors that share the ability to inhibit VEGF receptor and other receptor tyrosine kinases. Kinase inhibitor therapy should be considered in RAI-­refractory DTC patients with metastatic, rapidly progressive, symptomatic, and/or imminently threatening disease not otherwise amenable to local control using other approaches.29 Benefit has been demonstrated in the form of improved progression-­free survival (delay in time to disease progression or death) in three randomized, double-­blinded, placebo-­ controlled clinical trials of vandetanib,119 sorafenib,120 and lenvatinib.121 On this basis, sorafenib and lenvatinib were approved for use in the United States and the European Union for patients with advanced RAI-­refractory DTC. To date, no clinical trial has demonstrated an overall survival advantage or improved quality of life from use of any therapy in RAI-­refractory DTC.120,121 Advances in understanding the genomic and functional alterations contributing to the pathogenesis of thyroid cancers have opened new therapeutic opportunities and are beginning to improve patient outcomes. In addition to the multiple kinase inhibitor drug approvals for each subtype of thyroid cancer, there are several US Food and Drug Administration indications that are mutation-­ specific: dabrafenib/trametinib for BRAF-­mutated anaplastic thyroid cancer, larotrectinib and entrectinib for NTRK-­fusion thyroid cancer, and selpercatinib and pralsetinib for RET-­mutated thyroid cancer. Furthermore, other mutation-­specific drugs, immunotherapies, and novel strategies for advanced thyroid cancer are under investigation122 (Table 78.6). KEY POINTS  • Preoperative evaluation and completeness of surgical resection are important determinants of outcome, because residual metastatic lymph nodes represent the most common site of disease persistence/recurrence. • In properly selected lower-­risk patients, clinical outcomes are very similar following unilateral or bilateral thyroid surgery. • Radioactive iodine therapy (RAI) adjuvant therapy is indicated in most high-­ risk patients, given significant improvement in overall and disease-­specific mortality as well as disease-­free survival. In a majority of low-­risk patients, RAI ablation is not recommended, as its administration has no additional benefit after complete surgical resection. In patients at intermediate risk, RAI adjuvant therapy may be considered, but the decision must be individualized. • The large majority of recurrences are detected in the first 5 years after diagnosis. • Patients with a negative 131I–whole-­body scan and a positive (18F)-­ fluorodeoxyglucose–positron emission tomography have more aggressive and less differentiated disease, with a worse prognosis. • Advances in understanding the genomic and functional alterations contributing to the pathogenesis of thyroid cancers have opened new therapeutic opportunities and are beginning to improve patient outcomes. These include multiple kinase inhibitor drugs for each subtype of thyroid cancer, as well as the newest therapies, which are mutation-­specific.

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PART 6  Thyroid

TABLE 78.6  Molecular Markers for

­Therapeutic of Advanced Thyroid Cancer Genetic Alteration

Tumor Type

BRAFV600E

PTC

HRAS

PTC, FTC, PDTC

PAXS/PPARG ALK fusions NTRK1/2/3 fusions RET

FTC PTC, PDTC PTC, PDTC PTC

Available Targeted Therapeutics Vemurafenib, dabrafenib + trametinib Farnesyltransferase inhibitor tipifamib Pioglitazone Crizotinib, cerjtinib Entrectinib, larotrectinib Vandetanib, cabozantinib, selpercatinib, pralsetinib

*Approved by the US Food and Drug Administration. PTC, Papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma. Modified from Cabanillas ME, Ryder M, Jimenez C. Targeted therapy for advanced thyroid cancer: kinase inhibitors and beyond. Endocr Rev. 2019;40:1573–1604.

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18. Chereau N, Giudicelli X, Pattou F, et al. Diffuse sclerosing variant of papillary thyroid carcinoma is associated with aggressive histopathological features and a poor outcome: results of a large multicentric study. J Clin Endocrinol Metab. 2016;101:4603–4610. 19. Ghossein RA, Leboeuf L, Patel KN, et al. Tall cell variant of papillary thyroid carcinoma without extrathyroid extension: biologic behavior and clinical implications. Thyroid. 2007;17:655–661. 20. Silver C, Owen R, Rodrigo J, et al. Aggressive variants of papillary thyroid carcinoma. Head Neck. 2011;7:1052–1059. 21. Ito Y, Hirokawa M, Fukushima M, et al. Prevalence and prognostic significance of poor differentiation and tall cell variant in papillary carcinoma in Japan. World J Surg. 2008;32:1535–1543. 22. Morris LG, Shaha AR, Tuttle RM, et al. Tall-­cell variant of papillary thyroid carcinoma: a matched-­pair analysis of survival. Thyroid. 2010;20:153–158. 23. Dettmer MS, Schmitt A, Steinert H, et al. Tall cell papillary thyroid carcinoma: new diagnostic criteria and mutations in BRAF and TERT. Endocr Relat Cancer. 2015;22:419–429. 24. Kazaure HS, Roman SA, Sosa JA. Aggressive variants of papillary thyroid cancer: incidence, characteristics and predictors of survival among 43,738 patients. Ann Surg Oncol. 2012;19:1874–1880. 25. Sywak M, Pasieka JL, Ogilvie T. A review of thyroid cancer with intermediate differentiation. J Surg Oncol. 2004;86:44–54. 26. Bongiovanni M, Mermod M, Canberk S, et al. Columnar cell variant of papillary thyroid carcinoma: cytomorphological characteristics of 11 cases with histological correlation and literature review. Cancer Cytopathol. 2017;125:389–397. 27. Song E, Jeon MJ, Oh HS, et al. Do aggressive variants of papillary thyroid carcinoma have worse clinical outcome than classic papillary thyroid carcinoma? Eur J Endocrinol. 2018;179:135–142. 28. Uchino S, Ishikawa H, Miyauchi A, et al. Age-­and gender-specific risk of thyroid cancer in patients with familial adenomatous polyposis. J Clin Endocrinol Metab. 2016;101:4611–4617. 29. Haugen BR, Alexander EK, Bible KC, et al. 2015 American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American Thyroid Association Guidelines Task Force on thyroid nodules and differentiated thyroid cancer. Thyroid. 2016;26:1–133. 30. Lam AK, Saremi N. Cribriform-­morular variant of papillary thyroid carcinoma: a distinctive type of thyroid cancer. Endocr Relat Cancer. 2017;24:R109–R121. 31. Asioli S, Erickson LA, Righi A, et al. Papillary thyroid carcinoma with hobnail features: histopathologic criteria to predict aggressive behavior. Hum Pathol. 2013;44:320–328. 32. Liu Z, Kakudo K, Bai Y, et al. Loss of cellular polarity/cohesiveness in the invasive front of papillary thyroid carcinoma, a novel predictor for lymph node metastasis; possible morphological indicator of epithelial mesenchymal transition. J Clin Pathol. 2011;64:325–329. 33. Thomas GA, Bunnell H, Cook HA, et al. High prevalence of RET/PTC rearrangements in Ukrainian and Belarussian post Chernobyl thyroid papillary carcinomas: a strong correlation between RET/PTC3 and the solid-­follicular variant. J Clin Endocrinol Metab. 1999;84:4232–4238. 34. Nikiforov YE, Erickson LA, Nikiforova MN, et al. Solid variant of papillary thyroid carcinoma: incidence, clinical-­pathologic characteristics, molecular analysis, and biologic behavior. Am J Surg Pathol. 2001;25:1478–1484. 35. Pozdeyev N, Gay LM, Sokol ES, et al. Genetic analysis of 779 advanced differentiated and anaplastic thyroid cancers. Clin Cancer Res. 2018;24:3059–3068. 36. Ito Y, Hirokawa M, Masuoka H, et al. Prognostic factors of minimally invasive follicular thyroid carcinoma: extensive vascular invasion significantly affects patient prognosis. Endocr J. 2013;60:637–640. 37. Hugen N, Sloot YJE, Netea-­Maier RT, et al. Divergent metastatic patterns between subtypes of thyroid carcinoma results from the nationwide Dutch pathology registry. JCEM. 2020;105:e299–e306. 38. Ganly I, Ricarte Filho J, Eng S, et al. Genomic dissection of Hurthle cell carcinoma reveals a unique class of thyroid € malignancy. J Clin Endocrinol Metab. 2013;98:E962–E972.

CHAPTER 78  Differentiated Thyroid Cancer – Streamlining Diagnosis And Optimizing Management 39. Erickson LA, Jin L, Goellner JR, et al. Pathologic features, proliferative activity, and cyclin D1 expression in Hurthle cell neoplasms of the thyroid. Mod Pathol. 2000;13:186–192. 40. Volante M, Collini P, Nikiforov YE, et al. Poorly differentiated thyroid carcinoma: the Turin proposal for the use of uniform diagnostic criteria and algorithmic diagnostic approach. Am J Surg Pathol. 2007;31:1256– 1264. 41. de la Fouchardiere C, Decaussin-­Petrucci M, Berthiller J, et al. Predictive factors of outcome in poorly differentiated thyroid carcinomas. Eur J Cancer. 2018;92:40–47. 42. Agrawal N, Akbani R, Aksoy BA, et al. For the cancer genome atlas research network. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014;159:676–690. 43. Landa I, Ibrahimpasic T, Boucai L, et al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest. 2016;126:1052–1066. 44. Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014;159:676–690. 45. Xing M. Clinical utility of RAS mutations in thyroid cancer: a blurred picture now emerging clearer. BMC Med. 2016;14:12. 46. French CA, Alexander EK, Cibas ES, et al. Genetic and biological subgroups of low-­stage follicular thyroid cancer. Am J Pathol. 2003;162:1053–1060. 47. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med. 2011;135:569–577. 48. Tallini G, Santoro M, Helie M, et al. RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes. Clin Cancer Res. 1998;4:287–294. 49. Zhu Z, Ciampi R, Nikiforova MN, et al. Prevalence of RET/PTC rearrangements in thyroid papillary carcinomas: effects of the detection methods and genetic heterogeneity. J Clin Endocrinol Metab. 2006;91:3603–3610. 50. Wood WM, Sharma V, Bauerle KT, et al. PPARγ promotes growth and invasion of thyroid cancer cells. PPAR Res. 2011;2011:171765. 51. Musholt TJ, Musholt PB, Khaladj N, et al. Prognostic significance of RET and NTRK1 rearrangements in sporadic papillary thyroid carcinoma. Surgery. 2000;128:984–993. 52. Pozdeyev N, Gay LM, Sokol ES, et al. Genetic analysis of 779 advanced differentiated and anaplastic thyroid cancers. Clin Cancer Res. 2018;24:3059–3068. 53. Landa I, Ganly I, Chan TA, et al. Frequent somatic TERT promoter mutations in thyroid cancer: higher prevalence in advanced forms of the disease. J Clin Endocrinol Metab. 2013;98:E1562–E1566. 54. Vuong HG, Altibi AM, Duong UN, et al. Role of molecular markers to predict distant metastasis in papillary thyroid carcinoma: promising value of TERT promoter mutations and insignificant role of BRAF mutations-­a meta-­analysis. Tumour Biol. 2017;39:1010428317713913. 55. Moon S, Song YS, Kim YA, et al. Effects of coexistent BRAFV600E and TERT promoter mutations on poor clinical outcomes in papillary thyroid cancer: a meta-­analysis. Thyroid. 2017;27:651–660. 56. Liu R, Zhang T, Zhu G, et al. Regulation of mutant TERT by BRAF V600E/MAP kinase pathway through FOS/GABP in human cancer. Nat Commun. 2018;9:579. 57. Ganly I, Makarov V, Deraje S, et al. Integrated genomic analysis of Hürthle cell cancer reveals oncogenic drivers, recurrent mitochondrial mutations, and unique chromosomal landscapes. Cancer Cell. 2018;34:256–270.e5. 58. Charkes ND. On the prevalence of familial nonmedullary thyroid cancer in multiply affected kindreds. Thyroid. 2006;16:181–186. Erratum in: Thyroid. 2006;16:520. 59. Vriens MR, Suh I, Moses W, et al. Clinical features and genetic predisposition to hereditary nonmedullary thyroid cancer. Thyroid. 2009;19:1343–1349. 60. Son EJ, Nosé V. Familial follicular cell-­derived thyroid carcinoma. Front Endocrinol (Lausanne). 2012;3:61.

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61. Zambrano E, Holm I, Glickman J, et al. Abnormal distribution and hyperplasia of thyroid C-­cells in PTEN-­associated diseases. Endocr Pathol. 2004;15:55–64. 62. Ishikawa Y, Sugano H, Matsumoto T, et al. Unusual features of thyroid carcinomas in Japanese patients with Werner syndrome and possible genotype-­phenotype relations to cell type and race. Cancer Sci. 1999;85:1345–1352. 63. Ron E, Lubin JH, Shore RE, et al. Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiat Res. 1995;141:259–277. 64. Nikiforov YE. Is ionizing radiation responsible for the increasing incidence of thyroid cancer? Cancer. 2010;116:1626–1628. 65. Rabes HM, Demidchik EP, Sidorow JD, et al. Pattern of radiation-­ induced RET and NTRK1 rearrangements in 191 post-­chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications. Clin Cancer Res. 2000;6:1093–1103. 66. Ciampi R, Knauf JA, Kerler R, et al. Oncogenic AKAP9-­BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. J Clin Invest. 2005;115:94–101. 67. Holm LE, Hall P, Wiklund K, et al. Cancer risk after iodine-­131 therapy for hyperthyroidism. J Natl Cancer Inst. 1991;83:1072–1077. 68. Kitahara CM, Preston DL, Sosa JA, et al. Association of radioactive iodine, antithyroid drug, and surgical treatments with solid cancer mortality in patients with hyperthyroidism. JAMA Netw Open. 2020;3:e209660. 69. Davies L, Morris LG, Haymart M, et al. American Association of Clinical Endocrinologists and American College of Endocrinology disease state clinical review: the increasing incidence of thyroid cancer. Endocr Pract. 2015;21:686–696. 70. Demers LM, Spencer CA. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13:45–56. 71. Slough CM, Randolph GW. Workup of well-­differentiated thyroid carcinoma. Cancer Control. 2006;99–105. 72. Fundakowski CE, Hales NW, Agrawal N, et al. Surgical management of the recurrent laryngeal nerve in thyroidectomy: American Head and Neck Society Consensus Statement. Head Neck. 2018;40:663–675. 73. Sinclair CF, Bumpous JM, Haugen BR, et al. Laryngeal examination in thyroid and parathyroid surgery: an American Head and Neck Society consensus statement: AHNS Consensus Statement. Head Neck. 2016;38:811–819. 74. Randolph GW, Duh QY, Heller KS, et al. The prognostic significance of nodal metastases from papillary thyroid carcinoma can be stratified based on the size and number of metastatic lymph nodes, as well as the presence of extranodal extension. Thyroid. 2012;22:1144–1152. 75. Durante C, Haddy N, Baudin E, et al. Long-­term outcome of 444 patients with distant metastases from papillary and follicular thyroid carcinoma: benefits and limits of radioiodine therapy. J Clin Endocrinol Metab. 2006;91:2892–2899. 76. Grigsby PW, Gal-­or A, Michalski JM, et al. Childhood and adolescent thyroid carcinoma. Cancer. 2002;95:724–729. 77. Sebastian SO, Gonzalez JMR, Paricio PP, et al. Papillary thyroid carcinoma: prognostic index for survival including the histological variety. Arch Surg. 2000;135:272–277. 78. https://www.cancer.org/cancer.html#. 79. Shaha AR, Loree TR, Shah JP. Prognostic factors and risk group analysis in follicular carcinoma of the thyroid. Surgery. 1995;118:1131–1138. 80. Ruegemer JJ, Hay ID, Bergstralh EJ, et al. Distant metastases in differentiated thyroid carcinoma: a multivariate analysis of prognostic variables. J Clin Endocrinol Metab. 1988;67:501–508. 81. Tuttle M, Morris LF, Haugen B, et al. Thyroid-­differentiated and anaplastic carcinoma. In: Amin MB, Edge SB, Greene F, et al., eds. AJCC Cancer Staging Manual. 8th ed. New York: Springer International Publishing; 2017. 82. Tuttle RM, Haugen B, Perrier ND. Updated American Joint Committee on cancer/tumor-­node-­metastasis staging system for differentiated and anaplastic thyroid cancer (eighth edition): what changed and why? Thyroid. 2017;27:751–756.

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83. American Thyroid Association (ATA). Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009;19:1167–1214. Erratum in: Thyroid. 2010;20:942. Erratum in: Thyroid. 2010;20:674–675. 84. Tuttle RM, Tala H, Shah J, et al. Estimating risk of recurrence in differentiated thyroid cancer after total thyroidectomy and radioactive iodine remnant ablation: using response to therapy variables to modify the initial risk estimates predicted by the new American Thyroid Association staging system. Thyroid. 2010;20:1341–1349. 85. Vaisman F, Tala H, Grewal R, et al. In differentiated thyroid cancer, an incomplete structural response to therapy is associated with significantly worse clinical outcomes than only an incomplete thyroglobulin response. Thyroid. 2011;21:1317–1322. 86. Momesso DP, Vaisman F, Yang SP, et al. Dynamic risk stratification in patients with differentiated thyroid cancer treated without radioactive iodine. J Clin Endocrinol Metab. 2016;101:2692–2700. 87. Momesso DP, Tuttle RM. Update on differentiated thyroid cancer staging. Endocrinol Metab Clin North Am. 2014;43:401–421. 88. Elisei R, Molinaro E, Agate L, et al. Are the clinical and pathological features of differentiated thyroid carcinoma really changed over the last 35 years? Study on 4187 patients from a single Italian institution to answer this question. JCEM. 2010;95:1516–1527. 89. Jonklaas J, Nogueras-­Gonzalez G, Munsell M, et al. The impact of age and gender on papillary thyroid cancer survival. J Clin Endocrinol Metab. 2012;97:E878–E887. 90. Morris LG, Shaha AR, Tuttle RM, et al. Tall-­cell variant of papillary thyroid carcinoma: a matched-­pair analysis of survival. Thyroid. 2010;20:153–158. 91. Malandrino P, Russo M, Regalbuto C, et al. Outcome of the diffuse sclerosing variant of papillary thyroid cancer: a meta-­analysis. Thyroid. 2016;26:1285–1292. 92. Rivera M, Tuttle RM, Patel S, et al. Encapsulated papillary thyroid carcinoma: a clinico-­pathologic study of 106 cases with emphasis on its morphologic subtypes (histologic growth pattern). Thyroid. 2009;19:119–127. 93. Zhou X, Zheng Z, Chen C, et al. Clinical characteristics and prognostic factors of Hurthle cell carcinoma: a population based study. BMC Cancer. 2020;20:407. 94. Youngwirth LM, Adam MA, Scheri RP, et al. Extrathyroidal extension is associated with compromised survival in patients with thyroid cancer. Thyroid. 2017;27:626–631. 95. Podnos YD, Smith D, Wagman LD, et al. The implication of lymph node metastasis on survival in patients with well-­differentiated thyroid cancer. Am Surg. 2005;71:731–734. 96. Adam MA, Pura J, Goffredo P, et al. Presence and number of lymph node metastases are associated with compromised survival for patients younger than age 45 years with papillary thyroid cancer. J Clin Oncol. 2015;33:2370–2375. 97. Randolph GW, Duh QY, Heller KS, et al. The prognostic significance of nodal metastases from papillary thyroid carcinoma can be stratified based on the size and number of metastatic lymph nodes, as well as the presence of extranodal extension. Thyroid. 2012;22:1144–1152. 98. Adam MA, Pura J, Gu L, et al. Extent of surgery for papillary thyroid cancer is not associated with survival: an analysis of 61,775 patients. Ann Surg. 2014;260:601–605;discussion 605–607. 99. Matsuzu K, Sugino K, Masudo K, et al. Thyroid lobectomy for papillary thyroid cancer: long-­term follow-­up study of 1,088 cases. World J Surg. 2014;38:68–79. 100. Barney BM, Hitchcock YJ, Sharma P, et al. Overall and cause-­specific survival for patients undergoing lobectomy, near-­total, or total thyroidectomy for differentiated thyroid cancer. Head Neck. 2011;33:645–649. 101. Nixon IJ, Ganly I, Patel SG, et al. Thyroid lobectomy for treatment of well differentiated intrathyroid malignancy. Surgery. 2012;151:571–579. 102. Mendelsohn AH, Elashoff DA, Abemayor E, et al. Surgery for papillary thyroid carcinoma: is lobectomy enough? Arch Otolaryngol Head Neck Surg. 2010;136:1055–1061.

103. Haigh PI, Urbach DR, Rotstein LE. Extent of thyroidectomy is not a major determinant of survival in low-­or high-­risk papillary thyroid cancer. Ann Surg Oncol. 2005;12:81–89. 104. Schlumberger M, Catargi B, Borget I, et al. Strategies of radioiodine ablation in patients with low-­risk thyroid cancer. N Engl J Med. 2012;366:1663–1673. 105. Mallick U, Harmer C, Hackshaw A. The HiLo trial: a multicentre randomised trial of high-­versus low-­dose radioiodine, with or without recombinant human thyroid stimulating hormone, for remnant ablation after surgery for differentiated thyroid cancer. Clin Oncol (R Coll Radiol). 2008;20:325–326. 106. Cooper DS, Specker B, Ho M, et al. Thyrotropin suppression and disease progression in patients with differentiated thyroid cancer: results from the National Thyroid Cancer Treatment Cooperative Registry. Thyroid. 1998;8:737–744. 107. Pujol P, Daures JP, Nsakala N, et al. Degree of thyrotropin suppression as a prognostic determinant in differentiated thyroid cancer. J Clin Endocrinol Metab. 1996;81:4318–4323. 108. Malandrino P, Latina A, Marescalco S, et al. Risk-­adapted management of differentiated thyroid cancer assessed by a sensitive measurement of basal serum thyroglobulin. J Clin Endocrinol Metab. 2011;96: 1703–1709. 109. Robbins RJ, Wan Q, Grewal RK, et al. Real-­time prognosis for metastatic thyroid carcinoma based on 2-­[18F]fluoro-­2-­deoxy-­D-­glucose-­positron emission tomography scanning. J Clin Endocrinol Metab. 2006;91: 498–505. 110. Eskander A, Merdad M, Freeman JL, et al. Pattern of spread to the lateral neck in metastatic well-­differentiated thyroid cancer: a systematic review and meta-­analysis. Thyroid. 2013;23:583–592. 111. Shoup M, Stojadinovic A, Nissan A, et al. Prognostic indicators of outcomes in patients with distant metastases from differentiated thyroid carcinoma. J Am Coll Surg. 2003;197:191–197. 112. Zettinig G, Fueger BJ, Passler C, et al. Long-­term follow-­up of patients with bone metastases from differentiated thyroid carcinoma -­-­surgery or conventional therapy? Clin Endocrinol (Oxf). 2002;56:377–382. 113. Shah MD, Hall FT, Eski SJ, et al. Clinical course of thyroid carcinoma after neck dissection. Laryngoscope. 2003;113:2102–2107. 114. Steward DL. Update in utility of secondary node dissection for papillary thyroid cancer. J Clin Endocrinol Metab. 2012;97:3393–3398. 115. Ronga G, Filesi M, Montesano T, et al. Lung metastases from differentiated thyroid carcinoma. A 40 years’ experience. Q J Nucl Med Mol Imaging. 2004;48:12–19. 116. Hebestreit H, Biko J, Drozd V, et al. Pulmonary fibrosis in youth treated with radioiodine for juvenile thyroid cancer and lung metastases after Chernobyl. Eur J Nucl Med Mol Imaging. 2011;38:1683–1690. 117. Bernier MO, Leenhardt L, Hoang C, et al. Survival and therapeutic modalities in patients with bone metastases of differentiated thyroid carcinomas. J Clin Endocrinol Metab. 2001;86:1568–1573. 118. Schlumberger M, Brose M, Elisei R, et al. Definition and management of radioactive iodine-­refractory differentiated thyroid cancer. Lancet Diabetes Endocrinol. 2014;2:356–358. 119. Leboulleux S, Bastholt L, Krause T, et al. Vandetanib in locally advanced or metastatic differentiated thyroid cancer: a randomised, double-­blind, phase 2 trial. Lancet Oncol. 2012;13:897–905. 120. Brose MS, Nutting CM, Jarzab B, et al. Sorafenib in radioactive iodine-­ refractory, locally advanced or metastatic differentiated thyroid cancer: a randomised, double-­blind, phase 3 trial. Lancet. 2014;384:319–328. 121. Schlumberger M, Tahara M, Wirth LJ, et al. Lenvatinib versus placebo in radioiodine-­refractory thyroid cancer. N Engl J Med. 2015;372: 621–630. 122. Cabanillas ME, Ryder M, Jimenez C. Targeted therapy for advanced thyroid cancer: kinase inhibitors and beyond. Endocr Rev. 2019;40: 1573–1604.

79 Medullary Thyroid Cancer Rossella Elisei, Cristina Romei, and Antonio Matrone

OUTLINE Introduction, 1315 Clinical Presentation And Diagnosis, 1315 Sporadic Medullary Thyroid Cancer, 1315 Hereditary Forms (Multiple Endocrine Neoplasia Type II), 1317 Gene Carriers, 1318 Pathology And Pathogenesis, 1319 Histology, 1319 Genetics, 1319

Initial Therapy, 1320 Postsurgical Follow-­Up, 1321 Follow-­Up of Cured Medullary Thyroid Cancer Patients, 1321 Follow-­Up of Patients with Biochemical Persistent Disease, 1321 Follow-­Up of Patients with Structural Disease, 1322 Other Therapies, 1322 Local Treatment of Recurrent or Persistent Neck Disease, 1322 Conclusions, 1324

 

INTRODUCTION Two different types of cells are present in the thyroid gland: the follicular cells that secrete the thyroid hormones thyroxine (T4) and triiodothyronine (T3), and the parafollicular C cells that produce calcitonin (Ct). It is well recognized that these two thyroid cell types have unique embryonic origins, although details of their differentiation and development during organogenesis remain somewhat controversial.1,2 Parafollicular C cells represent only 2% to 4% of the thyroid cells, which explains the low frequency of medullary thyroid carcinoma (MTC) among all thyroid cancer types.3 However, tumors arising from the two cell types are clearly distinct entities, with different treatment and prognosis. MTC is the unique well-­differentiated thyroid tumor deriving from C cells and belongs to the group of neuroendocrine tumors. In contrast, several histotypes, such as papillary, follicular, insular, poorly differentiated, and anaplastic thyroid cancer, originate from follicular epithelial cells.4 Because of the absence of a dedicated registry, the incidence of MTC is not defined. Its prevalence is estimated at only around 5% to 10% of all thyroid cancers and 0.4% to 1.4% of all thyroid nodules, and it is found in less than 1% of thyroids at autopsy.5 Unlike papillary and follicular carcinomas, which are highly prevalent in females, no difference in gender distribution has been reported in MTC. The clinical appearance is mainly in the fourth and fifth decades, but there is a wide range of ages at onset.6 No risk factors, whether environmental or genetic, for the development of MTC have been identified, although associations with prior thyroid diseases and other unrelated disorders such as hypertension, allergies, and gallbladder disease have been reported in a metanalysis of epidemiological studies.7 In 1993, the pathogenesis of MTC was reported to be activation, mainly due to point mutations or small insertion/deletions, of the RET protooncogene.8,9 However, it remains unclear what causes RET activation, which in turn sets in motion the development of MTC. RET activation can be responsible for both the sporadic form, which accounts for approximately 75% of cases, and the hereditary or familial form of

MTC, which accounts for the remaining 25%. In sporadic cases the RET alteration is present only in the tumor tissue, at the somatic level, while the RET protooncogene alteration is transmitted in an autosomal dominant manner at the germline level.10 The biological behavior of MTC is much less favorable when compared with that of the other well-­differentiated thyroid carcinomas, although it is not as unfavorable as that of anaplastic carcinoma.11 While in older series a 10-­year survival of approximately 50% was frequently reported for patients with MTC,12 most recent series, including ours, show better overall survival, with 80% to 85% of patients surviving longer than 10 years, likely thanks to improvements in both diagnostic procedures and care (Fig. 79.1). Survival is dependent on several factors, such as age at diagnosis,13 but the stage at diagnosis remains the most relevant clinical prognostic factor for survival in these patients. When an early diagnosis is made, and the tumor is still intrathyroidal, 90% of patients can survive up to 35 years.12,14 KEY POINTS • MTC is a rare endocrine tumor that can be either sporadic (75%) or familial (25%). Males and females are equally affected. Children are affected only in the familial forms. No risk factors are known. An early diagnosis is desirable, since only intrathyroidal MTC can be completely cured with surgery.

CLINICAL PRESENTATION AND DIAGNOSIS Sporadic Medullary Thyroid Cancer The most common clinical presentation of sporadic MTC is a thyroid nodule, either as a single nodule or inside a multinodular goiter. Only in very advanced cases, diarrhea and/or flushing syndrome, due to high levels of serum Ct, can represent the first symptoms.15 In 5% to 10% of very advanced cases, symptoms due to ectopic adrenocorticotropic hormone (ACTH) production and a consequent paraneoplastic Cushing syndrome with severe hypercortisolism can be present.16 The simultaneous presence of a (multi)nodular thyroid and a lump in one of the laterocervical regions of the neck may lead the clinician to suspect a thyroid malignancy, but not specifically MTC.

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TABLE 79.1  Procedures for Calcium

Pisa’s series MTC patients survival (%)

100

Stimulation Test for Serum Calcitonin in Patients with Mildly Elevated Values (Between 20 and 100 pg/mL)

87 80 60

Steps: 1) 2)

40 20

3)

0

200 250 100 120 150 Time (months) Fig. 79.1  Survival curve of 538 medullary thyroid cancer (MTC) patients followed from 2000–2018 at the University Hospital of Pisa, Italy. In this relatively recent series the survival rate at 10 years from diagnosis was 87%. 0

50

A classical work-­ up for thyroid nodular disease is then performed.17 Neck ultrasound (US) is the first imaging to be performed to describe thyroid nodule features. MTC nodules are usually solid and hypoechoic, sometimes with microcalcifications. A few studies have clearly demonstrated that US can only identify some malignant features and suggest a general risk of malignancy without any specificity for MTC.18,19 Thyroid scintigraphy, either with 131I or Tc99, is commonly performed only if there is a large multinodular goiter, to map the entire thyroid and the several nodules, or if the thyrotropin-­ stimulating hormone level is low, suggesting hyperfunction of the gland/nodule. If performed, MTC appears as a cold nodule at neck scintigraphy. Fine-­needle aspiration cytology (FNAC) is the gold standard procedure for determining whether thyroid nodules are benign or malignant. In a typical MTC cytological smear, cells are usually isolated, with shape varying from oval to round, large polygonal or spindled. Cytoplasm may be abundant or scant and usually contains acidophilic granulation visible with specific stains (May-­ Grunwald-­ Giemsa). Nuclei are frequently multiple (i.e., two or three), preferentially round, and eccentrically localized.17 Amyloid is frequently detectable as clumps of amorphous material and revealed by Red Congo staining20: when present, it is highly suggestive of MTC. Immunocytochemistry for Ct and/or chromogranin, another neuropeptide secreted by MTC, is not routinely performed, but can be carried out if there is suspicion for MTC.21,22 Despite this apparently typical MTC cytological pattern, several series have shown a high rate of failure in making a cytological presurgical diagnosis.23,24 Missed presurgical diagnosis of MTC can lead to incorrect initial surgical treatment, which in principle should be total thyroidectomy and central neck node dissection. Recently, a multicentric international study involving 12 different referral centers across seven different continents and four nations showed that only 46% of 245 MTC cases received a correct cytological diagnosis of MTC, demonstrating that this is a worldwide pathology issue.25 Among other explanations, FNAC-­negative results might be due to the fact that MTC could be present in a nodule not subjected to FNAC, especially when multinodular goiter is the clinical diagnosis. In these cases, serum Ct measurement is more reliable, because this hormone is elevated even in the presence of MTC microfoci.23,26 However, there is some controversy in the different guidelines regarding routine measurement of serum Ct in all patients with thyroid nodule(s). The British Thyroid Association27 and the National Comprehensive Cancer Network28 share the view that serum Ct should be measured any time a presurgical diagnosis of MTC is suspected due to positive FNAC,

4) 5)

NOTES

Patient must be weighed (do not trust what the patient says) 2.5 mg of calcium element (or 25 mg of calcium gluconate) per 1 kg or 2.2 lbs of weight should be taken in a syringe The volume of calcium collected in step 2 must be diluted up to 50 mL with standard saline solution The intravenous infusion of the 50 mL containing calcium must be rapidly administered (in 5 minutes) Blood samples must be taken at baseline before the infusion and then 2, 5, 15 minutes after the complete infusion of the 50 mL containing calcium for calcitonin measurements Be careful to collect samples for calcitonin measurement from an intravenous access other than that used for the infusion, or aspirate at least 10 mL of liquid to be drained and then aspirate the next sample for calcitonin measurement to avoid dilution of the sample due to the liquid present in the drip tube.

familial history, or the simultaneous presence of other endocrine neoplasia. The American Thyroid Association guidelines for the management of thyroid nodules29 do not recommend for or against routine serum Ct testing. In contrast, the European Thyroid Association (ETA) consensus30 clearly states that this procedure should be applied in the work-­up of all thyroid nodules, because it is more sensitive than FNAC. In 2010, a new document signed by the American Association of Clinical Endocrinologists, the Associazione Medici Endocrinologici, and the ETA was published, declaring that routine serum Ct may be useful, especially before surgery, and strongly recommending Ct measurement in certain high-­risk groups.31 The major concerns about the routine measurement of serum Ct in all patients with thyroid nodules are related to both the possible “false-­ positive” cases and the “cost-­benefit” analysis. There is a real risk that mildly elevated serum Ct, between 20 and 100 pg/mL, could be due to C cell hyperplasia (CCH) or other neuroendocrine tumors, or even to assay artifact rather than authentic MTC.32,33 However, serum Ct values in this “grey zone” do raise suspicion for MTC, even though they are not diagnostic, and other diagnostic tools should be used to rule out this diagnostic doubt. Subjects with mildly elevated basal serum Ct could undergo a calcium stimulation test34,35 following a well-­defined procedure, as reported in Table 79.1. The degree of Ct increase can distinguish different clinical scenarios, because only serum Ct produced by C cells will significantly increase after stimulation, while serum Ct due to artifacts, such as heterophilic antibodies, or produced by other neuroendocrine tumors does not respond to the stimulus.36,37 Another useful diagnostic tool to apply when serum Ct is mildly elevated is measuring Ct in the washout fluid from the needle used for FNAC.38 This approach is of diagnostic utility to ascertain the nature not only of the thyroid nodule, if MTC is suspected, but also of neck lymph nodes, especially before thyroidectomy, to plan the surgical approach or the most appropriate therapeutic strategy. Moreover, in the presence of mildly elevated serum Ct, the pathologist can be alerted and asked to perform immunocytochemistry for Ct, which, as previously mentioned, is not a routine procedure. Although the routine measurement of serum Ct in all subjects with thyroid nodules is still controversial,39 there is evidence that this approach allows early diagnosis and treatment, thus significantly improving the outcome of this potentially lethal disease.40

CHAPTER 79  Medullary Thyroid Cancer

KEY POINTS • Both sporadic and familial MTC commonly appears as a thyroid nodule; the cytological diagnosis of MTC is feasible in only 50% of cases. The measurement of serum calcitonin is recommended in the presence of a thyroid nodule. Mildly elevated serum calcitonin is not diagnostic of MTC but should raise the suspicion and induce the use of other diagnostic tools.

MTC, like many other neuroendocrine tumors, can produce other peptides than Ct, such as chromogranin, somatostatin, gastrin-­ releasing peptide, vasoactive intestinal peptide, neuron-­ specific enolase, serotonin, prostaglandins, and other neuroendocrine substances, but none of them are useful for presurgical diagnosis.41 When elevated, they may all contribute to flushing and diarrhea syndrome.42 Serum carcinoembryonic antigen (CEA) is usually elevated when the disease is diffuse and distant metastases are present.43 In the few cases of dedifferentiated MTC that are unable to secrete Ct, CEA becomes the serum marker of the disease.44 Nevertheless, it is more useful in monitoring disease progression because its level increases when the disease becomes rapidly progressive. Because at least 5% to 7% of apparently sporadic cases of MTC are indeed hereditary cases,45,46 preoperative evaluation of both the adrenal and parathyroid morphology and function should always be performed. The familial history should also be carefully considered, with particular regard to the occurrence of pheochromocytoma (PHEO) and hyperparathyroidism (HyperPTH) in other family members. Nowadays, all subjects with MTC, independently ascertained to be sporadic or familial, must undergo RET genetic screening, which can be easily performed in a genetic laboratory by analyzing the sequence of RET gene in DNA extracted from the patient’s blood or saliva.10 KEY POINTS • A  ll MTC patients must undergo the RET genetic screening, since about 8% of apparently sporadic cases are indeed hereditary. All first-­degree relatives of a MTC patient carrying a germline RET mutation should be submitted to the RET genetic screening.

A. C.

Hereditary Forms (Multiple Endocrine Neoplasia Type II) In approximately 25% of cases, MTC is one of the components of multiple endocrine neoplasia type II (MEN II), which is an autosomal dominant inherited syndrome with a variable degree of expressivity and an age-­related penetrance. Three different hereditary syndromes can be distinguished, depending on the involved glands (Fig. 79.2): 1) multiple endocrine neoplasia type IIA (MEN IIA), a syndrome consisting of MTC, PHEO, and HyperPTH due to parathyroid multiple adenomatosis/hyperplasia47; 2) multiple endocrine neoplasia type IIB (MEN IIB), a syndrome consisting of MTC, PHEO, mucosal neuromas, ganglioneuromatosis, marfanoid features, skeletal alterations, and megacolon48; and 3) familial MTC (FMTC), which is characterized by the presence of heritable MTC with no apparent association with other endocrine neoplasia.49 After the introduction of the RET genetic screening, the relative prevalence of FMTC syndrome was found to be much higher than expected (accounting for 10%–50% of all MEN syndromes). The increase in FMTC is mainly due to the high number of apparently sporadic MTCs demonstrated to be familial cases by RET mutation analysis.46 The clinical course of MTC varies considerably in the three syndromes. It is very aggressive and almost invariably unfavorable in MEN IIB, with affected patients rarely surviving past adolescence.48 In contrast, MTC is almost indolent in most patients with the FMTC form, and shows variable degrees of aggressiveness in patients with MEN IIA. Different types of RET gene mutations account for different biological behavior, and separate therapeutic protocols have been defined for the treatment of MTC occurring in the three different syndromes.50 Between 10% and 30% of patients with MEN IIA develop HyperPTH during the third or fourth decade of life. The clinical findings are superimposable on those of the sporadic form of HyperPTH, and very often no specific symptoms are noted. Unlike the sporadic form, multiple hyperplasia or adenomatosis of parathyroid glands is most commonly found.47,51 HyperPTH has only occasionally been reported in patients with MEN IIB.52 Very recently, a peculiar case of simultaneous MEN IIA and IIB clinical features has been described.53 Approximately 50% of MEN IIA and 40% to 45% of MEN IIB patients develop PHEO, which shares the same characteristics in both syndromes. Contrary to the sporadic form of PHEO, the adrenal Parathyroids

B. D. F.

E.

Adrenal glands

G.

Anterior

Pinhole

Multiple adenomatosis and/or hyperplasia

H.

(30% of MEN IIA) (anedoctically in MEN IIB) I.

Thyroid C Cells

J.

MTC Pheochromocytoma (50% of MEN IIA) (40% of MEN IIB)

1317

CCH

Medullary Thyroid Cancer and C Cells Hyperplasia

(100% of both MEN II, any type)

Fig. 79.2  Different endocrine glands’ involvement in the three different phenotypes of multiple endocrine neoplasia type II (MEN II).

1318

PART 6  Thyroid

tumors seen in MEN syndromes are usually bilateral and multicentric. However, the two adrenal glands are rarely simultaneously involved, and a mean period of 10 years usually elapses between the development of the tumor in the two glands. MEN IIB is characterized by association with mucosal neuromas, which are mainly located on the distal tongue and subconjunctival areas, and ganglioneuromatosis affecting the gastrointestinal tract, which is responsible for the very commonly seen symptom of megacolon. MEN IIB patients should be easily recognized at physical examination by the typical marfanoid habitus characterized by thin and inappropriately long extremities and pectus excavatum.48 Thick lips and thick eyelids are frequently observed in the presence of mucosal neuromas and are usually clearly evident when the eyes and mouth are explored. Gastrointestinal disorders due to intestinal neuromas throughout the intestinal tract, including obstructive symptoms, cramping, and diarrhea, are frequently observed in early childhood. However, despite these very pathognomonic features, the diagnosis is usually late, when MTC has already spread to lung and/or liver.54 An association with cutaneous lichen amyloidosis (CLA), a characteristic pigmented and itchy skin lesion specifically localized in the interscapular region of the back, has been reported in less than 10% of MEN IIA families.55 The development of CLA may precede the development of MTC. For this reason, when present it is almost invariably diagnostic of MEN IIA and may be considered a predictor of the syndrome. Four specific RET mutations in exon 10 (i.e., C620, C618, C611, and C609), collectively referred to as “Janus,” are responsible for both MEN IIA and Hirschsprung disease. This latter syndrome is commonly due to RET mutations located in the first exons of the gene that inactivate the gene. Historically these mutations were believed to be able to act simultaneously as both gain-­of-­function mutations, responsible for the MEN IIA phenotype, and loss-­of-­function mutations, responsible for the intestinal aganglionosis.56 Recently, however, it has been demonstrated that RET overstimulation alone is enough to cause both phenotypes.57 The hereditary nature of MTC may be suspected based on a positive family history (other members already affected), or based on the presence of other endocrine neoplasia (PHEO and/or HyperPTH) or other disorders (neuromas, marfanoid features, CLA). However, at least in the Pisa series,10 index cases of 40% of all hereditary MTC kindreds presented as apparently sporadic cases, and their hereditary origin was only discovered through RET screening. The clinical appearance of MTC in MEN II syndromes is that of a thyroid nodular disease, similar to that of the sporadic form, with the exception that it is usually bilateral and multicentric and is almost invariably associated with CCH.58 Evaluation of the thyroid nodule in the hereditary form follows the same work-­up as that recommended for sporadic cases, but the hereditary forms require mandatory simultaneous examination of adrenal and parathyroid glands. With a few exceptions,59 development and diagnosis of PHEO usually follow development and diagnosis of MTC. Symptoms of PHEO are not specific and may be confused with those caused by anxiety. Hypertension is rare, especially at the beginning of the disease. The first alteration of catecholamine production is represented by an elevation of the daily urinary excretion of epinephrine, while norepinephrine usually increases only later. Nowadays the measurement of urinary metanephrines, the O-­methylated metabolites of catecholamines, is used for the early diagnosis of PHEO, because they are much more stable than catecholamines.60 Tests for metanephrines are more specific and sensitive than those for catecholamines: while normal concentrations of metanephrines exclude the diagnosis of PHEO, normal concentrations of catecholamines do not.61 Once the biochemical suspicion of PHEO has arisen, an abdomen US with or without computed tomography (CT)

and/or magnetic resonance imaging (MRI) may be useful for localization of the adrenal mass. If there is no demonstrable adrenal mass by CT or MRI, patients should undergo functional imaging, typically positron emission tomography (PET), which can be performed with different tracers (i.e., (18F)-­fluorodeoxyglucose, 68Ga-­DOTATATE, or (18F)-­dihydroxyphenylalanine [DOPA]).62 Parathyroid glands may also be involved in hereditary MTC, particularly in MEN IIA. Both adenomas and hyperplasia may be associated with an increase in parathyroid hormone (PTH) secretion, resulting in hypercalcemia and hypercalciuria in more advanced cases.63 The earliest serum abnormality detected is a moderately elevated level of serum PTH, with normal-­high levels of serum calcium levels. A deficit of vitamin D could also be responsible for PTH increase, and for this reason its level should always be verified to exclude secondary HyperPTH due to vitamin D deficit.64 Once the MTC case has been proven to be hereditary, based on the presence of a germline RET mutation, all first-­degree relatives should undergo testing to search for the same genetic alteration.65 This procedure is fundamental for the early discovery of gene carriers who should undergo clinical and biochemical evaluation immediately after discovery of the mutation in their DNA.

Gene Carriers By definition, gene carriers are family members of an index patient with hereditary MTC that are potentially already affected or may develop MTC in the future. The diagnosis is performed through RET screening of all first-­degree relatives of the index case. The mutational analysis can be limited to the RET mutation found in the index case: informed consent must be provided for the genetic analysis, and in the case of children either both parents or guardians should sign the consent form and agree that they will inform the child at a more appropriate age about their positive or negative status as a gene carrier.65 All first-­degree adult relatives should undergo RET genetic screening as soon as possible, while the timing of testing children can vary based on the type of RET mutation: as soon as possible if the mutation is the M918T mutation typical of MEN IIB, before 5 years of age if the mutation involves a cysteine residue, particularly at codon 634, and even later in cases with any other type of non-­cysteine RET mutations.50 Once the gene carrier has been identified, the diagnostic procedure involves a thyroid neck US and a serum Ct measurement. Some family members will already be unknowingly affected and must be immediately treated, while others will still be negative for both parameters. The timing of total thyroidectomy in these latter cases should be personalized, taking into account the type of RET mutation and also the levels of serum Ct.50,66 In the 1990s, soon after the discovery of the pathogenic relationship between the presence of a germline RET mutation and the development of MTC, prophylactic thyroidectomy was suggested for all gene carriers of any age.67 Over the years, increasing knowledge of the aggressiveness of different mutations and the awareness that serum Ct highly correlates with the presence of MTC led to rethinking of this approach and the development of a more relaxed algorithm for treatment of these subjects, who can safely undergo early, rather than prophylactic, thyroidectomy.65 Annual evaluation of basal serum Ct and a neck US should be performed, taking into account that only microMTC and/or CCH, with no evidence of metastatic lymph nodes, have been reported for serum Ct levels less than 30 to 40 pg/mL in large series of gene carriers.66,68 In this regard, it is worth noting that central neck compartment dissection can be avoided in gene carriers with serum Ct les than 30 to 40 pg/mL who undergo thyroidectomy, especially children, thus reducing the risk of postsurgical hypoparathyroidism.50

CHAPTER 79  Medullary Thyroid Cancer

1319

B1

A

B2

Fig. 79.3 Macroscopic (A) and microscopic (B1–2) histology of medullary thyroid cancer. A, The tumor appears as a solid and compact, red mass. B1, Hematoxylin-­eosin microscopic appearance of medullary thyroid cancer. B2, The same section after calcitonin immunohistochemistry.

KEY POINTS • Prophylactic thyroidectomy are not mandatory in gene carriers with no evidence of MTC, especially in children. Serum calcitonin and the type of RET mutation should guide the timing of thyroidectomy.

PATHOLOGY AND PATHOGENESIS Histology The macroscopic histological appearance of MTC is that of a solid and compact, red or calcareous-­white nodule (Fig. 79.3A). Microscopically it is pleiomorphic, with spindle-­shaped or rounded cells typically organized in a nested pattern (Fig. 79.3B1–2). Nuclei are uniform, mitoses are rare, and the cytoplasm is characterized by the presence of secretory granules. Amyloid substance can be present among MTC cells, and when present, it allows the diagnosis. Sometimes MTC can be erroneously diagnosed as anaplastic thyroid carcinoma, Huerthle cell carcinoma, or insular differentiated thyroid carcinoma, especially if pseudopapillary aspects or giant cells are present. Positive immunohistochemistry for Ct, and also for chromogranin A and CEA, is undoubtedly diagnostic of MTC.69 The histological description of MTC should include the number and distribution of tumoral foci. CCH, either diffuse or focal, should also be reported because, although it is the histological hallmark of the hereditary forms, it is also present in approximately 13% of sporadic cases.70 With the exception of the prevalence of CCH, no other major histological differences have been described between the sporadic and the hereditary forms of MTC.

Genetics In 1994, after discovering the transforming role of the RET oncogene, the correlation between the presence of a germline activating mutation of RET and the hereditary form of MTC was demonstrated in almost 500 kindreds affected by MEN II or FMTC.71 In the same study, somatic RET mutations were reported in approximately 40% of sporadic MTCs. Thereafter, both in vitro and in vivo studies confirmed the driver role of RET mutations in the development of MTC.72,73 Germline RET point mutations are mainly localized in exons 10 to 11 and 13 to 16, but other rare mutations have been reported in

other exons, such as 5 and 8, and all of these exons must be analyzed as part of the genetic screening procedure.65 A significant genotype–phenotype correlation has been reported over the years, with some RET mutations being almost exclusively associated with a specific MEN II syndrome.74 With a few exceptions, known as “variants of unknown significance,”75 the penetrance of RET mutations is complete, in that all gene carriers will develop the disease sooner or later. In contrast, the level of expressivity is different, in that MTC due to different RET mutations can have different degree of aggressiveness based on the transforming ability, which is the highest for the M918T mutation.75 According to these differences, the RET mutations have been distinguished in three risk levels (i.e., moderate, high, highest) of aggressiveness, which should be taken into account to identify the right time for the surgical treatment of RET gene carriers.76 Somatic RET mutations are mainly concentrated in exon 16, and have been reported in other exons in only a few cases. Moreover, RET somatic mutation prevalence is significantly higher in larger MTC tumors than in smaller MTC tumors,77 and is particularly high in advanced and progressive MTC.78 Although not yet introduced into routine clinical practice, it would be valuable to know if a patient with sporadic MTC is carrying a RET somatic mutation, as mutated cases have a worse prognosis79; thus, a more aggressive therapeutic strategy or a more stringent follow-­up could be provided for these patients. Moreover, new RET-­targeted therapies are under development and require the characterization of the molecular profile of the MTC.80,81 RAS oncogene somatic mutations, in particular in H-­RAS and K-­ RAS, are present in approximately 10% to 20% of cases of sporadic MTC, and they are mutually exclusive with RET mutations.82 No other driver mutations have been discovered in RET-­ and RAS-­negative MTCs, even when tissues were analyzed with next-­generation sequencing techniques.83,84 These studies did identify very rare mutations, but their prevalence was so low that they are considered “private” mutations that are likely relevant only in that specific case.85 A similar finding has been reported for hereditary cases: the ESR2 gene, which encodes the beta subunit of the estrogen receptor, has been found to be mutated in two individuals with FMTC but not in their unaffected relatives. However, the same ESR2 mutation was not found in other

1320

PART 6  Thyroid presurgical Ct serum level, independent of the results from the neck US.88 In this regard it is worth noting that, unfortunately, when the MTC has metastasized to neck lymph node, especially to the laterocervical ones, it is very difficult to achieve definitive surgical and biochemical cure of the disease.89 Measurement of serum CEA is also indicated in the preoperative phase, because elevated levels are strongly suggestive of advanced disease. Cases with advanced local disease demonstrated by neck US and associated with elevated serum CEA levels should be studied with computerized tomography (CT) scan to better evaluate the local extension of the tumor and to verify the relationship of the disease with the gross veins, trachea, and esophagus and plan the most appropriate surgical treatment.50

RET-­negative FMTC kindreds, thus suggesting that it was a “private mutation” of the first family analyzed.86 The next challenge is to identify the driver mutations in both RET-­negative familial cases and RET-­ and RAS-­negative sporadic cases. KEY POINTS • With a few exceptions, hereditary MTC are positive for a germline RET mutation. Almost 50% of sporadic MTC are positive for RET mutation at somatic level. Another 30% are positive for H-­ or K-­RAS somatic mutations. The remaining 20% of sporadic cases are negative for any other known oncogene.

INITIAL THERAPY

KEY POINTS • The initial therapy consisted of at least total thyroidectomy and lymph node dissection of the central neck compartment. The lymphadenectomy of the latero-cervical compartments should not be prophylactically performed but only when metastatic latero-cervical metastatic lymphnodes are present at the time of the diagnosis or when presurgical serum calcitonin is highly elevated.

Total thyroidectomy and central node compartment dissection is the minimal surgical treatment, even in intrathyroidal cases. The need for total thyroidectomy is related to the multicentricity and bilaterality of the MTC, which occur in 17% and 5.6%, respectively, of cases with the sporadic form.87 Moreover, CCH, which is a preneoplastic lesion, is almost invariably associated with the hereditary form of MTC (75%) and, to a lesser extent, with the sporadic form (13%).70 An additional factor in favor of total thyroidectomy is the fact that, as mentioned above, 5% to 7% of apparently sporadic cases are in fact hereditary forms and almost invariably have bilateral disease.45,46 Total thyroidectomy and central neck dissection will be extended to the laterocervical compartment(s) if metastatic lymph nodes are recognized in these regions. For this reason, an accurate neck US should be performed in order to identify suspicious metastatic lymph nodes to be subjected to FNAC, as well as measurement of Ct in the washout fluid from the needle used.38 However, not all authors agree with this strategy, and some prefer to plan extension of surgery based on the

Surgical removal of the primary tumor and neck lymph nodes metastases is also recommended in cases with distant metastases already present at diagnosis.50 In these latter cases, and in cases with tumor invasion of the vital structures of the neck, a multidisciplinary meeting must be held before surgery to plan for additional therapies.90 Before surgery, an evaluation of the adrenal gland function to exclude the presence of a PHEO must always be performed, except in genetically documented sporadic MTC. The reason for this procedure is in the need to perform the PHEO excision before the MTC surgery, in cases of the simultaneous presence of both tumors, to avoid unexpected hypertensive crisis during the MTC surgery.

A

B

C

Cured: still risk of recurrence (udetectable basal Ct: 10%) (undetectable stimulated Ct: 3%)

Biochemical disease (detectable basal Ct with no evidence of structural disease)

Structural disease: Evidence of disease at neck US and/or CT scan and/or F18 dopa PET or other

Clinical, biochemical and neck US evaluation Every 12-18 months

Basal CT scan and/or F18 dopa PET: if negative continue with clinical, biochemical and neck US evaluation once a year

Calculation of 6 months DT of serum Ct and CEA to plan the timing of other imagings

Recurrences: Start with other imagings*

If Ct stable: Continue annual evaluations If Ct increasing: Start with other imagings*

If DT < 6 months: repeat imaging every 6 months to intercept the progression of the disease and start with a therapy

* imagings are usually negative until serum Ct < 150 pg/ml Ct: calcitonin; CEA: carcino embryonic antigen; CT: computerized tomography; PET: positron emission tomography; US: ultrasound; DT: doubling time Fig. 79.4  Algorithm for follow-­up of medullary thyroid cancer (MTC) patients after initial surgical treatment based on the results from the first evaluation. A, Follow-­up of patients cured of MTC whose risk of recurrence varies from 3% to 10%. B, Follow-­up of MTC patients with persistent biochemical disease. C, Follow-­up of MTC patients with persistent structural disease.

CHAPTER 79  Medullary Thyroid Cancer

1321

14.25 mm 23.19 mm

15.04 mm

22.63 mm

A1

A2

32.91 mm

53.53 mm

B1

B2 Fig. 79.5  Two cases of medullary thyroid cancer metastatic disease treated with vandetanib (A) and cabozantinib (B). A, Liver metastatic lesions (arrows) before vandetanib treatment (A1) and after 6 months of therapy (A2). B, Right metastatic hilar lymph node (arrows) before cabozantinib treatment (B1) and after 2 months of therapy (B2). In both cases, significant shrinkage of the lesions is visible, and their devascularization is demonstrated by the significantly lower density of the lesions after the treatments.

POSTSURGICAL FOLLOW-­UP The first evaluation after surgery should be performed 3 months after the surgical treatment, including physical examination, neck US, and measurement of serum free T3 (FT3), free T4 (FT4), thyroid-­ stimulating hormone (TSH), Ct, and CEA. Measurement of FT3, FT4, and TSH is requested for monitoring levothyroxine replacement therapy. Serum Ct and CEA measurement and neck US are necessary for the follow-­up of MTC. Due to the relatively long half-­lives, if performed too early, serum values of Ct may be misleading, especially if a high serum concentration was present preoperatively.91 According to the results of this first evaluation after the initial therapy, MTC patients may either be classified as cured or still affected by either biochemical disease or structural disease. The timing and the type of the following evaluations depend highly on the results from this first evaluation (Fig. 79.4).

Follow-­Up of Cured Medullary Thyroid Cancer Patients Cured patients have a postsurgical Ct less than 10 pg/mL, a negative neck US, and low-­normal CEA values. These patients have a 10% risk of recurrence during follow-­up that becomes 3% in those patients who have both postsurgical basal and peak Ct after calcium stimulation of less than 10 pg/mL.92 Patients with a negative stimulation

test should be reevaluated every 12 to 18 months, with the aim of early interception of rare, but possible, recurrence.50 In patients with undetectable levels of serum Ct, measurement of CEA is not necessary (Fig. 79.3A). KEY POINTS • After the initial treatment patients can be cured or persistently affected. In the latter case they can be either biochemically or structurally affected. Serum calcitonin and carcinoembryonic antigen (CEA) doubling times are fundamental for planning the type and timing of the imaging techniques to be performed for following the development of the disease.

Follow-­Up of Patients with Biochemical Persistent Disease Patients with biochemical persistent disease are those who show a postsurgical basal Ct greater than 10 pg/mL in the absence of any structural disease as assessed by neck US, CT scan, and, whenever possible, DOPA PET/CT. These patients should be monitored with periodical measurements of serum Ct and CEA (every 6–12 months), because increasing levels are strongly suggestive of progressive disease.43,93 In most cases, the challenge is to find the source of production of Ct and CEA. This is particularly true in MTC cases with serum Ct less than

1322

PART 6  Thyroid

150 pg/mL, which are frequently negative on any type of imaging.94 An accurate neck US is the first localization technique to be performed, due to the high frequency of local recurrence and cervical node metastases. Total-­body CT scan and bone scintigraphy are also suggested in the follow-­up of a MTC patient with detectable and increasing values of serum Ct. Other functional imaging, and in particular DOPA PET/ CT, may be useful for identifying micrometastases not detectable with other techniques.95 Because a detectable serum Ct level is compatible with long-­term survival, during which Ct and CEA may remain stable with time or slowly increase, the most widely accepted therapeutic strategy in these patients is that of “wait and see,” thus postponing any type of therapy until structural disease is detected96 (Fig. 79.4B).

Follow-­Up of Patients with Structural Disease Approximately 10% of MTC patients already have distant metastases at diagnosis that will not be cured by thyroid surgery. Another 10% of those with initial biochemical disease will sooner or later develop local or distant metastases. These patients must be followed up with 6-­month evaluations of serum Ct and CEA and calculation of their doubling time. A rapid increase in serum Ct and/or CEA is a poor prognostic factor both for recurrence and death, especially if the doubling time is less than 0.5 to 1 year.97 A poor prognosis for survival has been recently demonstrated for serum carbohydrate antigen 19.9, particularly in advanced MTC.98 In this event, imaging evaluations must be intensified to verify the progression of the disease and to decide if it is time to start active therapy.99

OTHER THERAPIES Patients with metastatic disease can be treated with other therapies, both for symptomatic reasons and/or for the progression of the disease. In this latter case the progression should be calculated according to the Response Evaluation Criteria in Solid Tumors (RECIST)100: if progression of the disease is documented according to these criteria, therapeutic intervention should be started. However, if progression is present but with a slow growth rate, the indication is to wait and see. If the progression is of a single lesion or of multiple lesions in the same organ, local therapy can be applied, while if the progression is of several lesions and in different organs, systemic therapy should be initiated.101

Local Treatment of Recurrent or Persistent Neck Disease

Surgery. The regional lymph nodes of the neck and mediastinum are the most frequent sites of persistent or recurrent disease. In these cases, a second surgical treatment with curative intent is desirable, and to this purpose an extensive modified neck dissection involving microdissection of all node-­bearing compartments is recommended. Although only less than 10% of patients affected by MTC with extrathyroidal invasion can be cured by a second surgical treatment, cervical reexploration is safe, and in selected patients may limit MTC progression.102 A second surgery on the neck could be also indicated for palliative, other than curative, intent, for example in patients with compressive symptoms who can benefit from surgical debulking.103 Similarly, a second surgical treatment might be performed for symptomatic lesions or when their growth may cause significant morbidity, as may happen for lymph nodes of the mediastinum adjacent to the great vessels, tracheoesophageal groove, carotid sheath, and brachial plexus.

External Radiotherapy. External radiation therapy (ERT) after thyroidectomy and node dissection is not generally recommended on a prophylactic basis in MTC patients, and the procedure should be reserved for patients with local aggressive disease that is either recurrent or persistent after the first surgery. Although MTC has very low sensitivity to ERT, there is evidence of a potential benefit from

TABLE 79.2  Possible Types of Local

Treatments to Be Considered in Advanced Cases of Medullary Thyroid Cancer Before Starting Systemic Therapy and to be Chosen on the Basis of Both the Localization of the Growing Lesion(s) and Local Expertise Example of Growing Lesions Type of Local Treatment Isolated growing lesions in the lung, Surgery or thermal ablation: the latter bone, liver can be performed by radiofrequency ablation, laser ablation, or cryoablation, according to local experience Vegetating tumor tissue inside Endotracheal/bronchial laser removal trachea or bronchus Single, small brain metastases Radiosurgery or stereotaxic ablation Multiple growing brain metastases Whole-­brain irradiation for stabilization Painful bone metastases Antalgic external radiotherapy Growing neck and/or mediastinal Palliative external radiotherapy lymphnode metastases not more treatable with surgery Multiple liver metastases Intraarterial chemotherapy or intraarterial radioembolization

radiotherapy in terms of a lower risk (from 2-­to 4-­fold) of further progression in patients with residual disease.104 In patients who have undergone a less aggressive primary resection, ERT should be postponed to a second surgical treatment.

Radiofrequency and Laser Thermal Ablation. While the safety and efficacy of both laser ablation and radiofrequency thermal ablation (RFA) have been widely demonstrated in the treatment of benign thyroid nodules, evidence regarding local tumor control of thyroid malignancies is still limited. However, preliminary results are encouraging, and image-­ guided thermal ablation techniques can be considered a valid alternative to surgery for the treatment for recurrent thyroid cancers. Thermal ablation is based on the use of electromagnetic waves or lasers that produce a high degree of heat. A needle-­like RFA probe is introduced in the tumoral mass, and the radiofrequency waves passing through the probe increase the temperature within tumor tissue, resulting in destruction of the tumoral tissue.105,106 Similarly, when the laser light hits the target, a local increase in temperature occurs, causing permanent damage such as coagulative necrosis and tissue carbonization/vaporization. Both techniques are performed under US guidance, which allows real-­time monitoring of the procedure. Thermal ablation is of particular utility when the lesion to be treated is the only site of disease or the only one, among others, that is growing, and surgical treatment cannot be performed, as frequently happens in local para-­or retrotracheal recurrence of MTC. Treatment of Distant Metastases. Local treatments, as described earlier, are also indicated for single, growing, distant metastatic lesions or, if multiple, when only one of them represents a clinical problem, such as pain, local compression of other organs, risk of fracture, or risk of blood invasion. The possibility of local treatment should be always considered before starting systemic therapy, which should be reserved for cases with multiple metastatic lesions that involve multiple organs and are actively growing.107 As shown in Table 79.2, local treatments can vary according with the site and the number of the metastatic lesions, and decision-­making requires the involvement of a multidisciplinary team.

CHAPTER 79  Medullary Thyroid Cancer

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TABLE 79.3  More Frequent Adverse Events Reported in Clinical Trials of the Currently

Available Tyrosine Kinase Inhibitors Used for the Treatment of Advanced and Progressive Multimetastatic Medullary Thyroid Cancer Adverse Events (All Grade)#

Vandetanib (%)

Cabozantinib (%)

Selpercatinib (%)

Pralsetinib (%)

Hypertension Diarrhea Skin rash* Anorexia Fatigue Nausea Weight loss QT prolongation Hand-­foot syndrome Increased SGOT Increased SGPT

32 56 45 21 24 33 10 14 NR NR NR

33 63 19 46 41 43 48 NR 50 86 86

43 38 NR^ NR 38 35 NR 19 NR 57 51

40 34 24 15 38 17 NR NR NR 69 43

#For

selpercatinib and pralsetinib, the majority of adverse events were grade 1 or 2. *Rash includes dermatitis, dermatitis acneiform, eczema, palmar-­plantar, erythrodysaesthesia syndrome, erythematous rash, macular rash, maculopapular rash, papular rash, and pustular rash. NR, Not reported; SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate-­pyruvate transaminase.

KEY POINTS • Advanced MTC should be treated when progressing according to RECIST or according to clinical judgment. If the disease progression is related to one single lesion or to several lesions but belonging to the same organ, local therapies should be preferred.

Systemic Targeted Therapies. Once generalized disease progression has been documented according to RECIST, systemic therapy must be started. Traditional chemotherapy for advanced and progressing MTC has shown limited and temporary response rates in several small series published to date.108 Chemotherapy is no longer used in progressing MTC, and, if ever used, is reserved for those MTC patients who, for any reason, cannot be treated with tyrosine kinase inhibitors (TKIs), which represent the first choice for systemic therapy.101 At the present, there are two available oral drugs belonging to the TKI family: vandetanib and cabozantinib. They were approved by both the US Food and Drug Administration (FDA) and the European Medical Agency (EMA) after the results of the phase III clinical studies ZETA and EXAM,109,110 which both demonstrated a significant increase in progression-­free survival (PFS) compared with placebo. Both vandetanib and cabozantinib are small molecules able to block, with different activities and different patterns, multiple tyrosine kinases.111 Among these anti–tyrosine kinase activities, both of them are able to block the constitutively activated Ret receptor. The drugs should be initiated with evidence of disease progression according to RECIST or, in very advanced cases, based on clinical judgment. Although the two drugs are both citostatic more than citotoxic, a significant srinkage of the metastatic lesions can be observed (Fig. 79.5). Vandetanib can be used successfully also in symptomatic patients and those with ectopic Cushing syndrome.112,113 The choice to start with one drug or another is strictly dependent on the local availability of the drug, as not all countries have approved or reimburse the use of both drugs. However, if both vandetanib and cabozantinib can be prescribed, the choice must be dictated by the patient’s clinical features, location of metastatic lesions, and drug characteristics. According to the results of the phase III studies,109,110 cabozantinib exhibits more rapid action but is associated with more severe adverse events (AEs). Taking into account these factors,

cabozantinib should be preferred when rapid shrinkage of the metastatic lesion is needed, and when this need is sufficient to run the risk of AEs. In the phase III EXAM study, patients could be enrolled even if previously treated with other TKIs, and the results showed that cabozantinib works similarly both as first-­and second-­line treatment in terms of prolongation of PFS. Based on this result, vandetanib should be used as first-­line therapy to reserve the possibility of using cabozantinib as second-­line therapy when, for any reason, vandetanib needs to be stopped. Vandetanib, but not cabozantinib, has been successfully tested in children affected by advanced and metastatic MTC in MEN II, mainly MEN IIB.114 The outcomes in these children demonstrated that vandetanib can be safely and satisfactorily used.115 Moreover, several reports have shown that ectopic ACTH secretion and paraneoplastic Cushing syndrome are completely reverted and cured by vandetanib.112,113,116 Vandetanib, but not cabozantinib, can result in prolonged QTc (>440 ms in adult males and >460 ms in adult females)117; this adverse effect can be life-threatening, thus cabozantinib is the first-­choice drug in patient with QTc values that are already prolonged before starting therapy. Both drugs can induce very similar AEs, but the prevalence varies (Table 79.3), and this must be considered, as well patient co-­morbidities, when deciding which drug should be started first. Although both drugs result in a a significant prolongation in PFS, to date there is no evidence for an increase in overall survival. However, exploratory analyses suggest that patients with RET M918T–positive MTC may benefit more than those with RET M918T–negative from treatment with cabozantinib.118 As with all TKIs, both vandetanib and cabozantinib should be continued until there is evidence of clinical benefit. The reason for this is that they are cytostatic and not cytotoxic, meaning they can block cell proliferation and growth but cannot kill tumoral cells. Nevertheless, two studies and real-­life experience have demonstrated that the objective response of metastatic lesions can be significant, although complete response has never been observed.109,110 Both drugs can induce several AEs, sometimes of grade 3, and their management is fundamental for success of the treatment. They must be early recognized and immediately treated either with other drugs if possible (i.e., hypertension) or by reducing the daily dose of the drug (i.e., fatigue or anorexia). A multidisciplinary team at a referral center should take care of these patients during all treatment follow-­up.

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PART 6  Thyroid

The development of drug resistance and the consequent escape phenomenon are the major concerns associated with this treatment. Once the disease shows progression under treatment, clinicians must decide to continue or to stop the drug. Further studies analyzing the possibility of using the two drugs in an alternating modality, or combined with each other or with other drugs, are an important challenge for the future. A new generation of TKIs that are much more specific for RET mutations has been developed in recent years. LOXO-­ 292, also known as selpercatinib, and BLU-­667, also known as pralsetinib, have been successfully tested in phase I/II clinical trials and, after publication of the results,80,81 both were recently approved by the FDA and are under evaluation from the EMA. They are both potent KDR/VEGF2-­sparing RET inhibitors with preclinical specificity for RET alterations, both gene fusions and point mutations, including the V804M mutation, which showed in vitro resistance to vandetanib.119 These results were further confirmed in animal models and clinical studies,120,121 in which relevant tumor shrinkage was shown. Interestingly, in mouse models, selpercatinib showed antineoplastic activity against brain metastasis too.120 Both drugs have a very good safety profile, with few AEs that are generally of low grade (Table 79.3). Whenever possible, these drugs should be the first choice, but their prescription is determined by the presence of a RET mutation, either somatic or germline; thus, 15% of cases of advanced MTC cannot be treated with any of these drugs. When no other drugs are available, other TKIs that have shown positive results in phase II clinical trials122-­127 or in real life128,129 can prescribed “off label.” They may be used also as second-­or third-­line treatments after cabozantinib and/or vandetanib, especially in MTC with no evidence of RET mutations. KEY POINTS • Nowadays two tyrosine multikinase inhibitors, vandetanib and cabozantinib, are available for the systemic therapy. In the near future a second generation of RET-­ specific inhibitors, selpercatinib and pralsetinib, will likely reach clinical practice.

Radionuclide Systemic Therapies. Treatment with radioactive elements has been widely explored in the past, including treatment with radioiodine (131I) linked to meta-­iodobenzylguanidine130 and anti-­ CEA targeted radioimmunotherapy, but with very limited results, and nowadays these approaches are not used or are only used in cases that are refractory to any other type of systemic therapy. MTC is a neuroendocrine tumor, and 30% to 50% of cases express somatostatin (SMS) receptors, as documented by octreoscan and by 68Ga-­DOTATATE PET/CT.131,132 A few studies have been reported with promising, but not enthusiastic, results.133 However, a hypothesis to explain this failure could be the too-­advanced disease status at the time of the treatment, thus suggesting of that further studies should be performed with less-­advanced cases. Several radiolabeled SMS analogs have been explored as potential therapeutic agents. Most studies of radionuclide therapy in MTC have investigated the use of SMS analogs labeled with the radionuclide(s) 90 yttrium (90Y) and/or 177 lutetium (177Lu).133-­136 The use of 111 indium (111In) was also evaluated.137 A greater than or equal to 50% decrease in Ct values compared with baseline in one third of patients treated with 90Y135 and 177Lu133 has been reported, while others demonstrated a relevant reduction in tumor growth rate. Despite these promising results, these treatments are not yet part of clinical practice.

At present, radionuclide therapy is reserved for those cases that, for any reason, do not respond to or cannot be treated with targeted therapy. However, several ongoing clinical trials (NCT00002947, NCT03647657, NCT02088645, and NCT04106843) should provide more reliable data in the near future.

Other Systemic Therapies. Over the years, different types of octreotide, from the native to the long-­acting analogs, have been tested as potential therapeutic agents for MTC. In many cases, a significant reduction in serum Ct has been demonstrated,138 but no evidence of a parallel relevant reduction in structural disease has been shown. In patients with severe diarrhea that is uncontrolled with any other conventional drug, the use of SMS long-­acting analogs can be considered. Although there are no specific studies of MTC series, treatment with either bisphosphonates or the receptor activator of nuclear factor kappa-­B ligand (RANKL) inhibitor denosumab has been recognized as valid in the treatment of bone metastases.76 This therapeutic procedure has been demonstrated to be effective in controlling bone pain and delaying the occurrence of skeletal-­related events in patients with bone metastases due to differentiated thyroid cancer.139 Both pamidronate and zoledronate are administered intravenously on a monthly basis, while denosumab is administered subcutaneously every 4 weeks. The side effects of these potent antiresorptive agents, although rare, include osteonecrosis of the jaw,140 atypical subtrochanteric fractures,141 and hypocalcemia, and must be carefully taken into consideration before starting the therapy and during long-­term treatment.

CONCLUSIONS Medullary thyroid cancer is a rare cancer with a relatively poor prognosis, and the only possibility of achieving a definitive cure is an early diagnosis when the tumor is still intrathyroidal. The routine measurement of serum Ct in all patients with thyroid nodules is still debated. In 25% of cases, MTC is inherited as an autosomal dominant disease, and children can be affected. RET genetic screening is recommended in this latter form to identify gene carriers early and adequately plan the timing of surgery. Initial therapy is complete removal of the thyroid gland and central node neck dissection, but only one third of patients will be definitively cured by this procedure. Patients must be followed up over the years for the possibility of recurrences, especially when serum Ct is still detectable after surgery. Several modalities of local and systemic therapies are available for metastatic progressive lesions. Nowadays, several drugs belonging to the TKI family are available, and others are under development. MTC patients should be followed in referral centers and by a multidisciplinary team that should be led by an expert endocrinologist, especially when the disease involves other endocrine glands, as occurs in the familial forms.

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46. Wiench M, Wygoda Z, Gubala E, et al. Estimation of risk of inherited medullary thyroid carcinoma in apparent sporadic patients. J Clin Oncol. 2001;19:1374–1380. 47. Keiser HR, Beaven MA, Doppman J, et al. Sipple’s syndrome: medullary thyroid carcinoma, pheochromocytoma, and parathyroid disease. Studies in a large family. NIH conference. Ann Intern Med. 1973;78:561–579. 48. Castinetti F, Waguespack SG, Machens A, et al. Natural history, treatment, and long-­term follow up of patients with multiple endocrine neoplasia type 2B: an international, multicentre, retrospective study. Lancet Diabetes Endocrinol. 2019;7:213–220. 49. Farndon JR, Leight GS, Dilley WG, et al. Familial medullary thyroid carcinoma without associated endocrinopathies: a distinct clinical entity. Br J Surg. 1986;73:278–281. 50. Wells Jr SA, Asa SL, Dralle H, et al. Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid. 2015;25:567–610. 51. Carney JA, Roth SI, Heath 3rd H, et al. The parathyroid glands in multiple endocrine neoplasia type 2b. Am J Pathol. 1980;99:387–398. 52. Cunliffe WJ, Hudgson P, Fulthorpe JJ, et al. A calcitonin-­secreting medullary thyroid carcinoma associated with mucosal neuromas, marfanoid features, myopathy and pigmentation. Am J Med. 1970;48:120–126. 53. Giani C, Ramone T, Romei C, et al. A new MEN2 syndrome with clinical features of both MEN2A and MEN2B associated with a new RET germline deletion. Case Rep Endocrinol. 2020;2020:4147097. 54. Elisei R, Matrone A, Valerio L, et al. Fifty years after the first description, MEN 2B syndrome diagnosis is still late: descriptions of two recent cases. J Clin Endocrinol Metab. 2019;104:2520–2526. 55. Ceccherini I, Romei C, Barone V, et al. Identification of the Cys634-­-­> Tyr mutation of the RET proto-­oncogene in a pedigree with multiple endocrine neoplasia type 2A and localized cutaneous lichen amyloidosis. J Endocrinol Invest. 1994;17:201–204. 56. Coyle D, Friedmacher F, Puri P. The association between Hirschsprung’s disease and multiple endocrine neoplasia type 2a: a systematic review. Pediatr Surg Int. 2014;30:751–756. 57. Nagy N, Guyer RA, Hotta R, et al. RET overactivation leads to concurrent Hirschsprung disease and intestinal ganglioneuromas. Development. 2020;147(21):1–10. 58. Wolfe HJ, Delellis RA. Familial medullary thyroid carcinoma and C cell hyperplasia. Clin Endocrinol Metab. 1981;10:351–365. 59. Jadoul M, Leo JR, Berends MJ, et al. Pheochromocytoma-­induced hypertensive encephalopathy revealing MEN-­IIa syndrome in a 13-­year old boy. Implications for screening procedures and surgery. Horm Metab Res Suppl. 1989;21:46–49. 60. Lenders JW, Pacak K, Walther MM, et al. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA. 2002;287:1427–1434. 61. Eisenhofer G, Huynh TT, Hiroi M, et al. Understanding catecholamine metabolism as a guide to the biochemical diagnosis of pheochromocytoma. Rev Endocr Metab Disord. 2001;2:297–311. 62. Taieb D, Hicks RJ, Hindie E, et al. European Association of Nuclear Medicine Practice Guideline/Society of Nuclear Medicine and Molecular Imaging Procedure Standard 2019 for radionuclide imaging of phaeochromocytoma and paraganglioma. Eur J Nucl Med Mol Imaging. 2019;46:2112–2137. 63. Silverberg SJ, Shane E, Jacobs TP, et al. A 10-­year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N Engl J Med. 1999;341:1249–1255. 64. Lips P, Wiersinga A, van Ginkel FC, et al. The effect of vitamin D supplementation on vitamin D status and parathyroid function in elderly subjects. J Clin Endocrinol Metab. 1988;67:644–650. 65. Elisei R, Alevizaki M, Conte-­Devolx B, et al. 2012 European thyroid association guidelines for genetic testing and its clinical consequences in medullary thyroid cancer. Eur Thyroid J. 2013;1:216–231. 66. Elisei R, Romei C, Renzini G, et al. The timing of total thyroidectomy in RET gene mutation carriers could be personalized and safely planned on the basis of serum calcitonin: 18 years experience at one single center. J Clin Endocrinol Metab. 2012;97:426–435. 67. Brandi ML, Gagel RF, Angeli A, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab. 2001;86:5658–5671.

68. Rohmer V, Vidal-­Trecan G, Bourdelot A, et al. Prognostic factors of disease-­free survival after thyroidectomy in 170 young patients with a RET germline mutation: a multicenter study of the Groupe Francais d’Etude des Tumeurs Endocrines. J Clin Endocrinol Metab. 2011;96:E509–E518. 69. Lloyd RV, Sisson JC, Marangos PJ. Calcitonin, carcinoembryonic antigen and neuron-­specific enolase in medullary thyroid carcinoma. Cancer. 1983;51:2234–2239. 70. Yadav M, Agrawal V, Pani KC, et al. C-­cell hyperplasia in sporadic and familial medullary thyroid carcinoma. Indian J Pathol Microbiol. 2018;61:485–488. 71. Eng C, Clayton D, Schuffenecker I, et al. The relationship between specific RET proto-­oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. Jama. 1996;276:1575–1579. 72. Acton DS, Velthuyzen D, Lips CJ, et al. Multiple endocrine neoplasia type 2B mutation in human RET oncogene induces medullary thyroid carcinoma in transgenic mice. Oncogene. 2000;19:3121–3125. 73. Michiels FM, Chappuis S, Caillou B, et al. Development of medullary thyroid carcinoma in transgenic mice expressing the RET protooncogene altered by a multiple endocrine neoplasia type 2A mutation. Proc Natl Acad Sci U S A. 1997;94:3330–3335. 74. Niccoli-­Sire P, Murat A, Rohmer V, et al. Familial medullary thyroid carcinoma with noncysteine ret mutations: phenotype-­genotype relationship in a large series of patients. J Clin Endocrinol Metab. 2001;86:3746– 3753. 75. Cosci B, Vivaldi A, Romei C, et al. In silico and in vitro analysis of rare germline allelic variants of RET oncogene associated with medullary thyroid cancer. Endocr Relat Cancer. 2011;18(5):603–612. 76. Wells Jr SA, Asa SL, Dralle H, et al. Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid. 2015;25:567–610. 77. Romei C, Ugolini C, Cosci B, et al. Low prevalence of the somatic M918T RET mutation in micro-­medullary thyroid cancer. Thyroid. 2012;22:476– 481. 78. Romei C, Casella F, Tacito A, et al. New insights in the molecular signature of advanced medullary thyroid cancer: evidence of a bad outcome of cases with double RET mutations. J Med Genet. 2016;53(11):729–734. 79. Elisei R, Cosci B, Romei C, et al. Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-­year follow-­up study. J Clin Endocrinol Metab. 2008;93:682–687. 80. Wirth LJ, Sherman E, Robinson B, et al. Efficacy of selpercatinib in RET-­ altered thyroid cancers. N Engl J Med. 2020;383:825–835. 81. Hu M, Subbiah M, Wirth LJ, et al. Results from the registrational phase I/II ARROW trial of pralsetinib (BLU-­667) in patients (pts) with advanced RET mutation-­positive medullary thyroid cancer (RET+ MTC). Ann Oncol. 2020;31:S1026–S1033. 82. Ciampi R, Mian C, Fugazzola L, et al. Evidence of a low prevalence of RAS mutations in a large medullary thyroid cancer series. Thyroid. 2013;23:50–57. 83. Agrawal N, Jiao Y, Sausen M, et al. Exomic sequencing of medullary thyroid cancer reveals dominant and mutually exclusive oncogenic mutations in RET and RAS. J Clin Endocrinol Metab. 2013;98:E364– E369. 84. Ciampi R, Romei C, Ramone T, et al. Genetic landscape of somatic mutations in a large cohort of sporadic medullary thyroid carcinomas studied by next-­generation targeted sequencing. iScience. 2019;20:324–336. 85. Heilmann AM, Subbiah V, Wang K, et al. Comprehensive genomic profiling of clinically advanced medullary thyroid carcinoma. Oncology. 2016;90:339–346. 86. Smith J, Read ML, Hoffman J, et al. Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-­regulation of RET expression. Hum Mol Genet. 2016;25:1836–1845. 87. Essig Jr GF, Porter K, Schneider D, et al. Multifocality in sporadic medullary thyroid carcinoma: an international multicenter study. Thyroid. 2016;26:1563–1572.

CHAPTER 79  Medullary Thyroid Cancer 88. Machens A, Dralle H. Biomarker-­based risk stratification for previously untreated medullary thyroid cancer. J Clin Endocrinol Metab. 2010;95:2655–2663. 89. Modigliani E, Cohen R, Campos JM, et al. Prognostic factors for survival and for biochemical cure in medullary thyroid carcinoma: results in 899 patients. The GETC Study Group. Groupe d’etude des tumeurs a calcitonine. Clin Endocrinol (Oxf). 1998;48:265–273. 90. Viola D, Elisei R. Management of medullary thyroid cancer. Endocrinol Metab Clin North Am. 2019;48:285–301. 91. Fugazzola L, Pinchera A, Luchetti F, et al. Disappearance rate of serum calcitonin after total thyroidectomy for medullary thyroid carcinoma. Int J Biol Markers. 1994;9:21–24. 92. Franc S, Niccoli-­Sire P, Cohen R, et al. Complete surgical lymph node resection does not prevent authentic recurrences of medullary thyroid carcinoma. Clin Endocrinol (Oxf). 2001;55:403–409. 93. Rougier P, Calmettes C, Laplanche A, et al. The values of calcitonin and carcinoembryonic antigen in the treatment and management of nonfamilial medullary thyroid carcinoma. Cancer. 1983;51:855–862. 94. Raue F, Frank-­Raue K. Long-­term follow-­up in medullary thyroid carcinoma. Recent Results Cancer Res. 2015;204:207–225. 95. Terroir M, Caramella C, Borget I, et al. F-­18-­Dopa positron emission tomography/computed tomography is more sensitive than whole-­body magnetic resonance imaging for the localization of persistent/recurrent disease of medullary thyroid cancer patients. Thyroid. 2019;29:1457–1464. 96. Rajan N, Khanal T, Ringel MD. Progression and dormancy in metastatic thyroid cancer: concepts and clinical implications. Endocrine. 2020;70:24–35. 97. Meijer JA, le Cessie S, van den Hout WB, et al. Calcitonin and carcinoembryonic antigen doubling times as prognostic factors in medullary thyroid carcinoma: a structured meta-­analysis. Clin Endocrinol (Oxf). 2010;72:534–542. 98. Elisei R, Lorusso L, Piaggi P, et al. Elevated level of serum carbohydrate antigen 19.9 as predictor of mortality in patients with advanced medullary thyroid cancer. Eur J Endocrinol. 2015;173:297–304. 99. Schlumberger M, Bastholt L, Dralle H, et al. 2012 European thyroid association guidelines for metastatic medullary thyroid cancer. Eur Thyroid J. 2012;1:5–14. 100. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45:228–247. 101. Hadoux J, Pacini F, Tuttle RM, et al. Management of advanced medullary thyroid cancer. Lancet Diabetes Endocrinol. 2016;4:64–71. 102. Kebebew E, Kikuchi S, Duh QY, et al. Long-­term results of reoperation and localizing studies in patients with persistent or recurrent medullary thyroid cancer. Arch Surg. 2000;135:895–901. 103. Chen H, Roberts JR, Ball DW, et al. Effective long-­term palliation of symptomatic, incurable metastatic medullary thyroid cancer by operative resection. Annals of Surgery. 1998;227:887–895. 104. Schwartz DL, Rana V, Shaw S, et al. Postoperative radiotherapy for advanced medullary thyroid cancer–local disease control in the modern era. Head Neck. 2008;30:883–888. 105. Mazzeo S, Cervelli R, Elisei R, et al. mRECIST criteria to assess recurrent thyroid carcinoma treatment response after radiofrequency ablation: a prospective study. J Endocrinol Invest. 2018;41:1389–1399. 106. Mauri G, Gennaro N, Lee MK, et al. Laser and radiofrequency ablations for benign and malignant thyroid tumors. Int J Hyperthermia. 2019;36:13–20. 107. Schlumberger MBL, Dralle H, Jarzab B, et al. 2012 European Thyroid Association guidelines for metastatic medullary thyroid cancer. Eur Thyroid J. 2012;1:5–14. 108. Orlandi F, Caraci P, Mussa A, et al. Treatment of medullary thyroid carcinoma: an update. Endocr Relat Cancer. 2001;8:135–147. 109. Wells Jr SA, Robinson BG, Gagel RF, et al. Vandetanib in patients with locally advanced or metastatic medullary thyroid cancer: a randomized, double-­blind phase III trial. J Clin Oncol. 2012;30:134–141. 110. Elisei R, Schlumberger MJ, Muller SP, et al. Cabozantinib in progressive medullary thyroid cancer. J Clin Oncol. 2013;31:3639–3646. 111. Matrone A, Valerio L, Pieruzzi L, et al. Protein kinase inhibitors for the treatment of advanced and progressive radiorefractory thyroid tumors:

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From the clinical trials to the real life. Best Pract Res Clin Endocrinol Metab. 2017;31:319–334. 112. Paepegaey AC, Cochand-­Priollet B, Louiset E, et al. Long-­term control of hypercortisolism by vandetanib in a case of medullary thyroid carcinoma with a somatic RET mutation. Thyroid. 2017;27:587–590. 113. Pitoia F, Bueno F, Schmidt A, et al. Rapid response of hypercortisolism to vandetanib treatment in a patient with advanced medullary thyroid cancer and ectopic Cushing syndrome. Arch Endocrinol Metab. 2015;59:343–346. 114. Fox E, Widemann BC, Chuk MK, et al. Vandetanib in children and adolescents with multiple endocrine neoplasia type 2B associated medullary thyroid carcinoma. Clin Cancer Res. 2013;19:4239–4248. 115. Kraft IL, Akshintala S, Zhu YJ, et al. Outcomes of children and adolescents with advanced hereditary medullary thyroid carcinoma treated with vandetanib. Clin Cancer Res. 2018;24(4):753–765. 116. Nella AA, Lodish MB, Fox E, et al. Vandetanib successfully controls medullary thyroid cancer-­related Cushing syndrome in an adolescent patient. J Clin Endocrinol Metab. 2014;99:3055–3059. 117. American College of Cardiology/American Heart Association Task Force on Clinical Data S, Buxton AE, Calkins H, et al. ACC/AHA/HRS 2006 key data elements and definitions for electrophysiological studies and procedures: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Data Standards (ACC/AHA/ HRS Writing Committee to Develop Data Standards on Electrophysiology). Circulation. 2006;114(23):2534–2570. 118. Schlumberger M, Elisei R, Muller S, et al. Overall survival analysis of EXAM, a phase III trial of cabozantinib in patients with radiographically progressive medullary thyroid carcinoma. Ann Oncol. 2017;28:2813– 2819. 119. Carlomagno F, Guida T, Anaganti S, et al. Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors. Oncogene. 2004;23:6056–6063. 120. Subbiah V, Velcheti V, Tuch BB, et al. Selective RET kinase inhibition for patients with RET-­altered cancers. Ann Oncol. 2018;29:1869–1876. 121. Subbiah V, Gainor JF, Rahal R, et al. Precision targeted therapy with BLU-­667 for RET-­driven cancers. Cancer Discov. 2018;8:836–849. 122. Schlumberger M, Jarzab B, Cabanillas ME, et al. A phase II trial of the multitargeted tyrosine kinase inhibitor lenvatinib (E7080) in advanced medullary thyroid cancer. Clin Cancer Res. 2016;22:44–53. 123. Ravaud A, de la Fouchardiere C, Caron P, et al. A multicenter phase II study of sunitinib in patients with locally advanced or metastatic differentiated, anaplastic or medullary thyroid carcinomas: mature data from the THYSU study. Eur J Cancer. 2017;76:110–117. 124. Lam ET, Ringel MD, Kloos RT, et al. Phase II clinical trial of sorafenib in metastatic medullary thyroid cancer. J Clin Oncol. 2010;28:2323–2330. 125. Locati LD, Licitra L, Agate L, et al. Treatment of advanced thyroid cancer with axitinib: Phase 2 study with pharmacokinetic/pharmacodynamic and quality-­of-­life assessments. Cancer. 2014;120:2694–2703. 126. Bible KC, Suman VJ, Molina JR, et al. A multicenter phase 2 trial of pazopanib in metastatic and progressive medullary thyroid carcinoma: MC057H. J Clin Endocrinol Metab. 2014;99:1687–1693. 127. Schlumberger MJ, Elisei R, Bastholt L, et al. Phase II study of safety and efficacy of motesanib in patients with progressive or symptomatic, advanced or metastatic medullary thyroid cancer. J Clin Oncol. 2009;27:3794–3801. 128. Matrone A, Prete A, Nervo A, et al. Lenvatinib as a salvage therapy for advanced metastatic medullary thyroid cancer. J Endocrinol Invest. 2021;44(10):2139–2151. 129. Masaki C, Sugino K, Saito N, et al. Lenvatinib induces early tumor shrinkage in patients with advanced thyroid carcinoma. Endocr J. 2017;64:819–826. 130. Maiza JC, Grunenwald S, Otal P, et al. Use of 131 I-­MIBG therapy in MIBG-­positive metastatic medullary thyroid carcinoma. Thyroid. 2012;22:654–655. 131. Baudin E, Lumbroso J, Schlumberger M, et al. Comparison of octreotide scintigraphy and conventional imaging in medullary thyroid carcinoma. J Nucl Med. 1996;37:912–916.

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132. Tuncel M, Kilickap S, Suslu N. Clinical impact of (68)Ga-­DOTATATE PET-­CT imaging in patients with medullary thyroid cancer. Ann Nucl Med. 2020;34:663–674. 133. Bodei L, Handkiewicz-­Junak D, Grana C, et al. Receptor radionuclide therapy with 90Y-­DOTATOC in patients with medullary thyroid carcinomas. Cancer Biother Radiopharm. 2004;19:65–71. 134. Satapathy S, Mittal BR, Sood A, et al. Efficacy and safety of concomitant 177Lu-­DOTATATE and low-­dose capecitabine in advanced medullary thyroid carcinoma: a single-­centre experience. Nucl Med Commun. 2020;41:629–635. 135. Parghane RV, Naik C, Talole S, et al. Clinical utility of (177) Lu-­ DOTATATE PRRT in somatostatin receptor-­positive metastatic medullary carcinoma of thyroid patients with assessment of efficacy, survival analysis, prognostic variables, and toxicity. Head Neck. 2020;42:401–416. 136. Paganelli G, Zoboli S, Cremonesi M, et al. Receptor-­mediated radiotherapy with 90Y-­DOTA-­D-­Phe1-­Tyr3-­octreotide. Eur J Nucl Med. 2001;28:426–434.

137. Valkema R, De Jong M, Bakker WH, et al. Phase I study of peptide receptor radionuclide therapy with [In-­DTPA]octreotide: the Rotterdam experience. Semin Nucl Med. 2002;32:110–122. 138. Lupoli GA, Fonderico F, Fittipaldi MR, et al. The role of somatostatin analogs in the management of medullary thyroid carcinoma. J Endocrinol Invest. 2003;26:72–74. 139. Orita Y, Sugitani I, Takao S, et al. Prospective evaluation of zoledronic acid in the treatment of bone metastases from differentiated thyroid carcinoma. Ann Surg Oncol. 2015;22:4008–4013. 140. Khosla S, Burr D, Cauley J, et al. Bisphosphonate-­associated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research. J Bone Miner Res. 2007;22:1479–1491. 141. Abrahamsen B, Eiken P, Eastell R. Subtrochanteric and diaphyseal femur fractures in patients treated with alendronate: a register-­based national cohort study. J Bone Miner Res. 2009;24:1095–1102.

80 Anaplastic (Undifferentiated) Thyroid Carcinoma Robert C. Smallridge and Sarika N. Rao OUTLINE Presentation, 1329 Epidemiology, 1329 Clinical Characteristics, 1329 Diagnosis, 1329 Biopsy, 1329 Histopathology Subtypes, 1329 Differential Diagnoses, 1330 Molecular Genetics, 1330 Evaluation, 1331 Clinical and Labs, 1331 Imaging, 1331 Staging, 1331

Establish Goals of Care, 1332 Multidisciplinary Team, 1332 Prognosis, 1332 Predictors of Survival, 1332 Median and Overall Survival, 1333 Therapy, 1333 Treatment Strategies by Stage, 1333 Surgery, 1333 Radiation Therapy, 1333 Multimodal Therapy, 1333 Future Directions, 1335 Conclusions, 1335

 

PRESENTATION Epidemiology Undifferentiated (i.e., anaplastic) thyroid carcinoma (ATC) comprises a small percentage of all thyroid malignancies worldwide. In a review of frequency in 15 countries, the median percentage of ATC out of all thyroid cancer cases was 3.6% (range: 0.6%–9.8%), with a decrease in several countries after dietary iodide was increased.1,2 In the United States, the estimated number of all new thyroid cancer cases in 2021 will be 44,280 (32,130 female and 12,150 male) and estimated deaths to be 2,200 (1,150 female; 1,050 male). ATC occurs in only 1% to 2% of thyroid cancers in the United States,3 but accounts for almost half of thyroid cancer-related deaths, reflecting the extremely aggressive behavior of ATC. The incidence varies slightly among racial/ethnic groups in the United States, occurring in 0.6% of all thyroid cancers in Hispanic Whites, 1.0% in Asian-­Pacific Islanders, 1.1% in non-­Hispanic Whites, 1.3% in Native Americans, and 1.8% in Black patients.1

Clinical Characteristics In a review of 44 series in 2016 comprising 2881 patients, 1898 (66%) were female, and 983 (34%) were male.1 Although patients ranged in age from 15 to 98 years, ATC is primarily a disease of older persons, with mean age of 66.5 years (n =19 studies) and median age of 69 years (n = 26 studies). The median size of the primary tumor was 6.8 cm (range: 0.5–25 cm). ATC could arise de novo but was more likely to develop in patients who had a longstanding goiter or, in 30% of cases (range: 3%–58%), coexist with differentiated thyroid cancer. Distant metastases were present at initial diagnosis in 42% of patients (range: 11%–90%). Five of the 44 reports identified the causes of death: local disease in 15% (range: 5%–37%) of patients, distant metastases in 59% (range: 12%–68%) or both local and distant disease in 26% (range: 25%–51%). Since that review, ten additional studies (comprising 1953 patients) have reported similar results, mirroring the characteristics observed in the earlier reports.4-­13

Clinically, the most frequent initial symptom is a rapidly enlarging neck mass, observed in 86% of studies (range: 46%–100%).1 The mass is often invasive, leading to destruction to nearby structures, including the trachea, esophagus, cervical vessels, and recurrent laryngeal nerves, resulting in hoarseness or dysphonia (33% of cases), dysphagia (38%), dyspnea (27%), pain (16%), and, less frequently, cough, hemoptysis, or superior vena cava syndrome (10% or less). Distant metastases at presentation are detected most often in the lungs (37%), followed by mediastinum, liver, bone, brain, kidneys, heart, and adrenal glands. At autopsy, distant metastases are noted at two to four times the clinical detection rate.1

DIAGNOSIS Biopsy KEY POINTS • Anaplastic thyroid cancer is rare, accounting for approximately 3.6% of all thyroid cancers, and is extremely aggressive. • Median age of diagnosis is approximately at 66 years with median tumor size of 6.8 cm. • Patients most commonly present with a rapidly enlarging neck mass, with symptoms related to extent of disease involvement.

Fine-­needle aspiration biopsy and cytology can be attempted first but has a higher nondiagnostic rate than in other thyroid malignancies because of the undifferentiated nature of the cells, as well as extensive necrosis and infiltration of tumor macrophages. If unsuccessful, then expeditious core tissue biopsy or possibly even surgical biopsy may be necessary. ATC has no histologic features of thyroid tissue, although it may coexist with differentiated thyroid cancer. Several other tumors should be considered in the differential diagnosis, however.

Histopathology Subtypes The major subtypes of ATC include sarcomatoid (i.e., spindle cell), pleomorphic giant cell, and epithelial (i.e., squamoid) (Fig. 80.1).

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A

B

C Fig. 80.1  Histologic subtypes of anaplastic thyroid carcinoma. Spindle cell variant (A), epithelioid variant (B), and giant cell variant (C).

These subtypes are often mixed. Other rare variants include paucicellular, rhabdoid, osteoclastic, angiomatoid, lymphoepithelioma-­like, and small cell.9,14-­18 The specific histotype may influence survival, as long-­term survival (>1 year) is significantly more common in tumors with epithelial or squamous cell component, and short-­term survival (90% accumulated intrathyroidal radiodine during a perchlorate discharge test).37,40 Heterozygous TPO mutations and mutations with residual catalytic function have rarely been associated with mild hypothyroidism and partial iodide organification defects (PIOD), sometimes presenting after the neonatal period. In isolated cases, heterozygous TPO mutations have been associated with TIOD, perhaps due to monoallelic intrathyroidal expression of TPO.41,42 More than 100 TPO mutations have been reported that may disrupt folding and membrane insertion and/or catalytic activity, although few have undergone molecular characterization. DUOX2 and DUOXA2: DUOX2 is a thyroidal NADPH oxidase that generates the H2O2 required by TPO as the final electron acceptor for both iodination of TG and the coupling reaction during TH biosynthesis. Its accessory protein DUOXA2 facilitates the delivery of DUOX2 from the ER to the plasma membrane and subsequently forms a stable complex with DUOX2 that is required for its enzymatic activity. DUOX2 mutations occur frequently, especially in East Asian individuals,39 and the allele frequency of some DUOX2 mutations approaches 1% in certain populations (e.g., p.Q570L in South Asians) suggesting that DUOX2 mutations may contribute to CH even more frequently than currently appreciated. DUOXA2 mutations are a rare cause of DH, with less than 20 mono-­or biallelic loss-­of-­function variants reported, although some populations harbor recurrent mutations.7,34,39,43 Both biallelic and monoallelic DUOX2 mutations cause CH, which is usually transient or mild permanent CH. In transient CH, the H2O2 synthesis defect cannot be fully compensated in the neonate, where TH biosynthesis is at its peak, resulting in neonatal hypothyroidism. However, when TH requirements decline in later childhood, other H2O2-­ synthesizing enzymes may compensate fully for the defect, resulting in resolution of hypothyroidism and enabling cessation of levothyroxine treatment. Thyroidal iodide uptake is usually normal or increased with DUOX2 mutations, and patients exhibit a PIOD.43,44

CHAPTER 81  Resistance to Thyroid Hormone and Genetic Defects of the Thyroid Clinical data are sparse for individuals with DUOXA2 mutations, but reported cases also demonstrate both mild permanent and transient CH with or without goiter, and PIOD.39,43,44 DUOX2 mutations exhibit highly variable penetrance and expressivity, and even biallelic truncating mutations, as well as heterozygous mutations, may be associated with both transient and permanent CH. It is speculated that variants in other H2O2-­synthesizing enzymes may modulate CH severity, e.g., DUOX1, which encodes an additional NADPH-­oxidase, is contiguous with DUOX2 on chromosome 15, and is expressed at lower levels than DUOX2 in thyroid.43 In support of this hypothesis, cases with likely complete DUOX isoenzyme deficiency exhibited unusually severe CH consistent with a compensatory role for DUOX1.45 A series of UK cases harboring DUOX2 and DUOXA2 mutations exhibited borderline neonatal bloodspot screening TSH levels followed by a delayed but significant TSH rise with subnormal venous FT4 measurements, suggesting that clinically actionable CH associated with DUOX2 and DUOXA2 mutations could be missed on neonatal screening.44 DUOX2 is also expressed in respiratory epithelium and throughout the gastrointestinal tract, and rare loss-­of-­ function mutations increase risk of inflammatory bowel disease.46 IYD (previously known as DEHAL1): IYD is a DIO, catalyzing the deiodination of MIT and DIT residues, thereby releasing thyroidal iodide and tyrosine for further hormonogenesis. IYD mutations have only been formally characterized in a few families, although the description of goiter with impaired iodide recycling preceded the first molecular diagnosis by many years. Monoallelic and biallelic IYD mutations can both cause goitrous hypothyroidism although heterozygotes may be asymptomatic. Affected individuals exhibit rapid thyroidal uptake of iodide with a normal perchlorate discharge test and characteristically increased urinary concentrations of MIT and DIT. Hypothyroidism may manifest in childhood following a normal neonatal CH screening test result, resulting in neurodevelopmental delay if diagnosis is delayed.40 SLC5A5 (NIS): NIS mediates the basolateral uptake of iodide into of thyrocytes by the electrogenic symport of two sodium ions for one iodide ion, down an electrochemical gradient generated by the Na+/K+ ATPase. Biallelic NIS mutations are a rare cause of DH, and thyroid dysfunction ranges from severe CH to euthyroidism, especially if dietary iodine content is high. NIS transports both iodide and pertechnetate; therefore, mutations cause diminished or absent thyroidal radioiodine or pertechnetate uptake despite clinical or ultrasonographic evidence of a normally located, usually enlarged, thyroid gland. Tracer uptake by other NIS-­expressing tissues, e.g., gastric parietal cells and salivary glands, is also reduced, and following injection of radioiodine, the ratio of salivary:plasma radioiodine is decreased in affected patients.40 NIS-­ mediated CH may not manifest at the time of neonatal screening and may present with delayed TSH rise and hypothyroidism in childhood, potentially resulting in neurodevelopmental delay.47 Because NIS mediates iodide accumulation in breast milk, prophylactic iodine supplementation of affected females may be required to prevent neonatal iodide deficiency in exclusively breastfed offspring.48 NIS mutations may result in loss of function due to impaired transporter function despite normal plasma membrane expression, or alternatively, mutant NIS proteins with either preserved or abrogated symporter activity may be retained intracellularly.34,47 SLC26A4 (pendrin): SLC26A4 (pendrin) is a multifunctional anion transporter that mediates both chloride-­bicarbonate and chloride-­ iodide exchange. Biallelic mutations in pendrin cause Pendred syndrome, comprising congenital sensorineural hearing impairment with enlargement of the vestibular aqueduct, and diffuse or multinodular goiter with PIOD. These phenotypes reflect a role for pendrin in chloride-­bicarbonate transport in the inner ear and in maintaining

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endolymphatic fluid acid-­base homeostasis, as well as mediating iodine efflux at the apical membrane of the thyrocyte. A subset of cases also harbor a Mondini cochlear malformation (incomplete partition of the cochlea). Only approximately 50% patients with Pendred syndrome exhibit subclinical or overt biochemical hypothyroidism, and thyroid dysfunction or goiter are rare before puberty.49 SLC26A7: Biallelic loss of function mutations in SLC26A7 represent the most recently discovered genetic cause of DH, with 19 cases now reported worldwide. Of those cases evaluated, 12 exhibit goiter, and all have permanent CH, although one case from Japan presented with hypothyroidism in childhood following a normal newborn screening TSH measurement. SLC26A7 belongs to the same family of anion transporters as pendrin and has previously been shown to function as a chloride-­bicarbonate exchanger or chloride channel, mediating murine renal bicarbonate resorption and gastric acid secretion. The SlC26A7 transcript is highly expressed in human and murine thyroid, and all mutations thus far are truncating mutations, which disrupt the highly-­conserved STAS domain and C-­terminus of the translated protein, affecting plasma membrane localization if the truncated protein is expressed. Individuals who have undergone further evaluation exhibit normal thyroidal uptake of radioiodine with PIOD. Slc26a7 null mice also exhibit goitrous hypothyroidism with defective hormone biosynthesis. However, in contrast to affected humans, thyroidal radioiodine uptake is reduced in null mice with normal perchlorate discharge; additionally, null mice exhibit distal renal tubular acidosis, which was not recapitulated in affected humans.50,51 The role of SLC26A7 in thyroid and its location in thyroid follicular cells have not yet been conclusively ascertained. In vitro studies have yielded varying results, with one study reporting a role for SLC26A7 in iodide efflux, whereas another study did not observe this effect.50,51 Future studies in thyrocytes are required to address its localization and role in thyroidal iodide metabolism, including its putative role as a direct iodide transporter. Because SLC26A7 is thought to regulate acid-­ base balance in the renal tubule and gastric epithelium, it has also been hypothesized that it may have an analogous function in thyroid follicular cells, where optimal ionic milieu and pH are critical for enzyme (TPO, DUOX2) and anion exchanger activities. Species differences in thyroid cell electrophysiology may then mediate divergent effects of SLC26A7 deficiency on iodide metabolism in humans and mice. KEY POINTS  • Most individuals with dyshormonogenesis (DH) will have a mutation affecting genes involved in thyroid hormone biosynthesis, all of which cause isolated DH, except for Pendred syndrome. There is a broad spectrum of congenital hypothyroidism (CH) biochemical severity and goitrogenesis, but specific genetic mutations may be associated with specific biochemical or radiological hallmarks. Genetic testing may help predict transiency of CH or potential for delayed thyroid-­stimulating hormone rise and inform genetic counseling regarding recurrence risk in future offspring.

Genetic syndromes that may include CH: CH, or hyperthyrotropinaemia, may also be a feature of multisystem involvement in other genetic syndromes, including pseudohypoparathyroidism and related disorders where TSHR signaling is impaired, without direct disruption to the TSHR gene, e.g., due to GNAS defects or monoallelic defects in PRKAR1A or PDE4D. Depending on the molecular defect, individuals may exhibit characteristic physical features (e.g., short stature, skeletal defects, obesity) ,and endocrine abnormalities including resistance to TSH and parathyroid hormone. CH or nonautoimmune hypothyroidism may also be associated with di George syndrome, for which TBX1

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PART 6  Thyroid

is a candidate gene. Neonates and children with trisomy 21 (Down syndrome) often exhibit nonautoimmune hyperthyrotropinaemia or mild primary CH, for which the underlying mechanism is poorly understood but may involve DYRK1A. Say–Barber–Biesecker–Young– Simpson syndrome (KAT6B) and Johanson–Blizzard syndrome (UBR1) may also include CH, although the underlying mechanism has not been fully elucidated.7 Recent advances in identifying genetic determinants of CH: Historically, despite the low yield of genetic mutations identified in TD, evaluation of CH cohorts has supported a genetic basis in a larger subset of CH than is currently recognized. A large national survey of CH in France found that 2% of individuals with TD have an affected relative, which is 15-­fold greater than predicted by chance alone. Moreover, thyroidal developmental abnormalities were more common in euthyroid first-­degree relatives of CH cases than in controls. TD has been found to occur more frequently in consanguineous or less genetically diverse populations, and individuals with CH have an increased risk of extrathyroidal developmental malformations.52-­54 Conversely, the greater than 90% discordance between monozygotic twins with CH, the female preponderance of thyroid ectopy, and the low frequency of causative mutations in TD seem incompatible with simple Mendelian inheritance and have been used to support the argument that TD is a sporadic disease, for which the underlying basis may include somatic mutations restricted to the thyroid or epigenetic events.55,56 Attempts to reconcile these observations with data supporting a role for underlying genetic factors include the “two hit” hypothesis, in which a predisposing germline mutation occurs in conjunction with an additional genetic or epigenetic alteration within the thyroid gland or surrounding structures.57 However, the only study to investigate this showed that, although ectopic thyroid exhibited differing gene expression compared with normally sited tissue, this was not due to differences in somatic gene methylation patterns. Additionally, whole-­ exome sequencing in lymphocyte DNA from monozygotic twins discordant for TD did not demonstrate frequent somatic mutations.58,59 Some thyroidal genes do appear to exhibit autosomal monoallelic expression; however, there is only one confirmed case of monoallelic expression of a mutant allele (TPO) in association with dyshormonogenic CH.41,60 More recently, data from next-­generation sequencing (NGS) studies have facilitated the identification of monogenic causes for CH in a greater proportion of cases. Targeted NGS of genes previously known to be implicated in TD or DH, in a hypothesis-­free manner, has identified likely pathogenic variants in at least 50% individuals with GIS CH, while demonstrating significant overlap of genetic etiologies in GIS CH and TD morphological subgroups.61 Mutations in genes characteristically associated with TD (e.g., biallelic FOXE1 mutations) have been reported in association with GIS CH without extrathyroidal features. Conversely, pendrin mutations have been reported in TD, perhaps due to secondary atrophy of the thyroid due to increased oxidative stress, and TPO mutations have been reported in association with thyroid hypoplasia.62,63 N-­terminal DUOX2 mutations have also been reported in individuals with thyroid ectopy, raising the possibility of a role for DUOX2 and H2O2 production in thyroid development.64 Crossover between classical TD-­associated and DH-­associated genes in the pathogenesis of both morphological subtypes of CH may partly explain the previously low mutation detection rate in phenotype-­ driven targeted genetic studies. Additionally, there has been ongoing discovery of novel causative genes for CH in human studies (e.g., SLC26A7, TUBB1, CDCA8). A further major insight from NGS studies has been the confirmation of the role of oligogenicity in CH. In particular, NGS evaluation of 11 CH-­associated genes in 177 Italian patients with GIS CH and TD identified a likely pathogenic variant in more than one gene in 25% of

cases, across both morphological subgroups, that cosegregated with CH or nonautoimmune hypothyroidism in several pedigrees.61 Similar NGS studies have implicated oligogenicity in the pathogenesis of CH across a range of ethnicities. This suggests that CH may occur due to the additive effect of multiple rare variants influencing both thyroid morphogenesis or hormonogenesis, which exert only modest effects on thyroid function when expressed in isolation. Such oligogenic inheritance may go some way towards explaining both the apparently sporadic occurrence of CH and the variable expressivity and penetrance of causal mutations.65 Clinical significance of genetics in congenital hypothyroidism: Molecular genetic testing, where available, may be helpful in cases of GIS CH where DH is suspected or in CH where there is a strong family history or other system involvement, and is recommended in these contexts.6 A genetic diagnosis may assist genetic counseling and enable tailored treatment where multisystem involvement is anticipated, or where levothyroxine treatment may not be required lifelong (e.g., where hypothyroidism is likely to be transient, such as with DUOX2/ DUOXA2 mutations, and with heterozygous TSHR mutations where treatment may not be required). Additionally, for etiologies known to be associated with a delayed TSH rise, such that hypothyroidism may present in childhood despite a normal neonatal screening TSH (NIS, DUOX2, DUOXA2, SLC26A7, or IYD mutations), establishing the genetic etiology may enable prompt diagnosis in affected siblings to prevent neurodevelopmental delay. An NGS-­based approach to genetic testing in CH is most likely to yield positive results. Future studies: The advent of NGS has improved genetic diagnosis in CH, both by identifying known and novel monogenic causes in a nonhypothesis driven manner and by identifying oligogenic causes for CH. However, conclusions from NGS studies have been limited by lack of functional characterization of the rare, likely pathogenic variants identified, and future studies are required both to substantiate the functional consequences of these variants and to investigate the mechanisms by which they result in CH. Whole-­exome or genome sequencing has the potential to identify further novel genetic causes of CH and to delineate novel genes involved in thyroid development or TH biosynthesis. The recent success in differentiating murine and human pluripotent stem cell-derived endodermal precursors into functional thyroid follicular structures, as well as technological advances in thyrocyte culture, present opportunities for validating the roles of such genes in the future. Additionally, the contribution of environmental factors, e.g., iodine and micronutrient status and the role of endocrine disruptors in the pathogenesis of CH, have yet to be established. KEY POINTS  • Although monogenic causes for thyroid dysgenesis (TD) remain rare, next-­ generation sequencing has played a significant role in identifying genetic causes for congenital hypothyroidism (CH) by i.) highlighting a role for oligogenicity in both TD and gland in situ CH; ii.) confirming overlap of genetic causes across different morphological subgroups of CH; iii.) identifying novel causative genes for CH; and iv.) identifying mutations in known CH-­ associated genes in individuals with atypical phenotypes. Future studies are required to substantiate the functional consequences of novel variants predicted to be pathogenic in silico and to investigate the mechanism by which novel genes and oligogenic variants cause CH.

CLASSIFICATION OF DISORDERS OF THYROID HORMONE ACTION Resistance to TH (RTH), first described in 1967 and characterized by elevated circulating THs (T4, T3) and nonsuppressed TSH levels

CHAPTER 81  Resistance to Thyroid Hormone and Genetic Defects of the Thyroid

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TABLE 81.3  Genetic Disorders Associated with Elevated Thyroid Hormones

Disorder

Familial Dysalbuminaemic Dystransthyretinaemic Hyperthyroxinaemia (FDH) Hyperthyroxinemia*

Resistance to Thyroid Resistance to Hormone beta Thyroid Hormone Allan–Herndon– SBP2 (RTHbeta) alpha (RTHalpha) Dudley Syndrome Deficiency**

Gene Free T4 Free T3 TSH Reverse T3 SHBG

TTR Raised Normal or raised Normal Raised Normal

THRB Raised Raised Normal (or raised) Raised Normal

ALB Raised Normal or raised Normal Raised Normal

THRA Low-­normal or low High-­normal or raised Normal (or raised) Low or low-­normal Normal or raised

MCT8 Normal or low Raised Normal (or raised) Low Raised

SBP2 Raised Normal or low Normal (or raised) Raised Raised

*Elevated thyroid hormone measurements can be intermittent. **Low circulating selenium levels are also characteristic of this disorder. SHBG, sex hormone-binding globulin; T3, triiodothyronine; T4, thyroxine; TSH, thyroid-­stimulating hormone.

together with variable tissue refractoriness to hormone action, is mediated by defects in TRβ.66 However, it is now recognized that other disorders, due to defective transport of TH into cells, intracellular metabolism of T4 to generate T3 (see Chapter 66), or hormone action via TRα, are also characterized by altered tissue sensitivity to TH. Accordingly, a consensus statement now defines RTH more broadly, to encompass all defects that can interfere with the biological action of TH secreted in normal or excessive amounts.67

DIFFERENTIAL DIAGNOSIS OF ELEVATED THYROXINE AND/OR TRIIODOTHYRONINE WITH NONSUPPRESSED THYROID-­STIMULATING HORMONE A number of inherited and acquired disorders are associated with differing patterns of elevated TH (T4 and/or T3) together with nonsuppressed TSH levels (see Table 9.1 in Chapter 9). The first step in making a diagnosis is to verify the validity of hormone measurements: confirmation of elevated free TH levels in two-­step or equilibrium dialysis assays excludes abnormal circulating binding proteins or interfering antibodies; preservation of linearity when TSH is assayed in dilution suggests that this measurement is not artefactual. Many causes (nonthyroidal illness, psychiatric disorder, neonatal period, drugs) can be excluded by clinical context. Genetic disorders associated with elevated TH levels and reduced sensitivity to hormone action can also be distinguished on the basis of different thyroid function test and metabolite patterns (Table 81.3).

RESISTANCE TO THYROID HORMONE β Clinical Features Resistance to TH due to defective TRβ (RTHβ) was first described in two siblings who were clinically euthyroid despite high circulating TH levels and who exhibited several other abnormalities, including deaf-­mutism, stippled femoral epiphyses with delayed bone maturation and short stature, dysmorphic facies, winging of the scapulae, and pectus carinatum.66 It is now clear that some of these features are unique to this kindred, in which the disorder was recessively inherited. The majority of RTHβ cases that have been subsequently described are dominantly inherited, with a highly variable clinical phenotype. Affected subjects are either asymptomatic or have nonspecific symptoms and may be noted to have a goiter, prompting thyroid function tests that suggest the diagnosis. In these individuals, classified as exhibiting generalized RTH (GRTH), the high TH levels are thought

to compensate for ubiquitous tissue resistance, resulting in a euthyroid state. In contrast, a smaller number of individuals (≈15%) who share the same biochemical phenotype exhibit clinical features of thyrotoxicosis. In adults these can include weight loss, tremor, palpitations, insomnia, and heat intolerance; in children, failure to thrive, abnormal growth (either accelerated, or more commonly retarded), and hyperkinetic behavior have also been noted. When this clinical entity was first described, patients were thought to exhibit “selective” pituitary RTH (PRTH) action with preservation of normal hormonal responses in peripheral tissues.68 However, it is now recognized that peripheral resistance (typically hepatic) to hormone action is present even in these subjects. Less commonly, hypothyroid features such as growth retardation, delayed dentition and bone age in children, or asthenia and hypercholesterolemia in adults have been observed in RTHβ and may even coexist with thyrotoxic symptoms in the same individual. Taken together, these observations suggest that the clinical features of RTHβ are variable, being governed by either resistance or retention of sensitivity to high circulating levels of free THs in different tissues. The estimated prevalence of RTHβ is 1 in 40,000 to 50,000 live births, and the disorder can be diagnosed neonatally by screening with a combination of TSH and free T4 measurements.69,70 Over 900 cases of RTHβ (from more than 400 families) have now been described worldwide, enabling clinical characteristics of this disorder to be defined more precisely. Goiter. A palpable goiter has been documented in 65% of individuals, particularly adult females. The enlargement is usually diffuse, but benign multinodular change is recognized, particularly in goiters that recur following partial thyroidectomy. Development of toxic multinodular goiter on the background of RTHβ has been documented in a single case.71 Interestingly, it has been noted that fewer children with RTHβ born to affected mothers exhibit thyroid enlargement (35%) than offspring of unaffected mothers (87%), suggesting that fetal exposure to maternal hyperthyroxinemia may protect against goiter formation.72 The bioactivity of circulating TSH has been shown to be significantly enhanced in RTHβ, perhaps accounting for the goiter and markedly elevated serum THs observed in many cases, despite normal immunoreactive TSH levels.73 Rarely, RTHβ cases with coexistent thyroid cancer (usually small foci of papillary carcinoma, but one metastatic) have been described74,75; following thyroid ablation, despite inability to fully suppress TSH levels with T4 therapy due to hormone resistance, clinical outcomes are not unfavorable.76 Cardiovascular system. Palpitations and resting tachycardia have been reported in approximately 75% of cases with GRTH and almost all cases of PRTH, and atrial fibrillation has been reported in 6% of

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patients with mutations.72 The incidence of cardiac symptoms is notably higher in RTHβ patients than in unaffected relatives or in the general population, although still less frequent compared with patients with classic hyperthyroidism.77 In one study, 30% of RTHβ subjects showed echocardiographic features of increased myocardial contractility and impaired diastolic relaxation, with a greater incidence of mitral valve prolapse.72 In a prospective study of cardiovascular involvement in a large cohort of children and adults with RTHβ, resting heart rate was significantly higher, and some indices of cardiac systolic and diastolic function (e.g., stroke volume, cardiac output, maximal aortic flow velocity) were intermediate between values in normal and hyperthyroid subjects, whereas other parameters (e.g., ejection and shortening fractions of the left ventricle, systolic diameter, and left ventricle wall thickness) were not different, indicating a partially hyperthyroid response of the heart in this disorder.77 Clinically significant mixed dyslipidaemia (total cholesterol >5 mmol/L and triglyceride >1.7 mmol/L) has been documented in 49% of an RTHβ patient cohort, together with increased hepatic and intramyocellular lipid and systemic insulin resistance, but normal carotid intima media thickness78; however, the hypercoagulable state associated with conventional hyperthyroidism is not present.79 Musculoskeletal system. Stippled epiphyses and winged scapulae were noted in the original RTHβ kindred, but have not been observed in other cases, such that these features may represent a specific manifestation of the known gene deletion (THRB) or an unrelated genetic abnormality in this consanguineous kindred.66 In contrast, growth retardation and delayed skeletal maturation are more common in childhood RTHβ patients, with height below the fifth percentile in 18% and delayed bone age (>2 standard deviations) in 29%,72 with no significant differences between GRTH and PRTH cases. However, despite these abnormalities, final adult height is often not affected.80 Bearing in mind the known adverse effects of untreated hyperthyroidism on bone mineralization, we conducted a cross-­sectional survey of approximately 80 adult subjects with RTHβ and observed a reduction in bone mineral density in the femoral neck (mean Z score –0.71) and lumbar spine (mean Z score –0.73) but with normal markers of bone turnover (Moran & Chatterjee, unpublished observations). Energy intake and expenditure. We have documented raised resting energy expenditure (REE) (metabolic rate), mediated by uncoupling of oxidative phosphorylation and mitochondrial ATP synthesis, in skeletal muscle of RTHβ patients. Energy intake is also increased, particularly in childhood.81 Indeed, an imbalance between such hyperphagia and increased total energy expenditure (reflecting raised REE and increased physical activity due to attention deficit hyperactivity disorder [ADHD]; see later) may account for the abnormally low body mass index seen in approximately one third of children. Central nervous system. Two studies have documented neuropsychologic abnormalities in RTHβ. First, a history of ADHD in childhood was elicited more frequently in patients with RTHβ (75%) compared with their unaffected relatives (15%).82 A second study showed that both children and adults with RTHβ exhibited problems with language development, manifested by poor reading skills and problems with articulation, including speech delay and stuttering.83 However, frank mental retardation (intelligence quotient [IQ] corticosterone

Zona reticularis (zR) androgens ->DHEA and DHEA-S

X zone (fetal derived) progesterone catabolism?

Chromaffin cells catecholamines ->noradrenaline ->adrenaline

Chromaffin cells catecholamines ->noradrenaline ->adrenaline

Zona fasciculata (zF) glucocorticoids ->corticosterone

Chromaffin cells catecholamines ->noradrenaline ->adrenaline

Fig. 85.3  Adrenal gland structure across different species. The structure of the adrenal gland, and particularly the adrenal cortex, is highly conserved between species. However, there are some key differences in mice and rats as compared with humans. The majority of mouse species (middle) do not express CYP17A1 after fetal development. Consequently, these animals synthesize corticosterone rather than cortisol as the main glucocorticoid, and they do not make adrenal androgens (i.e., they lack a functional zona reticularis). The mouse adrenal cortex contains an additional zone called the X zone, which is hypothesized to function in progesterone catabolism. The X zone is derived from the fetal cortex and regresses in males during puberty and in females after pregnancy. The rat adrenal cortex (right) contains a unique region between the zona golmerulosa (zG) and zone fasciculata (zF) called the undifferentiated zone (zU). This region is defined by a lack of expression of zG or zF differentiation markers and is proposed to contain progenitor cells that repopulate the cortex.

while CYP11B2-­negative cells in the zG constitute a reservoir of cortical progenitor cells.17

Zona Fasciculata. Directly adjacent to the zG, the zona fasciculata (zF) is composed of cells organized into radial cords (Latin fascicle, bundle) that function to produce glucocorticoids. Cortisol is the main glucocorticoid in humans and is produced in response to adrenocorticotropin hormone (ACTH) as part of the hypothalamic–­pituitary–­ adrenal axis. Cortisol has a wide range of effects throughout the body, including increased blood sugar through stimulation of gluconeogenesis and suppression of the immune system.18 Cells within the zF are characteristically polygonal and lipid-­rich, with fenestrated capillaries encasing each cord to facilitate rapid hormone circulation. The production of glucocorticoids is driven by pituitary ACTH, which acts through the MC2R receptor in complex with the accessory protein MRAP.19 The ultimate production of cortisol depends largely on two key enzymes, CYP17A1 and CYP11B1. CYP17A1 hydroxylase activity converts pregnenolone into 17α-­OH-­ pregnenolone, which is then converted into 17α-­OH-­progesterone by 3βHSD. This is then converted to 11-­deoxycortisol by CYP21 and into cortisol by CYP11B1. However, the majority of rodent species do not express Cyp17a1 in the adrenal cortex after fetal development.20 As a result, CYP11B1 instead converts DOC into corticosterone, which acts as the main glucocorticoid in these animals.

Zona Reticularis. The innermost zona reticularis (zR) contains cells spread across a mesh or net-­like structure (Latin rete, net), which function to produce androgens. These include dehydroepiandrosterone

(DHEA) and DHEA-­sulfate (DHEA-­S). DHEA is a weak androgen and estrogen receptor agonist itself but can be converted peripherally into more potent steroids, including testosterone and estradiol, and to a lesser extent, glucocorticoids.21 The zR is composed of polyhedral cells and first becomes morphologically distinguishable at 3 years of age in humans. It forms a continuous layer of cells by 6 years, when adrenal androgens synthesis significantly increases with the onset of adrenarche, and further enlarges until 13 years of age.21,22 The production of DHEA is mediated in large part by CYP17A1. As a result, rodents deficient for adrenal Cyp17a1 lack a functional zR and do not produce adrenal androgens. While CYP17A1 is expressed in both the zF and zR, lower expression of 3βHSD in the zR and high expression of cytochrome B5 (CYB5) and P450 oxydoreductase (POR) favors CYP17A1 lyase activity, thus rerouting 17α-­OH-­pregnenolone into DHEA synthesis. zR specific expression of SULT2A1 further allows production of DHEA-­S.21

Mouse X Zone and Rat Undifferentiated Zone. Mice and rats each contain an additional cortical region not found in the human adrenal gland (Fig. 85.3). The mouse adrenal cortex contains a transient inner zone called the “X zone” that is located at the cortical-­ medullary boundary.23 The origin of the X zone was debated for decades, but lineage-­tracing studies have now demonstrated that this region is a remnant of fetal cortex.24 The X zone is initially quite narrow just after birth and becomes most evident by 3 weeks of age before ultimately regressing in males after puberty and in females during pregnancy.23 The X zone is distinguished based on the expression of

CHAPTER 85  Adrenal Development and Homeostasis specific molecular markers, including 20-­α-­hydroxysteroid dehydrogenase (20αHSD)25 and phosphatidylinositol-­4-­phosphate 3-­kinase c2 domain-­containing gamma polypeptide (PIK3C2G)26. The function of the X zone remains poorly understood, though it has been hypothesized to participate in progesterone catabolism because 20αHSD catalyzes the reduction of progesterone and deoxycorticosterone into their 20α-­hydroxylated derivatives.25 However, the role of this enzymatic activity in the adrenal cortex of rodents remains unclear, as 20αHSD knockout adrenals are not distinguishable from their wild-­type counterparts.25 The rat adrenal cortex contains a unique region between the zG and zF known as the “undifferentiated zone” (zU).27 The zU is defined by a lack of zG or zF differentiation markers and contains undifferentiated progenitor cells that may help replenish the adrenal cortex.28 In mice, stem and progenitor cells are contained within the outer mesenchymal capsule and underlying zG, respectively.17,29,30

Adrenal Medulla. The adrenal medulla is derived from ectodermal neural crest cells and functions to produce catecholamines.31 Unlike steroid hormones produced by the adrenal cortex, catecholamines are derived from the amino acid tyrosine and are water-­soluble. Tyrosine is converted through a multistep process into dopamine followed by the predominant catecholamines, noradrenaline, and adrenaline (also called norepinephrine and epinephrine, respectively). Catecholamines are produced in chromaffin cells that are grouped around venous sinusoids, which facilitate their rapid circulation during the “fight-­or-­ flight” response. The release of catecholamines is directly controlled by preganglionic nerve fibers that provide a neural stimulus and connect chromaffin cells to the sympathetic division of the autonomic nervous system. Adrenaline acts as a hormone to enhance cardiac output through increased heart rate and blood pressure, and to increase glucose metabolism in the liver and muscle tissue. Noradrenaline is released in smaller amounts and acts as a neurotransmitter to promote vasoconstriction, thus increasing blood pressure. Vasculature. To facilitate the rapid delivery of hormones into the bloodstream, the adrenal gland contains a rich vascular network. Blood is supplied from three main arteries: the superior, middle, and inferior adrenal artery, which derive from the inferior phrenic artery, the abdominal aorta, and the renal artery, respectively.32 These major vessels form a plexus, or branched network of arteries, in the capsule, which then descend centripetally through the cortical layers. In the medulla, arteries form a capillary network that directs blood flow into the medullary vein before ultimately draining into either the inferior vena cava on the right side or the left renal vein. This extensive vascular network ensures that most cells are in close proximity (1–2 cells) to vascular endothelial cells, thus facilitating rapid hormone distribution throughout the body. Nerve Supply. In contrast to the cortex, where steroid production is controlled in large part by hormonal stimulation, catecholamine production from the medulla is regulated by synaptic stimulation. The medulla is innervated by preganglionic nerve fibers of the sympathetic nervous system that originate in the intermediolateral cell column of the spinal cord.33 These fibers form the greater splanchnic nerve, from which some fibers directly enter the adrenal gland to synapse at the membrane of chromaffin cells. Other fibers from the greater splanchnic nerve synapse at the celiac ganglion, whose postsynaptic fibers innervate blood vessels in the adrenal gland. Together, this network of nerve fibers enables the medulla to release its neurohormones in response to activation of the sympathetic nervous system.

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KEY POINTS  • The adrenal gland is composed of two distinct endocrine organs: the inner medulla derived from ectodermal neural crest cells that produces catecholamine hormones, and the outer cortex derived from intermediate mesoderm that produces steroid hormones. • The cortex is subdivided into three structurally and functionally distinct zones: the outer zona glomerulosa produces mineralocorticoids in response to angiotensin II as part of the renin-­angiotensin-­aldosterone system, the intermediate zona fasciculata produces glucocorticoids in response to adrenocorticotropic hormone as part of the hypothalamic-­pituitary-­adrenal axis, and the innermost zona reticularis produces androgens. • Much of our understanding about development and homeostasis of the adrenal gland is derived from studies in mice. While the mouse adrenal gland is similar in many respects to the human gland, there are some important differences: (1) the mouse adrenal cortex contains a transient inner zone not found in humans called the “X zone,” which is a remnant of fetal cortex, and (2) many strains of mice do not express CYP17A1 after fetal adrenal development. As a result, these animals synthesize corticosterone rather than cortisol as the main glucocorticoid, and they do not produce adrenal androgens.

EMBRYONIC ADRENAL DEVELOPMENT Overview The adrenal cortex is derived from the adrenogonadal primordium (AGP), which arises from thickening of the coelomic epithelium. The AGP extends from the dorsal aorta to the dorsal coelomic epithelia of the urogenital ridge.34 This bipotent cell population serves as a common embryological origin for both the adrenal gland and somatic cells of the gonad. The AGP is identified during development based on the expression of steroidogenic factor 1 (SF-­1, encoded by the gene NR5A1), which is observed in humans starting at approximately 4 weeks postconception35,36 and at embryonic day (E) 9 in mice.37 Cells on the dorsal aorta side progressively separate from the AGP to form the nascent adrenal cortex, while cells on the coelomic epithelial side give rise to the gonadal anlage.34 AGP separation begins during the fifth week of human gestation or between E10.0 and E10.5 in mice. Following AGP separation, neural crest cells invade the adrenal primordium (∼6 weeks human gestation or E13 in mice) to give rise to the adrenal medulla.38 These cells are initially dispersed but eventually coalesce after birth and differentiate into catecholamine-­producing chromaffin cells in the presence of glucocorticoids.39 Mesenchymal cells encapsulate the gland at approximately 9 weeks of human gestation or E14 to E14.5 in mice,38,40 and the cortex proceeds with functional zonation and differentiation (Fig. 85.4).

Adrenal Fate Determination AGP development and specification of the adrenal primordium is dependent upon a number of important factors, including several key nuclear hormone receptors and transcription factors. Because the AGP is a common precursor to both the adrenal and the gonad, disruption of these factors in humans or mice is typically associated with both adrenal and gonadal defects. The nuclear hormone receptors SF-­1 and DAX-­1 are among the most central and well-­studied. Their expression and activity are highly interdependent and must be tightly regulated. In addition, GATA family transcription factors, and particularly the balance between GATA6 and GATA4, helps direct adrenal versus gonadal fate. Following adrenal fate determination, growth factor signaling, including Wnt/β-­catenin, sonic hedgehog (SHH), and fibroblast growth factor (FGF), plays a critical role supporting the survival and further progression of the developing gland.

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PART 7  Adrenal 1. AGP formation Mouse: Human:

E9.0 ~4 weeks p.c.

2. AGP separation E10-10.5 ~5 weeks p.c.

3. Neural crest cell invasion E13 ~6 weeks p.c.

4. Encapsulation

E18.5 ~24-32 weeks p.c.

E14-14.5 ~9 weeks p.c. Capsule

Neural crest Adrenal anlage cells

Adreno-gonadal primordium (AGP)

5. Zonation

Zona Glomerulosa

Medulla Zona Fasciculata

Fetal adrenal cells Definitive adrenal cells

X zone

Primordial germ cells Bipotent gonad anlage Fig. 85.4  Embryonic adrenal development. The adrenal gland is derived from the adrenogonadal primoridum (AGP). The AGP forms at embryonic day (E) 9 in mouse and ∼4 weeks postconception (p.c.) in humans and serves as a common embryological origin for both the adrenal gland and somatic cells of the gonad. Following AGP separation (E10–10.5 in mice, ∼5 weeks p.c. in humans), neural crest cells invade the adrenal primordium and ultimately coalescence to form the medulla. The gland is encapsulated by surrounding mesenchyme (E14–14.5 in mice, 9 weeks p.c. in humans) before proceeding with functional zonation and differentiation (E18.5 in mice, ∼24–32 weeks p.c. in humans). Fetal adrenal cells give rise to definitive adrenal cells that comprise the adult cortex. In mice, some fetal adrenal cells remain in the cortex and form the X zone, which regresses in males during puberty and in females after pregnancy. (Adapted from Val, P and Swain, A. Molecular and Cellular Endocrinology. 2010;323:105–114.)

Steroidogenic Factor 1 (NR5A1). SF-­1 (also called adrenal 4 binding protein [Ad4BP]) is a master regulator of steroidogenic cell differentiation. Consistent with its expression early in AGP formation, Sf-­1deficient mice fail to develop adrenal glands or gonads and die soon after birth due to adrenal insufficiency.41,42 Rudimentary structures initially form in Sf-­1 null animals, but adrenal and gonadal tissues are fully extinguished by E13 through apoptotic cell death, suggesting that SF-­1 is necessary for the survival of early progenitor cell populations. Importantly, the actual dosage of Sf-­1 gene expression is a critical determinant of adrenocortical growth and differentiation. This is evident in Sf-­1 heterozygous mice, which have significantly smaller adrenal glands compared with controls,43 as well as impaired compensatory adrenal growth following unilateral adrenalectomy.44 Consistent with these observations in mouse models, patients with SF-­1 mutations have been found to have a broad spectrum of phenotypes, often including gonadal dysgenesis, and in some cases adrenal insufficiency.45-­48 Because the dosage of Sf-­1 is critical, tissue-­specific enhancers and promoter elements tightly control Sf-­1 activation and expression. Early in AGP formation, a 647-­bp fragment in the Sf-­1 promoter is sufficient for its expression.49 This region contains key Wilm’s tumor suppressor (WT1) response elements that are required for WT1-­mediated transcriptional activation (see the following section). Following AGP separation, tissue-­specific enhancer activity independently maintains Sf-­1 expression in the adrenal and gonad.50,51 In the adrenal primordium, a regulatory element located in the fourth intron drives expression. This element, termed the fetal adrenal enhancer (FAdE), is highly conserved

between mouse and human.50 However, FAdE shuts off by E14.5 in mouse, and Sf-­1 expression is then maintained in the adrenal cortex by a proposed, but yet to be identified, definitive adrenal enhancer.52

Wilm’s Tumor Suppressor. Expression of SF-­1 in the AGP is directly controlled by WT1.49 WT1 is a transcriptional regulator expressed early in the urogenital ridge.29,35,53 Consistent with SF-­1 as a direct WT1 target, Wt1 knockout (KO) mice fail to express SF-­1 and display adrenal and gonadal agenesis.54,55 The ability of WT1 to activate Sf-­ 1 expression is dependent on a transcription cofactor called Cited2.56 WT1 and Cited2 act together directly on the Sf-­1 promoter to drive expression above the critical threshold required for adrenal development.57 However, despite its critical role activating Sf-­1 expression early in AGP formation, WT1 is repressed in the adrenal primordium soon after separation, which is required for further adrenocortical maturation.29 While WT1 is not expressed in steroidogenic cells after AGP separation, it is expressed in a high proportion of mesenchymal cells that encapsulate the gland. This population of WT1+ capsular cells was identified experimentally using a tamoxifen-­inducible Wt1:CreERT2 driver combined with the R26RmTmG reporter.29 This approach permanently labels WT1-­expressing cells and their descendants after the administration of tamoxifen. Following tamoxifen at E11.5 and E12.5 (i.e., after AGP separation), a portion of the capsule is green fluorescent protein (GFP)+.29 Importantly, these capsular WT1+ cells are multipotent and have the capacity to give rise to adrenocortical cells, which can be

CHAPTER 85  Adrenal Development and Homeostasis visualized experimentally using lineage tracing. Tamoxifen injection at E12.5 followed by analysis at E18.5 onwards revealed that GFP-­labeled (i.e., WT1-­derived) steroidogenic cells (GFP+/SF-­1+/WT1-­) expand centripetally over time.29 These capsular WT1+ stem cells contribute at relatively low levels to normal development and homeostasis, but can be activated in response to extreme stress, including gonadectomy29 (see section on Tissue Stress Progenitors and Adrenal Aging).

DAX-­1 (NR0B1). One of the most highly studied SF-­1-­target genes in the developing adrenal is DAX-­1.47 This atypical nuclear hormone receptor is expressed in the AGP shortly after the onset of SF-­1 expression.58,59 DAX-­1 alters the transcriptional activity of SF-­1 in a context-­ dependent manner, most traditionally through the recruitment of corepressors that function to inhibit steroidogenesis.60 Mutations in DAX-­1 in humans cause X-­linked adrenal hypoplasia congenita, which often presents with adrenocortical failure.61,62 Dax-­1-deficient mice initially have a mild phenotype characterized by retention of the X zone.63 However, loss of Dax-­1 partially rescues Sf-­1 haploinsufficiency,64 and aged animals display significant adrenocortical dysplasia.65 These results are consistent with DAX-­1 as a key mediator of SF-­1-dependent adrenal development and function.

GATA factors. GATA4 and GATA6 are both expressed in the AGP and are essential transcriptional regulators of embryonic development. However, whereas GATA6 expression is maintained in the adrenal cortex throughout development and postpartum, GATA4 expression in steroidogenic cells is extinguished as early as E12.5.29,66,67 Combined inactivation of both Gata4 and Gata6 using Sf-­1:Cre results in adrenal aplasia.68 In contrast, mice with isolated inactivation of Gata6 have adrenal hypoplasia characterized by a thin cytomegalic cortex, overt hypoaldosteronism, and subclinical hypocorticosteronism.26 A striking observation in these mice is upregulation of GATA4 and gonadal-­like markers at the expense of adrenal markers, which is consistent with the idea that GATA6 promotes adrenocortical differentiation, whereas GATA4 drives gonadal differentiation.69-­71 Interestingly, mice lacking the histone methyltransferase EZH2 in steroidogenic cells also show adrenal hypoplasia and glucocorticoid insufficiency.67 Although GATA6 expression is unaltered in these mice, GATA4 and a number of gonadal differentiation markers (e.g., Pdgfrα, Foxl2, and Lhr) are also markedly upregulated. Altogether, these data demonstrate that the balance between GATA6 and GATA4 expression has a profound impact on adrenal versus gonadal differentiation. Wnt/β-­catenin signaling. Canonical Wnt/β-­ catenin signaling is one of the main pathways required for early adrenal development. This pathway, which plays an essential role in the development and homeostasis of multiple tissues, relies on extracellular secreted Wnt ligands and the multifunctional protein β-­catenin.72 In the absence of ligands, the cytoplasmic pool of β-­catenin is maintained at low levels. This is mediated by phosphorylation of key β-­catenin residues by serine/threonine kinases CK1α and GSK3β that are part of the so-­called destruction complex, which also comprises the tumor suppressors APC and AXIN. Phosphorylated β-­catenin is recognized and ubiquitinylated by the E3 ubiquitin ligase βTrCP, which promotes proteasomal degradation of the protein. This inhibits activation of the pathway by preventing nuclear translocation of β-­catenin and stimulation of TCF/ LEF-­dependent transcriptional activity. Upon Wnt ligand binding to Frizzled (FZD)/LRP5-­6 receptor complexes, the destruction complex is sequestered to the plasma membrane, thus preventing phosphorylation and subsequent degradation of β-­catenin. Stabilization of β-­ catenin facilitates its nuclear translocation and interaction with TCF/

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LEF family members, which triggers expression of canonical Wnt target genes, such as Axin2, Lef1, or Apcdd1.73-­75 Further levels of fine-­tuning of the pathway also exist at the plasma membrane. Specifically, FZD receptors are ubiquitinylated by membrane E3 ubiquitin ligases ZNRF3 and RNF43,76,77 which are targets of the canonical Wnt pathway.76 This allows establishment of a negative feedback loop, preventing deregulated pathway activation. This negative regulation mechanism can be counteracted by secreted R-­Spondins (RSPOs),78 which induce membrane clearance of ZNRF3 and RNF43 through both LGR-­ dependent and LGR-­ independent mechanisms.79,80 In addition to the RSPO-­ZNRF3/RNF43 signaling module, canonical Wnt/β-­catenin activation can also be negatively controlled by other mechanisms, including secreted FZD-­related proteins (SFRPs) and (DKKs) glycoproteins.81 Activation of canonical Wnt/β-­catenin signaling begins early in adrenal development. In mice, immunohistochemistry for β-­catenin and TCF/LEF-­LacZ reporter activity both demonstrate that signaling is first initiated between E12.5 and E14.5.82 Pathway activation is highest just beneath the capsule in the underlying zG. Complete inactivation of Ctnnb1 (the gene encoding β-­catenin) using a high efficiency Cre expressed in all SF-­1–positive cortical cells (Sf-­1-­Crehigh)83 results in severe loss of adrenocortical cells by E14.5 and complete adrenal aplasia by E16.5.82 These results suggest that canonical Wnt/β-­catenin signaling is required for early adrenal development. Interestingly, the converse experiment in which β-­catenin is constitutively activated throughout the adrenal cortex early in development results in a similar outcome. Approaches to either directly (Sf-­1-­Crehigh:Catnblox(ex3) mice) or indirectly (Sf-­1-­Crehigh:Apcfx/fx) stabilize Ctnnb1 result in significant adrenal hypoplasia by E16.5.84,85 These results suggest that the dosage of Wnt/β-­catenin activation is critically important for proper adrenal development. Several additional models that alter the level of Wnt activation have been subsequently generated that bypass the early β-­catenin-dependent developmental defects and offer insight into the role of Wnt/β-­catenin signaling during postnatal adrenal development and homeostasis (see section on Adrenal Cortex Regeneration).

Sonic Hedgehog Signaling. Similar to Wnt/β-­catenin signaling, the hedgehog (HH) pathway is a paracrine signaling mechanism driven by secreted factors that is required for the growth and patterning of a wide range of tissues.86 The three mammalian orthologs, sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH), act by binding to the transmembrane receptor Patched1 (PTCH1). In the absence of these ligands, pathway activation is blocked by PTCH1, which represses the transmembrane protein smoothened (SMO). Ligand binding derepresses SMO through PTCH1 inactivation, resulting in intracellular signal transduction and activation of the GLI family of transcription factors. These include GLI1, which is a transcriptional target of active HH signaling and functions in a feedforward loop, as well as GLI2 and GLI3, which are primarily regulated posttranscriptionally. The activation of GLI transcription factors ultimately stimulates a wide range of target genes that promote cell proliferation and help direct tissue patterning. In the adrenal gland, the HH pathway was first implicated in adrenal development when mutations in SHH and other components were identified in patients with holoprosencephaly and Pallister–­Hall syndrome.87-­90 These patients have endocrine disorders, including adrenal hypoplasia, which suggested that SHH signaling may be critical for adrenal specification and growth. Consistent with this hypothesis, in situ hybridization studies found strong expression of SHH mRNA as early as E12.5 throughout the peripheral cortex of the developing gland.17,91,92 Functional studies targeting SHH for deletion throughout the cortex (Sf-­1-­Crehigh:Shhfx/fx) resulted in significantly smaller adrenal glands in mice, consistent with a critical role for SHH signaling in

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PART 7  Adrenal

early adrenal development.17,92,93 Expanding upon these KO models, lineage-­tracing studies further demonstrated that SHH-­expressing cortical cells in the periphery of the gland give rise to cells of the inner cortex through centripetal migration and differentiation. In these studies, a tamoxifen-­inducible SHH:CreERT2 driver was combined with an enhanced GFP (EGFP)-­based lineage reporter to label and track the SHH lineage over time.17 Following the administration of tamoxifen at E14.5, SHH-­derived steroidogenic cells (EGFP+/SCC+) were observed in the peripheral cortex after 5 days and extended into longer vertical columns by 28 days. These results demonstrate that, during embryonic development, Shh-­expressing cells help to populate the inner cortex. Although SHH is expressed in the peripheral cortex, its receptor, PTCH1, is expressed in the capsule.93,94 Consequently, SHH ligands produced in the zG act in a paracrine fashion on capsular cells to activate downstream signaling. Consistent with this paradigm, GLI1, which acts in a feedforward loop to promote active SHH signaling, is also expressed in the capsule.17,93,94 Gli1+ capsular cells act as multipotent stem cells to give rise to adrenocortical cells during embryonic development, which has been visualized by lineage tracing experiments. Using Gli1:CreERT2 combined with an EGFP-­based reporter, cortical cells derived from the embryonic Gli1 lineage were tracked over time and found to progressively extend from the capsule into the cortex.17 Notably, SHH signaling and the GLI1 lineage also significantly contribute to homeostatic maintenance of the adrenal cortex (see section on Adrenal Cortex Regeneration).

Fibroblast Growth Factor Signaling. The FGF signaling pathway is critical for the growth and development of many tissues.95 The pathway is comprised of 22 secreted FGF ligands and four FGF receptors (FGFRs), with an additional level of complexity through alternative splicing of FGFR1 to 3. Notably, FGFs bind heparan and heparan sulfate, which results in their sequestration to the extracellular matrix. This allows for local release of ligands by heparan sulfate proteoglycans during tissue morphogenesis or in response to injury. Once activated, FGF signaling is generally transduced through activation of RAS/MAP kinase, PI3/AKT, or PLCγ, which promote mitogenic growth, cell survival, and cell migration. In the developing adrenal gland, all four receptors have been detected,96,97 along with three primary ligands.97 These include Fgf2 and Fgf9, which are expressed in the capsule, and Fgf1, which is expressed in the cortex. Functional studies have primarily focused on loss of Fgfr2, which results in significantly impaired adrenal growth. Mouse models of germline loss of Fgfr2-­IIIb,97,98 as well as conditional Fgfr2 deletion,15,99,100 consistently show a significant reduction in adrenal size that is often associated with lower rates of proliferation and increased apoptosis. Analysis of glomerulosa structure has further demonstrated that the loss of Fgfr2 impairs the formation of rosettes and reduces zG function.15 Together, these studies demonstrate that FGF signaling is critical for adrenocortical cell growth and proliferation, particularly in the early stages of development. KEY POINTS  • The adrenal cortex is derived from the adrenogonadal primordium (AGP), which separates to give rise to both the adrenal gland and somatic cells of the gonad. • Following AGP separation, neural crest cells invade the adrenal primordium to give rise to the medulla. These cells progressively coalesce and differentiate into catecholamine-­producing chromaffin cells. • Finally, mesenchymal cells encapsulate the gland and the cortex proceeds with functional zonation and differentiation. • Proper specification and development of the adrenal gland is dependent upon a number of important factors, including nuclear hormone receptors

(e.g., steroidogenic factor 1, Wilm’s tumor suppressor, and DAX-­1), transcription factors (e.g., GATA4 and GATA6), and growth factor signaling (e.g., Wnt/β-­catenin, sonic hedgehog, and fibroblast growth factor). • The initial adrenal primordium is composed of a large fetal zone that functions to produce high levels of dehydroepiandrosterone, dehydroepiandrosterone sulfate, and cortisol, and a smaller definitive zone that ultimately becomes the adult cortex after fetal zone involution.

Fetal Adrenal Development

Fetal Zone Versus Definitive Zone. The initial adrenal blastema is composed of two distinct zonal compartments: a large region of fetal zone (FZ) cells surrounded by a smaller adult, definitive zone (DZ). In humans, the fetal adrenal cortex grows disproportionately after encapsulation.38,101 This expansion within the FZ enables the adrenal gland to significantly increase in size (nearly 10-­fold), becoming one of the largest organs at term and comparable in size to the kidney. Surrounding the FZ, there is a narrow band of definitive cortex. The DZ initially contains densely packed basophilic cells that are lipid poor. However, these cells are proliferative and begin to accumulate more cytoplasmic lipid after midgestation as they become increasingly steroidogenic. A so-­called transitional zone (TZ) takes shape between the DZ and FZ and is capable of producing cortisol. By late gestation, the fetal adrenal begins to resemble the adult cortex, with the DZ and TZ taking on the morphology and function of the zG and zF, respectively. The FZ undergoes involution after birth (see section on Fetal Zone Involution), causing a significant decrease in adrenal weight just after birth.

Function of the Fetal Zone. The FZ is comprised of large eosinophilic cells with high steroidogenic activity. Specifically, FZ cells strongly express CYP17A1, which converts pregnenolone to DHEA. This results in the production of large amounts of DHEA and DHEA-­S (up to 200 mg per day) that are then converted by the placenta into estrogens.101 As the human placenta cannot make estrogens de novo, the generation of estrogens through this process is critically important to help maintain normal pregnancy. In addition to DHEA, CYP17A1 also mediates cortisol production in the FZ, which promotes maturation of the fetal organ system. Thus, the FZ primarily functions to produce steroids that help maintain intrauterine development. Adrenocorticotropic Hormone-Driven Fetal Zone Growth. The principal regulator of fetal adrenocortical development is ACTH. This tropic regulator of the postnatal adrenal cortex is known to promote both hypertrophy and hyperplasia of adrenocortical cells. However, rapid FZ growth predominately results from hypertrophy, whereby the size of each cell rather than the number of cells increases.101 Unexpectedly, rather than increasing during FZ expansion, plasma ACTH levels decrease by nearly 50% during prenatal adrenal development.102 This paradox is partially explained by the progressive sensitization of FZ cells. While not maximally sensitive initially, FZ cells become more ACTH-­responsive over time through mechanisms that include increased expression of the ACTH receptor (and thus enhanced ACTH binding capacity).103,104 This adaptive ACTH response is thought to help prepare the fetus for the stress of birth and postnatal development by increasing the steroid-­producing capacity of the FZ.

POSTNATAL ADRENAL DEVELOPMENT Fetal Zone Involution

Human Adrenal. The first notable event in postnatal adrenals is the rapid involution of the FZ, followed by progressive expansion and zonation of the adult definitive cortex (Fig. 85.5). Whereas the FZ

CHAPTER 85  Adrenal Development and Homeostasis

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Mouse adrenal cortex E14.5

E18.5-P0

Establishment of zonation Medulla Fetal cortex Definitive cortex Zona Reticularis (zR) Zona Fasciculata (zF) Zona Glomerulosa (zG) Capsule 9 weeks

Puberty/pregnancy

Adult

Regression of X zone

Human adrenal cortex 24-32 weeks

Birth to 8 months

6 to 8 years

Regression of Adrenarche Establishment of zonation fetal zone Fig. 85.5  Pre-­and postnatal adrenal development in mice and humans. Functional zonation of the adrenal cortex starts at embryonic day 18.5 in mouse and between 24–32 weeks in humans. Whereas the fetal zone immediately regresses in humans after birth, its regression is delayed until puberty in male mice or first pregnancy in female mice. In human adrenals, zona reticularis develops during adrenarche (6–8 years), between the zona fasciculata and the medulla. There is no zona reticularis in the mouse adrenal cortex.

represents up to 70% of total adrenal volume at birth (up to 85% of the cortex), it only takes up 3% of the gland after 8 months.105 FZ involution is evident immediately after birth and can be divided in two phases.105,106 The first phase is characterized by rapid regression of FZ until the end of the second week postpartum. This phase is associated with hemorrhage and high levels of apoptosis, which is maximal during the second week of life (30% of FZ cells). The second phase is characterized by slower involution and relatively constant levels of apoptosis (20% of FZ cells). The FZ mainly synthesizes androgens, and during fetal life, concentrations of DHEA follow the increase in FZ size in the last two trimesters of pregnancy.107 Following FZ involution, adrenal androgen levels rapidly decline until the onset of adrenarche at around 6 to 9 years.108,109 The mechanisms involved in FZ regression in the human adrenal gland remain elusive. Early reports showed that levels of plasma DHEA-­S and urinary 3β-­OH-­5ene steroids remained elevated for many weeks following parturition in preterm babies, consistent with persistence of the FZ in this context. This suggested that FZ involution was controlled by the duration of gestation rather than by parturition itself.110 However, more recent evidence relying on ultrasonography and measurement of serum cortisol and androstenedione concentrations reported similar adrenal involution, and endocrine activity in preterm and full-­term babies.109 This suggests that parturition itself triggers FZ involution. A number of factors have been proposed to control human FZ maintenance and involution (Fig. 85.6). Anencephalic fetuses display marked adrenal atrophy, and in vitro treatment of human fetal adrenal cells with ACTH stimulates their growth and endocrine activity.111–115

Although fetal ACTH is required to support normal FZ growth and activity during fetal life, there is no decrease in ACTH at the time of FZ involution,110,115 suggesting that this process is dependent on other mechanisms. Experimental evidence suggests involvement of transforming growth factor β (TGFβ) signaling, and in particular of activins and inhibins, in this process. These are peptide hormones formed by the dimerization of α, βA, and βB inhibin subunits, encoded by three distinct genes. Activins A or B are formed by homodimerization of βA or βB subunits, respectively, whereas inhibins A or B are formed by heterodimerization of α subunits with either βA or βB subunits, respectively. Activins signal through ACVRI and ACVRII activin receptors, which results in phosphorylation of SMAD2 and SMAD3. These interact with SMAD4 and migrate to the cell nucleus, where they trigger expression of target genes that can be involved in apoptosis. Inhibins, on the other hand, can sequester ACVRII by interacting with betaglycan. This results in inhibition of activin signaling.116 βA and βB subunits are expressed throughout human adrenal cortex, with predominant expression in zG, whereas the α subunit is mostly found in the zR and in the fetal adrenal.117-­120 ACVRI/II receptors and betaglycan are expressed in the adult and fetal adrenal cortex.121 Fetal adrenal cells also express SMADs3/4/6/7, suggesting that inhibin/ activin signaling may play a role in FZ involution. Consistent with this idea, TGFβ and activin A were shown to specifically inhibit growth and induce apoptosis of human FZ cells in culture.106,118,122 This suggests that these TGFβ/activin/inhibin family members may play a role in FZ involution. However, this would require induction of activin signaling in the human fetal adrenal after birth. Interestingly, evaluation of

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PART 7  Adrenal Spgl1 Acd T3

Fetal zone maintenance

Activin

Androgens Parturiton

Fetal zone regression

Inhibin

SF-1 SUMO DAX-1 Fig. 85.6  Factors involved in maintenance or regression of the fetal cortex. Hormonal manipulations and mouse genetics have shown that activins, androgens, and parturition favor fetal cortex regression. This can be counteracted by inhibin, which can be stimulated by steroidogenic factor 1 (SF-­1). This effect of SF-­1 is inhibited by its sumoylation and by the antagonistic activity of DAX-­1. In contrast, Spgl1, Acd, and triiodothyronine may be involved in maintenance of the fetal cortex. Most of the molecular mechanisms involved in these processes remain elusive.

activins and inhibins in the artery and vein of the umbilical cord of term infants showed that the placenta was the major source of inhibin B, whereas the fetus was the main producer of activin A.123 It is therefore tempting to speculate that parturition, by removing a major source of inhibin, could alter the activin/inhibin ratio to favor activin signaling and FZ involution. Although these mechanisms are difficult to study in humans, studies in mouse models have provided further insight into potential regulators of FZ homeostasis (Fig. 85.6).

Mouse Adrenal. The mouse X zone (see section on Mouse X Zone and Rat Undifferentiated Zone) is exclusively derived from fetal adrenal cells that express the fetal enhancer of Sf-­1,24,50,124 and may thus be the mouse equivalent to the human FZ. However, it does not express CYP17,20 which does not allow production of adrenal androgens. Rather, the X zone is characterized by expression of 20αHSD,25 which appears at around 3 weeks of life.25,125,126 Another key feature that distinguishes X zone from the human FZ is the kinetics of its involution (Fig. 85.5). Indeed, whereas the FZ regresses postnatally in both sexes in humans, X zone involution is observed after around 4 months or during the first pregnancy in females and after puberty in males.23,25,127 Role of Sex Hormones and Prolactin. The sexual dimorphism associated with X zone regression suggested an involvement of sex hormones in this phenomenon. Indeed, injection of testosterone propionate or the nonaromatizable androgen dihydrotestosterone (DHT) to nulliparous adult females results in X zone regression, which can be reversed upon removal of DHT after 7 days of treatment. Conversely, gonadectomy in prepubertal males prevents X zone regression and results in growth of a secondary X zone in postpubertal males.23,25,128,129 This indicates that androgens, but not estrogens, directly induce involution of the X zone in male mice at puberty. These observations have been further confirmed by the targeted ablation of the androgen receptor (AR) in steroidogenic cells of the adrenal cortex, which resulted in a dramatic reduction in apoptosis and X zone maintenance in male adrenals at puberty. Interestingly, AR ablation resulted in maintenance of a larger X zone than castration alone, suggesting that AR may exert androgen independent effects on X zone homeostasis129 (Fig. 85.6).

The mechanisms associated with X zone regression during pregnancy are a lot more elusive. Concordant observations have shown a rapid decline in 20αHSD activity and morphological X zone during pregnancy, which are not restored after parturition.23,25,125,127 Ovariectomy is associated with transient X zone thickening,130 and progesterone injection induces rapid regression of the X zone.125,131 However, this effect is only observed in females with intact ovaries,131 suggesting that the effect of progesterone is dependent on its conversion to androgens by the ovary, particularly during pregnancy.132 Consistent with this, testosterone peaks at 9 days postcoitum (dpc), which is coincident with onset of X zone regression between 8 and 12 dpc in gravid females.25,133 Induction of pseudopregnancy by mating female mice with vasectomized males has no effect on X zone morphology and 20αHSD activity, compared with nulliparous females.25 This suggests that the elevation of progesterone and estrogens induced during the first 8 days of pseudopregnancy is not sufficient to trigger the first phases of X zone involution, even though hormonal levels are comparable with pregnancy. Interestingly, removal of pups, which results in decreased prolactin secretion in deprived dams, is associated with partial recovery of 20αHSD activity and partial regrowth of the X zone.25 This suggests that the rise in prolactin secretion during pregnancy and before weaning may be involved in X zone involution. However, prolactin injection to nulliparous females has no impact on X zone homeostasis. Similarly, inactivation of STAT5 signaling pathway, which plays a central role in prolactin response, has no impact on the X zone of either males or females.25 Altogether, these data suggest that the prolactin increase induced by suckling may have an impact on X zone involution in primiparous females, but does not significantly contribute to regression during pregnancy or at puberty in males.

Role of Thyroid Hormones. Thyroxin and thyroid extracts induce enlargement of a histological X zone in the adrenal cortex of mice.134,135 Conversely, hypothyroid Hyt mutant mice that harbor a mutation of the thyrotropin receptor, have a poorly developed X zone,136 suggesting that thyroid hormones and their receptors may play a role in X zone homeostasis. Recent evidence, based on a LacZ reporter inserted at the Thrb locus, showed that thyroid hormone receptor beta 1 (TRβ1) is expressed in mouse inner adrenal cortex from E14.5 into the postnatal period. Its expression pattern is reminiscent of and partly overlaps with that of 20αHSD. However, it is more broadly expressed, expanding in the inner zona fasciculata, and although it declines in males by postnatal day 28, it persists after pregnancy in females. Interestingly, hormonal treatments at different time points showed that triiodothyronine (T3) could accelerate appearance of the 20αHSD+ zone after birth, induce hypertrophy of the X zone before puberty, and counteract X zone regression at puberty in males, although it could not induce regeneration after regression.126 Analysis of Thrb-­deficient mice showed that these effects of exogenous T3 were dependent on the thyroid hormone receptor. However, Thrb-­/-­ adrenals were indistinguishable from wild-­type at baseline.126 Although this may reflect compensation by other thyroid hormone receptors, it suggests that thyroid hormones are not required for normal development of the X zone, but that they may be involved in modulating its homeostasis and function. Whether this has relevance to the human fetal adrenal cortex is currently unknown. Role of Activin/Inhibin Signaling. Consistent with findings in human adrenals, the activin/inhibin/TGFβ signaling pathway may also control fetal adrenal cell growth in mouse adrenals (Fig. 85.6). Indeed, all four subunits of activin receptors (ACTR-­IA, ACTR-­IB, ACTR-­IIA, and ACTR-­IIB) are expressed in the mouse adrenal cortex, and ACTR-­IA, ACTR-­IIB, and SMAD2, an essential component

CHAPTER 85  Adrenal Development and Homeostasis of the TGFβ signaling pathway, are expressed more specifically in the X zone.137 Interestingly, activin treatment has a specific growth inhibitory effect on primary cultures of adrenals containing X zone cells and is able to induce apoptosis in the presumptive X zone of whole adrenals in culture.137 The potential role of activin in controlling X zone homeostasis is further supported by accelerated X zone regression in inhibin-­deficient mice that show increased levels of gonadal and adrenal activin secretion.137,138 This effect is counteracted by gonadectomy, which induces unrestricted growth of presumptive X zone cells and SMAD3-­dependent adrenal cortex tumor development.137,139 However, there is no reported adrenal gland phenotype in mice deficient for activin.140

Genetic Models Associated with Failure to Develop an X Zone. A number of genetically engineered or mutant mice display X zone aplasia. The adrenocortical dysplasia gene (ACD) encodes TPP1, which plays a central role in telomere protection within the TRF1 complex.141 Mice harboring a spontaneous homozygous splice donor mutation in the Acd gene display multiple developmental defects, including cytomegalic adrenocortical dysplasia and glucocorticoid insufficiency. They are also characterized by X zone aplasia.142,143 Adrenal aplasia is associated with massive induction of cellular senescence and can be partly reversed by genetic inactivation of Tp53.144 However, it is unclear if reduction of senescence following Tp53 inactivation also rescues X zone differentiation.144 Sgpl1 mutant mice also show X zone aplasia.145 Sgpl1 encodes the enzyme responsible for the last step of sphingosine 1 phosphate (S1P) catabolism, which works as a signaling molecule. Interestingly, SGPL1 is expressed in the human fetal adrenal (from Carnegie stage 19 onwards) in both the FZ and DZ. In the adult adrenal, SGPL1 expression is found throughout the cortex, but is more concentrated in the zR. Besides X zone aplasia, Sgpl1-­/-­ mice also show altered cortical zonation and atrophic zF, associated with lower CYP11A1 and CYP11B2 expression. This is consistent with the primary adrenal insufficiency observed in patients with homozygous inactivating mutations in SGPL1.145,146 The molecular mechanisms underpinning adrenal insufficiency and X zone aplasia in this context are unclear. However, Sgpl1 inactivation results in accumulation of S1P,145,147 which can be converted back to sphingosine by S1P phosphatases. Whereas S1P has been shown to stimulate steroidogenesis in H295R cells in culture,148 sphingosine is an inhibitory ligand for SF-­1 and acts to maintain it in an inactive conformation.149,150 This suggests that alterations of SF-­1 activity may be involved in X zone homeostasis (Fig. 85.6).

Genetic Models Associated with Persistent X Zone. Activity of the nuclear receptor SF-­1, the master regulator of steroidogenic tissues development and differentiation,151 can be regulated by a number of posttranslational modifications, including phosphorylation,152 acetylation,153 and sumoylation.154-­156 This modification, which consists in the reversible addition of SUMO modules on two lysine residues of SF-­1 (K119 and K194), does not have a global effect on SF-­1-mediated transcription, but rather dampens recognition of a subset of SF-­1 target genes, coined SUMO-­sensitive targets, which are essentially transcribed when SF-­1 is hyposumoylated.156 In vivo, the replacement of the two lysine residues by nonsumoylatable arginine residues, through homologous recombination (SF-­12KR), results in abnormal adrenal development. This is characterized by aberrant expression of gonadal markers (Sox9, Amhr2, Aldh1a1), upregulation of HH signaling, hypoaldosteronism, and persistence of a 20αHSD-­positive X zone after puberty, even though it eventually regresses later in life.157,158 Although the molecular mechanisms underlying persistence of the FZ were not evaluated in this model, previous data had shown that inhibinα is a

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SUMO-­sensitive target of SF-­1, the expression of which is upregulated by nonsumoylatable SF-­1 mutants.156 This may account for an imbalance in activin/inhibin signaling that would prevent normal apoptosis of the X zone (Fig. 85.6). DAX-­1 is an atypical non-DNA-­binding nuclear receptor that has been shown to repress SF-­1 transcriptional activity159-­161 (see section on DAX-­1 (NR0B1)). DAX-­1 mutations in patients are associated with X-­ linked adrenal hypoplasia congenita (AHC).61,62 Patients with AHC present with neonatal adrenal insufficiency associated with cytomegalic hypoplastic adrenals.162 Surprisingly, mice with Dax-­1 gene ablation have no signs of adrenal insufficiency in early life, but show temporary persistence of the X zone after puberty.63,158 Interestingly, analysis of the fetal adrenal lineage using the fetal adrenal enhancer of Sf-­1 to drive expression of a LacZ reporter gene (FAdE:LacZ) shows persistence of FAdE activity in 20αHSD+ cells of both Sf-­12KR and Dax-­1 KO adrenals, confirming their fetal rather than adult origin.158 Altogether, these data suggest that stimulation of SF-­1 transcriptional activity, either by inhibiting its sumoylation or by inactivation of a key repressor, can result in temporary persistence of the X zone. Regression of the X zone may thus be mediated by inhibition of SF-­1 activity (Fig. 85.6). In cell culture, sumoylation of SF-­1 can increase interactions between SF-­1 and DAX-­1, resulting in almost complete inactivation of Sf-­1 fetal enhancer activity.158 This may provide a mechanism that would explain extinction of Sf-­1 expression in fetal adrenal cells, resulting in their death, consistent with the role of SF-­1 in the survival of steroidogenic cells.41,163,164 The recent observation that PKA signaling inhibits sumoylation in the adrenal cortex may add another level of control to these processes.165 KEY POINTS  • The fetal cortex regresses after birth in both mouse and human. Whereas human fetal cortex regresses immediately after birth, regression is delayed until puberty in male mice and after the first pregnancy in female mice. • Involution of the fetal cortex involves apoptosis and is mostly controlled by activin/inhibin/transforming growth factor β signaling. This may also involve sex hormones, particularly in mice. • Genetically engineered mouse models show that tampering with SF-­1 dosage has a key role in controlling homeostasis of the fetal zone. • Lineage-­tracing studies in mice have shown that the definitive cortex, which takes over the entire gland after fetal zone regression, originates from cells of the fetal cortex during embryonic development.

Origin of the Definitive Cortex. Lineage-­tracing studies relying on FAdE have shown that most of the definitive cortex is derived from FAdE+ fetal adrenal cells at E11.5. However, the capacity of FAdE+ cells to give rise to adult cells is lost after E14.5.24,50 Stronger expression of DAX-­1 in cells of the definitive cortex is thought to play a role in the extinction of FAdE in this lineage. In these cells, Sf-­1 expression is potentially maintained through mobilization of an adult enhancer that has yet to be identified.24,50 In addition, analysis of the role of SHH signaling in the adrenal cortex showed that SF-­1-­/GLI1+ capsular cells could give rise to some definitive cortical cells at E14.5 and onwards, albeit with low efficiency.17,93 Further lineage-­tracing studies suggest that a very small subset of SF-­1-­/GLI1+ cells (∼7%) may be derived from FAdE+ cells.166 However, such capsular lineage is not found with Sf-­1-­Cre, suggesting that this may be the result of ectopic expression of the short FAdE construct. Altogether, these observations suggest that both fetal SF-­1+/FAdE+ and capsular SF-­1-­/GLI1+ cells can give rise to the definitive cortex. Whether similar mechanisms also exist in human

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adrenals is unknown, but the high conservation of FAdE sequences in humans is in support of this hypothesis.

Establishment of Adrenal Cortex Zonation Functional zonation is established in cells of the definitive cortex in the perinatal period. It manifests as the establishment of three (primates) or two (most rodents) concentric zonae with distinct histological and functional features, that undertake the endocrine activity of the gland167 (see section on Adrenal Cortex). In rodents’ adrenals, zonal restriction of steroidogenic enzymes expression is established during embryonic development. CYP11B1 (zF) is expressed in the entire cortex from E13.5 onwards. In contrast, CYP11B2 and angiotensin receptor AT1b (zG) are expressed at the periphery of the gland after E18.5 (Fig. 85.5). At this stage, CYP11B1 and CYP11B2 are expressed in mutually exclusive areas of the cortex.17,168-­170 In human adrenals, steroidogenic enzymes responsible for glucocorticoids and androgens synthesis are readily detected during the first trimester, mostly in the fetal and transition zones. Their expression markedly increases during the second trimester, concomitant with increased production of glucocorticoids.171-­173 Consistent with the lack of aldosterone production during the first and second trimester,172 CYP11B2 appears in the DZ at 24 weeks of gestation and is maximal at 32 weeks172,174 (Fig. 85.5). Establishment of these zonal expression patterns is the basis for functional adult adrenal cortex zonation, which is maintained throughout life.169,175-­177 Genetic and cell lineage tracing studies in mouse models have allowed understanding of the molecular mechanisms involved in establishing and maintaining these differentiation patterns at homeostasis and in response to hormonal stimuli and cell renewal.

Differentiation of Zona Glomerulosa. Ang II and plasma potassium concentrations are the main regulators of zG endocrine activity.178 Activation of the renin-­angiotensin system following hyponatremia or hyperkaliemia induces a rapid expansion of zG, an increase in the number of CYP11B2+ cells, and an induction of CYP11B2 expression, allowing for increased aldosterone secretion. Conversely, inhibition of the renin-­angiotensin system using drugs such as Captopril results in a rapid atrophy of zG.179-­181 However, mice deficient for the two type I receptors of Ang II (Agtr1a-­/-­;Agtr1b-­/-­) have normal adrenal histology.182 This indicates that Ang II signaling is not involved in the establishment and initial differentiation of zG, even though it plays a central role in remodeling the zone to modulate the supply of mineralocorticoids. Consistent with this idea, ectopic chemogenetic activation of Gαq signaling, which is downstream of angiotensin receptors, results in ectopic expression of CYP11B2 within the zF and increased renin-­independent aldosterone production.183 Over the last decade, analysis of genetically engineered mouse models has allowed identification of the canonical Wnt/β-­catenin signaling pathway as one of the key regulators of zG differentiation and homeostasis15,85,184-­186 (Fig. 85.7). Whereas β-­catenin is activated throughout the developing adrenal cortex at E12.5, its activity is highest in the presumptive zG by E18.5.82 In the adult, β-­catenin is expressed and accumulated at high levels throughout the zG.82,85,184,185 This area is also characterized by accumulation of the Wnt targets LEF1 and AXIN2 and by expression of Wnt reporter constructs.82,85,184,185 This suggests that strong Wnt pathway activation in zG cells may play a role in the differentiation of this zone. Consistent with this idea, conditional inactivation of Ctnnb1 (the gene encoding β-­catenin) within zG cells, using Cyp11b2-­Cre as the driver, results in smaller and morphologically altered glomeruli.15 Conversely, constitutive activation of β-­catenin within CYP11B2+ cells (Cyp11b2-­Cre driver) results in continuous expansion of zG, increased rosette frequency, and a significant increase in plasma aldosterone

(albeit without renin suppression).15,186 This is further confirmed by stochastic activation of β-­catenin throughout the entire cortex (Akr1b7-­Cre), which results in zG expansion, ectopic differentiation of scattered zG cells within the zF, and elevated plasma aldosterone levels.85,187 The observation of primary aldosteronism in these models suggested that deregulated Wnt pathway activity could be involved in hyperaldosteronism in patients. Consistent with this idea, 33/47 patients with aldosterone-­producing adenomas (APA) had constitutive Wnt pathway activation.188 This can result from activating mutations of CTNNB1, found in approximately 5% of APA patients189,190 or from downregulation of Wnt inhibitors such as SFRP2.188 Altogether, these data show that β-­catenin signaling plays a central role in zG differentiation. Mechanistic investigations have shown that β-­catenin, either directly or indirectly through NURR1 and NUR77 nuclear receptors, stimulates transcription of CYP21, CYP11B2, and AT1R.188 This provides an explanation for increased aldosterone production following Wnt pathway activation. However, this does not account for increased rosette formation. Interestingly, the growth factor receptor FGFR2 is upregulated in response to constitutive β-­catenin activation and its genetic inactivation in CYP11B2+ cells disrupts rosette morphology and frequency.15 This suggests that FGF signaling may play a role downstream of Wnt signaling to establish the specific three-­ dimensional structure of the zG. How is Wnt/β-­catenin activated to allow for zG differentiation? A number of Wnt ligands, including WNT2b, WNT4, WNT5a, WNT5b, WNT9a, and WNT11, are expressed at the periphery of the adrenal cortex during embryonic development and/or in the adult adrenal.85,184,185,191 However, only the role of WNT4 has been extensively studied so far (Fig. 85.7). It is expressed in the developing adrenal glands of mice as early as E11.5 and shows restricted expression at the periphery of the cortex by E14.5192 and within zG in the adult adrenal,85 suggesting that it may be involved in zG differentiation. Indeed, full-­body ablation of Wnt4 is associated with reduced zG differentiation and decreased plasma aldosterone concentration.192 This is further confirmed by conditional ablation of Wnt4 in adrenal steroidogenic cells using Sf-­1-­Cre.85,184 A role for WNT4 in human adrenals is suggested by the observation that patients with mutations in WNT4 associated with SERKAL (SEx Reversion, Kidneys, Adrenal and Lung dysgenesis) syndrome have signs of adrenal dysgenesis.193 Conversely, overexpression of WNT4 in human adrenocortical H295R cell lines induces CYP11B2 expression and aldosterone production.194 Another important aspect of Wnt pathway control is modulation of FZD receptor availability at the plasma membrane. This involves the membrane E3 ubiquitin ligases ZNRF3 and RNF43 that target FZDs for degradation76,77 and the RSPOs (RSPO1–4) that antagonize ZNRF3 and RNF43 by inducing their membrane clearance.79,80,195 Rspo3, and to a lesser extent Rspo1, are expressed in the adrenal capsule,184,185 and genetic ablation of Rspo3 in the capsule results in a marked reduction of Wnt pathway activity and zG differentiation, associated with decreased expression of WNT4.184 This suggests that RSPO3 from the capsule plays a key paracrine function to stimulate Wnt signaling in the underlying zG (Fig. 85.7). Whereas RNF43 is barely detectable in the adrenal, ZNRF3 is expressed throughout the cortex.185 Consistent with its low level of expression, adrenal-­specific ablation of Rnf43 does not alter adrenal homeostasis. In contrast, mice with Znrf3 inactivation show robust hyperproliferation and hyperplasia, resulting in a nearly 8.5-­fold increase in adrenal size by 6 weeks. This phenotype can be significantly reversed by ablation of Porcupine (Porcn), which is an enzyme required for Wnt ligand secretion, indicating that the hyperplasia is at least in part Wnt-­dependent. However, in contrast to mice bearing constitutive activation of β-­catenin, there is no alteration of zonation patterns, and the hyperplasia is focused on zF rather than

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CHAPTER 85  Adrenal Development and Homeostasis

WNTs

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Fig. 85.7  Pathways involved in functional zonation of the adrenal cortex. A,WNT signaling and adrenocorticotropic hormone (ACTH) signaling control differentiation of the zona glomerulosa (zG) and zona fasciculata (zF), respectively. Schematic representation of the mouse adrenal cortex. Canonical WNT signaling pathway activated by WNT4 (produced by zG cells) and RSPO3 (produced by capsule cells) stimulates differentiation of the zG and production of aldosterone. ACTH/cAMP/PKA signaling pathway stimulates differentiation of the zF and production of corticosterone in rodents or cortisol in humans. AT1R and CYP11B2 are specifically expressed in the zG, whereas MC2R, MRAP, and CYP11B1 are specifically expressed in the zF. B, Molecular mechanisms of zG differentiation. In the zG, canonical WNT signaling is activated by RSPO3, which inhibits the membrane E3 ubiquitin ligase ZNRF3, preventing degradation of Frizzled (FZD) receptors. This allows stimulation of FZD by WNT4 produced within the zG, which results in accumulation and nuclear translocation of β-­catenin (β-­cat). β-­catenin, by interacting with LEF/TCF transcription factors, stimulates expression of its target genes, including WNT4. This results in reinforcement of WNT pathway activation within zG cells. β-­catenin also stimulates expression of AT1R, allowing response to Angiotensin II, of CYP21 and of the zG-­specific CYP11B2 to allow production of aldosterone. This is achieved directly (AT1R and CYP21) and indirectly (CYP11B2 and CYP21) through stimulation of NURR1 and NUR77 expression (NURs). ZNRF3 is also a direct target of β-­catenin, which ensures a negative feedback on WNT pathway activation to maintain tightly controlled levels of activation. C, Molecular mechanisms of zF differentiation. Expression of MC2R and the accessory protein MRAP allows response of zF cells to pituitary ACTH. This induces recruitment of the Gα protein to the adenylate cyclase, which converts ATP to cAMP. By binding to the regulatory subunits of PKA (R), cAMP allows release of the catalytic subunits (C), which phosphorylate the transcription factor CREB. Phosphorylated CREB migrates in the nucleus, where it stimulates transcription of a number of steroidogenic enzymes, among which the zF-­specific CYP11B1, which allows production of corticosterone (mouse) or cortisol (human).

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zG.185 This is correlated with normal, high levels of Wnt activation in the zG. Znrf3-­/-­ adrenals instead display sustained low Wnt pathway activity throughout the zF. Moreover, heterozygous inactivation of Ctnnb1 partially rescues the hyperplastic phenotype. These results suggest that, whereas high levels of canonical Wnt signaling achieved by direct β-­catenin stabilization primarily drive zG differentiation, and to a lesser extent hyperproliferation, moderate levels resulting from Znrf3 inactivation promote proliferation and are not sufficient for glomerulosa differentiation. This is consistent with the observation of moderate Wnt activation and high levels of proliferation in the normal outer zF.185

Differentiation of Zona Fasciculata. The pituitary hormone ACTH is a key regulator of zF differentiation. It acts through MC2R and the accessory protein MRAP, which are expressed at higher levels in the zF compared with the zG, to stimulate cAMP/PKA signaling.196-­198 In turn, PKA through direct phosphorylation of CREB, and indirect effects on SF-­1 and some other transcription factors, stimulates expression of zF-­specific genes such as CYP11B1, Akr1b7, or HSD11B2199-­201 (Fig. 85.7). Consistent with a specific role of ACTH/cAMP/PKA signaling in zF differentiation, chronic administration of ACTH induces hypertrophy of zF and increases glucocorticoid secretion, whereas dexamethasone administration induces zF atrophy and inhibition of glucocorticoid production, without alteration of zG.124,202-­204 In further support of this concept, mutations in either MC2R or MRAP in patients are associated with zF degeneration and unresponsiveness to ACTH, resulting in familial glucocorticoid deficiency.197,205,206 The key role of the ACTH/cAMP/PKA pathway in zF differentiation is further confirmed by massive hypoplasia of the zF and failure to produce glucocorticoids in Pomc, Mc2r, and Mrap KO mice.207-­210 Although Pomc and Mc2r KO mice also have mild mineralocorticoid insufficiency, consistent with a minor role of ACTH in acutely controlling aldosterone secretion,211 Mrap KO mice have normal circulating aldosterone levels.210 This suggests that MRAP plays a more zF-­specific role in the ACTH response. Isolated glucocorticoid insufficiency and aberrant zF differentiation are also observed in mice with adrenal-­specific ablation of the histone-­methyltransferase EZH2, which results in overexpression of phosphodiesterases PDE1B, 3A, and 7B, and a subsequent reduction in cAMP/PKA signaling.67 At the other end of the spectrum, patients with inactivating mutations of the regulatory subunit RIα of PKA (encoded by PRKAR1A)212 or of the phosphodiesterases PDE11A213 and PDE8B214 have increased cAMP/PKA signaling, which is associated with hypercortisolism and ACTH-­independent Cushing syndrome.215 This is also the case for patients with activating mutations of the α-­subunit of the Gs protein (encoded by GNAS1)216 or of the catalytic subunit of PKA (encoded by PRKACA).217 In most cases, the endocrine defect is restricted to hyperproduction of cortisol, confirming the prominent role of PKA signaling in zF differentiation and homeostasis. Consistent with this idea, mice with conditional ablation of Prkar1a in the adrenal cortex have expansion of zF and autonomous corticosterone production.85,128,218 Similarly, Pde8b KO mice have elevated urinary corticosterone, as a result of hypersensitivity to ACTH stimulation.219 Differentiation of Zona Reticularis. Adrenarche, which occurs between 6 and 8 years in humans, corresponds to a marked increase in DHEA and DHEA-­S production by cells of the zR, which is morphologically apparent by 3 years of age but only reaches its maximum size at around 13 years.22,220,221 The molecular mechanisms that control differentiation of zR remain elusive. Interestingly, constitutive activation of PKA, resulting from conditional inactivation of Prkar1a in cells of the definitive cortex, results in development of a reticularis-­like

zone in genetically engineered mice.128 Indeed, in contrast to wild-­type mice, which do not develop any zR features, Cyp11b2-­Cre;Prkar1afl/ fl mice express high levels of CYP17 in cells located between the zF and medulla and produce cortisol, DHEA, and DHEA-­S, which are normally not found in mice. As in humans, this is associated with high levels of CYB5 and low levels of HSD3B expression in the reticularis-­ like zone.128 This suggests that increased PKA activity may be sufficient to trigger zR development. This is consistent with data showing that dexamethasone can suppress adrenal androgens production222-­224 and failure of adrenarche in children with ACTH receptor deficiency.225 However, there is no evidence of a concomitant rise in DHEA and ACTH levels during adrenarche,226-­228 suggesting that some other, unidentified factors may trigger zR differentiation. KEY POINTS  • Functional zonation of the adrenal cortex is established perinatally by zone-­ restricted expression of Angiotensin II receptor (zona glomerulosa), MC2R/ MRAP (zona fasciculata), CYP11B2 (zona glomerulosa), and CYP11B1 (zona fasciculata). • Zona glomerulosa differentiation is dependent on canonical WNT/β-­ catenin signaling and is modulated by Angiotensin II. • Zona fasciculata differentiation is dependent on adrenocorticotropic hormone/cAMP/PKA signaling. • The mechanisms involved in zona reticularis differentiation remain elusive, although the process may involve PKA signaling.

MAINTENANCE OF ADULT ADRENAL CORTEX FUNCTION BY CORTICAL CELL RENEWAL Function of the adult adrenal cortex is maintained throughout life by a constant renewal process relying on stem and progenitor cells in the outer capsule and peripheral cortex, centripetal cell migration, and sequential differentiation of migrating cells as a function of their position along the corticomedullary axis167 (Fig. 85.8). Some of the first evidence for the presence of progenitor cells within the capsule and outer cortex was provided by experiments showing that, following rat adrenal enucleation, remaining tissues were able to form a fully differentiated and hormonally active cortex after autotransplantation to the ovary.229,230 This suggested that adrenal cortex progenitor cells were present in the capsular/subcapsular compartment. Consistent with this, most proliferation was observed in the outer cortex and could be strongly stimulated by unilateral adrenalectomy to allow for compensatory growth of the remaining adrenal.44,230,231 Labeling of cells with a single injection of BrdU, a synthetic nucleoside incorporated during DNA replication, followed by different periods of chase allowed demonstration of a continuous stream of BrdU+ cells from the outer to the inner cortex, associated with cell death as they reached the medulla.232-­236 Interestingly, whereas BrdU+ cells at the periphery did not express steroidogenic differentiation markers, BrdU+ cells within the cortex were differentiated.236 Altogether, these experiments suggested that undifferentiated progenitor cells in the capsule and outer cortex could proliferate and migrate centripetally towards the medulla to reconstitute pools of differentiated cells throughout the cortex. This theory was demonstrated by lineage-­tracing and genetic studies that have identified a number of stem and progenitor cell populations and the molecular pathways involved in their recruitment.17,29,124

Homeostatic Renewal The first formal evidence for a pool of capsular stem and subcapsular progenitor cells came from analyses of the HH signaling pathway17

CHAPTER 85  Adrenal Development and Homeostasis

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Tissue stress Capsule WT1+ stress prog.

SHH+ zG progenitor zG cell zF cell Apoptotic cell

Centripetal migration & differentiation

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Medulla cell Major contribution

Death

12 wks 28 wks

Minor contribution Fig. 85.8  Adrenal cortex renewal. Adrenal cortex homeostasis is maintained by constant renewal. This involves stem/progenitor cells located within the capsule and the zona glomerulosa (zG). Sonic hedgehog– positive (SHH+) zG cells ensure the majority of cortical renewal in the normal postpubertal adrenal cortex. After recruitment, SHH+ cells and their progeny initially differentiate as CYP11B2+ zG cells. They subsequently adopt a CYP11B1+ zF cell phenotype along their centripetal displacement within the cortex. When reaching the corticomedullary boundary, migrating cells enter apoptosis and are eliminated. GLI1+ cells can also contribute, albeit with low efficiency, to adrenal cortex renewal in both males and females before puberty. However, their contribution ceases in postpubertal males and is maintained in females. This sexual dimorphism is also reflected in the speed of renewal: full cortical replacement takes approximately 12 weeks in females and more than 7 months in males. WT1+ capsular cells are only mobilized following tissue stress, such as after gonadectomy. They may be mobilized in aging adrenals following the natural decline in sex steroids following menopause and andropause.

(see section on Sonic Hedgehog Signaling). The only HH ligand in the adrenal cortex is SHH, which is expressed in SF-­1+ cells within zG rosettes.17 Elegant lineage-­tracing studies using Shh-­gfpCre and Shh-­CreERT2 showed that SHH+ cells and their progeny migrate centripetally throughout the cortex. They differentiate as zG (CYP11B2+/ SF-­1+) cells and zF (CYP11B1+/SF-­1+) cells. This showed that SHH+ cells are progenitors for both zG and zF17 (Fig. 85.8). Lineage tracing also showed that SHH-­responsive undifferentiated GLI1+/SF-­1-­capsular cells could also contribute to all steroidogenic lineages of the cortex and to a subset of SHH+ progenitors. However, their contribution to the different lineages is rather low during homeostasis and is sexually dimorphic17,237 (see section on Sexual Dimorphism in Renewal). Early experimental evidence relying on trypan blue injections in the capsular and subcapsular space had shown that labeled cells could transit from the zG to the zF.238 This, together with the observation that GLI1+ and SHH+ cells could contribute to both terminally differentiated lineages, suggested that cortical renewal occurs by sequential differentiation resulting from conversion of capsular cells to zG cells that subsequently adopt a zF phenotype during their centripetal displacement in the cortex. This was formally demonstrated by using the Cyp11b2-­Cre driver and the mTmG reporter for lineage tracing. Indeed, whereas EGFP+ CYP11B2-­derived cells were initially restricted to the zG (CYP11B2+/CYP11B1-­), by 5 weeks, descendants of the CYP11B2 lineage were found in the zF (CYP11B1+/CYP11B2-­). By 12 weeks in females, the entire zF was recombined, and all the cells in the zF had lost CYP11B2 expression and gained CYP11B1 expression.124 Therefore, although it is technically challenging to prove that descendants of CYP11B2+ cells are also descendants of SHH+ and/ or GLI1+ cells, these data demonstrate that the majority of adrenal cortex renewal occurs through sequential transdifferentiation from zG

to zF, during migration within the cortex (Fig. 85.8). However, while this is the major homeostatic pathway, cells of the inner cortex can be derived independent of the CYP11B2 lineage under some extreme conditions.124 These findings raise the question of the mechanisms involved in transdifferentiation from zG to zF (Fig. 85.9). Analysis of models of constitutive activation of PKA throughout the cortex showed that this pathway plays an essential role in blocking Wnt pathway activation in adrenal steroidogenic cells, to allow conversion from a zG to zF phenotype. This inhibitory mechanism relies on inhibition of WNT4 expression and destabilization of β-­ catenin, through direct phosphorylation and indirect activation of GSK3β within the destruction complex.85 Conversely, Wnt pathway activation was found to inhibit PKA signaling. Indeed, pharmacological activation of Wnt signaling in adrenocortical cells in culture resulted in repression of MC2R and CYP11B1 expression.239 This was further confirmed in mouse models of constitutive β-­catenin activation in which expression of CYP11B1 and AKR1B7 was inhibited187,188 and conversion from fasciculata to glomerulosa was blocked, resulting in hyperaldosteronism.186 This may involve overexpression of the phosphodiesterase PDE2A, a direct target of WNT/β-­catenin signaling in the adrenal cortex,186 but also inhibition of both SF-­1 expression and transcriptional activity, presumably through titration and displacement of SF-­1 from the promoters of steroidogenic genes, by β-­catenin.239 Altogether, these findings strongly suggest that, after subcapsular SHH+ progenitor cells are recruited, they initially differentiate as zG cells under the influence of Wnt signaling, activated by WNT4 and further potentiated by RSPO3. This stimulates expression of AT1R and CYP11B2 and blocks expression of MC2R and CYP11B1. As cells migrate within the cortex, they progressively escape the positive

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A A

Fig. 85.9  Antagonistic interaction between WNT and PKA signaling pathways control zonal differentiation during renewal. After recruitment, sonic hedgehog–positive progenitor cells initially differentiate as zona glomerulosa cells under the influence of Wnt signaling, activated by WNT4 and further potentiated by RSPO3, which inactivates ZNRF3, allowing accumulation of Frizzled (FZD; Wnt receptor). This is reinforced by induction of WNT4 expression by β-­catenin, which also stimulates expression of AT1R and CYP11B2 and blocks expression of MC2R and CYP11B1. Aldosterone is then produced under the control of Angiotensin II, acting through AT1R. As cells migrate within the cortex, they progressively escape the positive influence of RSPO3/WNT4. This prevents Wnt pathway activation by allowing ZNRF3-­mediated clearance of FZD, resulting in a further decrease in WNT4 expression. This reduction in Wnt activation allows expression of MC2R, which in turn stimulates the PKA signaling pathway. This further inhibits expression of WNT4, prevents β-­ catenin accumulation, and stimulates expression of CYP11B1, allowing zona fasciculata differentiation and production of glucocorticoids.

CHAPTER 85  Adrenal Development and Homeostasis influence of RSPO3/WNT4, which is further suppressed by ZNRF3-­ mediated downregulation of Wnt receptors.185 This reduction in Wnt activation allows expression of MC2R, which in turn stimulates the PKA signaling pathway. This further inhibits expression of WNT4, prevents β-­catenin accumulation, and stimulates expression of CYP11B1, allowing zF differentiation (Fig. 85.9). Whether these mechanisms are also involved in the human adrenal cortex is unknown. However, consistent with mouse adrenals, proliferation in the human adrenal is also more concentrated in the outer cortex, whereas apoptosis is localized at the corticomedullary boundary.240 There is also more intense staining for SHH at the periphery of the cortex and GLI1 expression in the capsule.241,242 Interestingly, analysis of telomere length within the different zones of human adrenal cortices showed that telomeres were shorter in zF than in zG in subjects between 22 and 68 years of age, whereas they were identical in younger individuals.243 This age-­and zone-­dependent telomeric attrition suggests that the human adrenal cortex also undergoes constant renewal, and that zF cells are older than zG cells, consistent with the sequential differentiation model. However, zR cells were found to have longer telomeres than either zG or zF.243 This suggests that the zR may be established and/or maintained independently from the zG and zF.

Role of Renin-­Angiotensin-­Aldosterone System and Adrenocorticotropic Hormone/PKA Signaling in Regulation of Renewal The RAAS and ACTH/PKA signaling pathways, which control endocrine activity and proliferation in the adrenal cortex, also play a role in progenitors’ homeostasis. Indeed, in the context of genetic ablation of Cyp11b2, which results in a 5-­fold increase in renin-­angiotensin activation, lineage conversion from zG to zF is significantly accelerated, which is correlated with increased proliferation and expansion of the zG. This results in complete zF colonization by the descendants of zG cells after 5 weeks, compared with 12 weeks in wild-­type mice.124,186 This effect can be partially reversed by treatment with candesartan, indicating that cortical cell renewal can be stimulated by increased renin-­angiotensin activity.186 Interestingly, cortical cell renewal can also be accelerated by PKA signaling. Indeed, whereas 2-­week treatment with dexamethasone to suppress pituitary ACTH secretion results in inhibition of zG-­to-­zF conversion,124 constitutive activation of PKA using Cyp11b2-­Cre is associated with a marked acceleration of cortical cell renewal.128 Altogether, these data show that the major adrenal regulators of adrenal endocrine activity are able to stimulate adrenal cortex renewal to allow for sustained response to potentially chronic stimuli. How these hormones can stimulate differentiation, proliferation, and renewal remains unclear.

Sexual Dimorphism in Renewal The adrenal cortex is a sexually dimorphic tissue. In mice, female adrenals are larger after puberty,244-­246 and sex-­specific transcriptomic signatures have been described.247,248 Interestingly, analysis of proliferation using BrdU injections showed that mature female adrenals had a much higher proliferation rate (6-­fold) than male adrenals. This was partially compensated by a 2-­fold increase in apoptosis at the corticomedullary junction, suggesting that female adrenals undergo a faster cell turnover than male adrenals.237 Consistent with this hypothesis, lineage tracing using Cyp11b2-­Cre, Axin2-­CreERT2, or Wnt4-­CreERT2 showed full replacement of the adult female cortex by zG-­derived cells after 3 months, whereas only 42% and 75% of cortical cells were derived from the zG after 3 and 7 months, respectively, in males (Fig. 85.8).128,237 Lineage tracing using Gli1-­CreERT2 to identify the descendants of capsular progenitors at 3 weeks showed a similar

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contribution of these cells to differentiated cortical cells in males in females. However, when similar experiments were conducted after puberty, the contribution of GLI11+ cells to the differentiated cortex almost ceased in males, whereas it was maintained in females.237 This shows that, whereas both prepubertal male and female adrenals use GLI11+ and SHH+ progenitors for cortical renewal, GLI11+ progenitors are only used in females after puberty. This suggests that male adrenals may be more sensitive to aging than female adrenals. Consistent with this idea, human aging male zF cells have shorter telomeres than age-­matched female zF cells.249 How are these sexually dimorphic processes regulated? To evaluate a potential contribution of chromosomal sex, Grabek et al. used models of sex reversal consisting of XX males (overexpressing SOX9 under the control of Wt1 regulatory regions) and XY females (bearing Sf-­1-­Cre mediated inactivation of SOX9). These experiments showed that XY females had increased capsular proliferation compared with control males, whereas XX males had decreased proliferation compared with control females. In contrast to control XX females, GLI1+ cells did not contribute to the pool of steroidogenic cells, which was reminiscent of XY males.237 This suggested that sex-­specific proliferation and recruitment were not dependent on sex chromosomes, but were rather controlled by sex hormones. Indeed, gonadectomy in male mice induced a significant increase in capsular proliferation and a 3-­fold induction in recruitment of capsular cells to the steroidogenic lineage. Conversely, administration of the nonaromatizable androgen DHT to female mice inhibited proliferation and almost completely blocked GLI1+ progenitor recruitment.237 Similar experiments using Cyp11b2-­Cre to identify descendants of zG cells also showed that androgen deprivation in male mice could dramatically accelerate renewal, which was reversed by supplementation with DHT.128 Altogether, these experiments show an all-­encompassing role of androgens in inhibition of capsular progenitor cell recruitment and overall cortical cell renewal in male mice. Although the molecular mechanisms underlying this effect of androgens are not completely understood, transcriptomic analyses of castrated and supplemented mice suggest that they may involve stimulation of Wnt/β-­catenin signaling, which would in turn counteract the prorenewal effects of PKA signaling.128 These sexually dimorphic proliferation and renewal capacities may account for the female prevalence of adrenal diseases such as Cushing syndrome250 and adrenocortical carcinomas.251

Adrenal Cortex Regeneration Chronic administration of glucocorticoids is used for treatment of a number of medical conditions. By suppressing pituitary ACTH, these treatments result in adrenal cortex atrophy, which can be reversed by a regenerative process, following withdrawal of corticoids.252,253 Recent experimental evidence in mice has shown that this process involves mobilization of adrenal progenitors in response to both HH and Wnt signaling pathways. Indeed, following chronic dexamethasone treatment removal, SHH expression is increased, and SHH+ and GLI1+ cells are mobilized to reconstitute a functional cortex.204 This process can be efficiently blocked using GANT61, a specific inhibitor of GLI1 and GLI2 DNA-­binding activity. Conversely, constitutive activation of HH signaling within GLI1+ cells using a SmoM2 mutant facilitates regeneration.204 Wnt signaling is also actively engaged during adrenal cortex regeneration and inactivation of β-­catenin in AXIN2+ Wnt-­ responsive cells inhibits the regeneration process.204 This may involve direct stimulation of SHH expression by canonical Wnt signaling to trigger progenitor cell recruitment.184,191,204 Failure of these processes may result in persistent adrenal insufficiency despite glucocorticoid withdrawal.252,253

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PART 7  Adrenal

Tissue Stress Progenitors and Adrenal Aging Although SHH+ cells are the key progenitors for adrenal homeostasis, lineage-­tracing studies have identified some other populations of cells that can be mobilized to respond to tissue stress. In the adult, lineage tracing with Wt1-­CreERT2 showed that WT1+ cells can give rise to fully differentiated steroidogenic cells, albeit at very low frequency (Fig. 85.8).29 Interestingly, though, gonadectomy increases recruitment and differentiation of WT1-­derived cells into the steroidogenic lineage, in both males and females. However, these cells are not fully differentiated and express features of gonadal steroidogenic cells, such as CYP17 and LHR, that are not found in normal adrenals.29 Gonadectomy also induces subcapsular accumulation of GLI1-­derived aberrantly differentiated steroidogenic cells that express the gonadal markers PDGFRα, FOXL2, and GATA4.254 This gonadectomy-­ induced phenomenon is further amplified in the context of loss of adrenal inhibin (Inha-­ /-­) and results in the development of adrenocortical tumors characterized by ovarian identity.255 Further work has demonstrated that adrenal inhibin normally acts in an LH-­dependent manner to oppose GATA4-­mediated gonadal differentiation by antagonizing TGFβ2.256 Interestingly, GLI1-­derived subcapsular cells also accumulate in intact aging mice of the B6D2F2 strain,254 which is reminiscent of the GATA4-­ positive subcapsular hyperplasia observed following gonadectomy or in intact aging mice.67,257 On the basis of these observations, it is tempting to speculate that these GLI1/WT1-­derived GATA4+ cells could accumulate in the adrenal, following the natural decline of sex steroids after menopause and andropause. Whether GLI1+, WT1+, and/or GATA4+ cells could contribute to suboptimal adrenal cortex renewal following late exhaustion of SHH+ progenitors remains to be investigated. KEY POINTS  • Steroidogenic cells of the adrenal cortex are continuously renewed by the sexually dimorphic recruitment of stem and progenitor cells located in the capsule and within the zona glomerulosa (zG). • Both GLI1+ capsular and sonic hedgehog–positive (SHH+) zG cells can contribute to cortex renewal in males and females before puberty, but GLI1+ contribution ceases in postpubertal males. • SHH+ progenitors are the major source of steroidogenic cells during homeostasis in the adult. • The vast majority of progenitor cells undergo sequential differentiation as zG cells and subsequent transdifferentiation into zone fasciculata cells during their centripetal migration within the cortex. • Recruitment and sequential differentiation are tightly controlled by antagonistic interactions between Wnt/β-­catenin and PKA signaling pathways. • Progenitors are mobilized to allow adrenal regeneration following withdrawal from chronic glucocorticoid treatment. • Some distinct Wilm’s tumor suppressor-positive progenitors can be mobilized in response to abnormal tissue stress and may participate in renewal of the aging adrenal cortex.

CONCLUSION AND FUTURE DIRECTIONS Since the first description of the adrenal gland by Bartholomeus Eustachius in 1563, significant progress has been made in understanding its role as a central regulator of the stress response, through secretion of corticoids and catecholamines. Over the last 25 years, a combination of human and mouse genetics has identified the key molecular mechanisms controlling the development, differentiation, and renewal of the adrenal cortex. This has translated into a better understanding of adrenal diseases ranging from insufficiency to aggressive tumor development. However, these novel findings have raised

many questions that will need to be addressed to further our understanding of adrenal pathophysiology. Indeed, we still do not know how deregulation of adrenal cortex renewal, in particular in the aging gland, may play a role in development of adrenal diseases. In this respect, the appearance of abnormally differentiated subcapsular cell clusters such as aldosterone-­producing cell clusters and DLK1+ clusters in the aging human adrenal is particularly intriguing.258-­261 Understanding the molecular underpinnings of the sexual dimorphism of adrenal cortex function and homeostasis will also undoubtedly shed light on the dimorphic nature of many adrenal conditions. Another key aspect that has been underestimated so far, is the multiple interactions between steroidogenic cells and their tissue microenvironment. The key role of such interactions has recently been illustrated by the demonstration of an important role of the neuropeptide substance P in controlling aldosterone secretion in human adrenals.262 Answering these questions will help development of novel therapeutic approaches, such as regenerative therapies to treat adrenal insufficiency or targeted therapies to treat adrenocortical cancer patients.

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194. Chen M, Hornsby PJ. Adenovirus-­delivered DKK3/WNT4 and steroidogenesis in primary cultures of adrenocortical cells. Horm Metab Res. 2006;38:549–555. 195. de Lau W, Peng WC, Gros P, et al. The R-­spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev. 2014;28:305–316. 196. Mountjoy KG, Robbins LS, Mortrud MT, et al. The cloning of a family of genes that encode the melanocortin receptors. Science. 1992;257:1248– 1251. 197. Metherell LA, et al. Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nat Genet. 2005;37:166–170. 198. Gorrigan RJ, Guasti L, King P, et al. Localisation of the melanocortin-­2-­ receptor and its accessory proteins in the developing and adult adrenal gland. J Mol Endocrinol. 2011;46:227–232. 199. Wang XL, et al. Transcriptional regulation of human 11beta-­hydroxylase (hCYP11B1). Endocrinology. 2000;141:3587–3594. 200. Aigueperse C, et al. SF-­1 (steroidogenic factor-­1), C/EBPbeta (CCAAT/ enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-­like gene (AKR1B7) expression in adrenocortical cells. Mol Endocrinol. 2001;15:93–111. 201. Nishimoto K, et al. Transcriptome analysis reveals differentially expressed transcripts in rat adrenal zona glomerulosa and zona fasciculata. Endocrinology. 2012;153:1755–1763. 202. Dallman MF. Control of adrenocortical growth in vivo. Endocr Res. 1984;10:213–242. 203. Thomas M, Keramidas M, Monchaux E, et al. Dual hormonal regulation of endocrine tissue mass and vasculature by adrenocorticotropin in the adrenal cortex. Endocrinology. 2004;145:4320–4329. 204. Finco I, Lerario AM, Hammer GD. Sonic hedgehog and WNT signaling promote adrenal gland regeneration in male mice. Endocrinology. 2018;159:579–596. 205. Clark AJ, McLoughlin L, Grossman A. Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor. Lancet. 1993;341:461–462. 206. Novoselova TV, et al. ACTH signalling and adrenal development: lessons from mouse models. Endocrine Connections. 2019;8:R122–R130. 207. Yaswen L, Diehl N, Brennan MB, et al. Obesity in the mouse model of pro-­opiomelanocortin deficiency responds to peripheral melanocortin. Nat Med. 1999;5:1066–1070. 208. Coll AP, et al. The effects of proopiomelanocortin deficiency on murine adrenal development and responsiveness to adrenocorticotropin. Endocrinology. 2004;145:4721–4727. 209. Chida D, et al. Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis. Proc Natl Acad Sci U S A. 2007;104:18205–18210. 210. Novoselova TV, et al. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation. Faseb J. 2018;32:6186–6196. 211. El Ghorayeb N, Bourdeau I, Lacroix A. Role of ACTH and other hormones in the regulation of aldosterone production in primary aldosteronism. Front Endocrinol. 2016;7:72. 212. Kirschner LS, et al. Mutations of the gene encoding the protein kinase A type I-­alpha regulatory subunit in patients with the Carney complex. Nat Genet. 2000;26:89–92. 213. Horvath A, et al. A genome-­wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia. Nat Genet. 2006;38:794–800. 214. Horvath A, et al. A cAMP-­specific phosphodiesterase (PDE8B) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: a novel PDE8B isoform in human adrenal cortex. Eur J Hum Genet. 2008;16:1245–1253. 215. Bonnet-­Serrano F, Bertherat J. Genetics of tumors of the adrenal cortex. Endocr Relat Cancer. 2018;25:R131–R152. 216. Weinstein LS, et al. Activating mutations of the stimulatory G protein in the McCune-­Albright syndrome. N Engl J Med. 1991;325:1688–1695. 217. Beuschlein F, et al. Constitutive activation of PKA catalytic subunit in adrenal Cushing’s syndrome. N Engl J Med. 2014;370:1019–1028.

CHAPTER 85  Adrenal Development and Homeostasis 218. Sahut-­Barnola I, et al. Cushing’s syndrome and foetal features resurgence in adrenal cortex-­specific Prkar1a knockout mice. PLoS Genet. 2010;6:e1000980. 219. Tsai LCL, Shimizu-­Albergine M, Beavo JA. The high-­affinity cAMP-­ specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland. Mol Pharmacol. 2011;79:639–648. 220. Dhom G. The prepuberal and puberal growth of the adrenal (adrenarche). Beiträge zur Pathologie. 1973;150:357–377. 221. Endoh A, Kristiansen SB, Casson PR, et al. The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex resulting from its low expression of 3 beta-­hydroxysteroid dehydrogenase. J Clin Endocrinol Metab. 1996;81:3558–3565. 222. Abraham GE. Ovarian and adrenal contribution to peripheral androgens during the menstrual cycle. J Clin Endocrinol Metab. 1974;39:340–346. 223. Kim MH, Hosseinian AH, Dupon C. Plasma levels of estrogens, androgens and progesterone during normal and dexamethasone-­treated cycles. J Clin Endocrinol Metab. 1974;39:706–712. 224. Rich BH, Rosenfield RL, Lucky AW, et al. Adrenarche: changing adrenal response to adrenocorticotropin. J Clin Endocrinol Metab. 1981;52:1129– 1136. 225. Weber A, Clark AJ, Perry LA, et al. Diminished adrenal androgen secretion in familial glucocorticoid deficiency implicates a significant role for ACTH in the induction of adrenarche. Clin Endocrinol. 1997;46:431–437. 226. Mellon SH, Shively JE, Miller WL. Human proopiomelanocortin-­(79–96), a proposed androgen stimulatory hormone, does not affect steroidogenesis in cultured human fetal adrenal cells. J Clin Endocrinol Metab. 1991;72:19–22. 227. Penhoat A, et al. Human proopiomelanocortin-­(79–96), a proposed cortical androgen-­stimulating hormone, does not affect steroidogenesis in cultured human adult adrenal cells. J Clin Endocrinol Metab. 1991;72:23– 26. 228. Rege J, Rainey WE. The steroid metabolome of adrenarche. J Endocrinol. 2012;214:133–143. 229. Ingle DJ, Higgins GM. Autotransplantation and regeneration of the adrenal gland. Endocrinology. 1938;22:458–464. 230. Ennen WB, Levay-­Young BK, Engeland WC. Zone-­specific cell proliferation during adrenocortical regeneration after enucleation in rats. Am J Physiol Endocrinol Metab. 2005;289:E883–E891. 231. Mitani F. Functional zonation of the rat adrenal cortex: the development and maintenance. Proc Jpn Acad Ser B Phys Biol Sci. 2014;90:163–183. 232. Bertholet JY. Proliferative activity and cell migration in the adrenal cortex of fetal and neonatal rats: an utoradiographic study. J Endocrinol. 1980;87:1–9. 233. Zajicek G, Ariel I, Arber N. The streaming adrenal cortex : direct evidence of centripetal migration of adrenocytes by estimation of cell turnover rate. J Endocrinol. 1986;111:477–482. 234. McNicol AM, Duffy AE. A study of cell migration in the adrenal cortex of the rat using bromodeoxyuridine. Cell Prolif. 1987;20:519–526. 235. Carsia RV, Macdonald GJ, Gibney JA, et al. Apoptotic cell death in the rat adrenal gland: an in vivo and in vitro investigation. Cell Tissue Res. 1996;283:247–254. 236. Chang SP, et al. Cell proliferation, movement and differentiation during maintenance of the adult mouse adrenal cortex. PLoS One. 2013;8:e81865. 237. Grabek A, et al. The adult adrenal cortex undergoes rapid tissue renewal in a sex-­specific manner. Cell Stem Cell. 2019;25:290–296.e2. 238. Salmon T, Zwemer R. A study of the life history of cortico-­adrenal gland cells of the rat by means of trypan blue injections. Anat Rec. 1941;80:421– 429. 239. Walczak EM, et al. Wnt-­signaling inhibits adrenal steroidogenesis by cell-­autonomous and non-­cell-­autonomous mechanisms. Mol Endocrinol. 2014;28:1471–1486.

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240. Sasano H, Imatani A, Shizawa S, et al. Cell proliferation and apoptosis in normal and pathologic human adrenal. Mod Pathol. 1995;8:11–17. 241. Boulkroun S, et al. Aldosterone-­producing adenoma formation in the adrenal cortex involves expression of stem/progenitor cell markers. Endocrinology. 2011;152:4753–4763. 242. Gomes DC, et al. Sonic hedgehog signaling is active in human adrenal cortex development and deregulated in adrenocortical tumors. J Clin Endocrinol Metab. 2014;99:E1209–E1216. 243. Nonaka K, et al. Correlation between differentiation of adrenocortical zones and telomere lengths measured by Q-­FISH. J Clin Endocrinol Metab. 2019;104:5642–5650. 244. Moog F, Bennett CJ, Dean CM. Growth and cytochemistry of the adrenal gland of the mouse from birth to maturity. Anat Rec. 1954;120:873–891. 245. Badr FM, Spickett SG. Genetic variation in adrenal weight in young adult mice. J Endocrinol. 1971;49:105–111. 246. Bielohuby M, et al. Growth analysis of the mouse adrenal gland from weaning to adulthood: time-­and gender-­dependent alterations of cell size and number in the cortical compartment. Am J Physiol Endocrinol Metab. 2007;293:E139–E146. 247. El Wakil A, Mari B, Barhanin J, et al. Genomic analysis of sexual dimorphism of gene expression in the mouse adrenal gland. Horm Metab Res. 2013;45:870–873. 248. Jopek K, et al. Transcriptome profile of rat adrenal evoked by gonadectomy and testosterone or estradiol replacement. Front Endocrinol. 2017;8:26. 249. Nonaka K, et al. Correlation between telomere attrition of zona fasciculata and adrenal weight reduction in older men. J Clin Endocrinol Metab. 2020;105:dgz214. 250. Steffensen C, Bak AM, Rubeck KZ, et al. Epidemiology of Cushing’s syndrome. Neuroendocrinology. 2010;92(suppl 1):1–5. 251. Audenet F, Méjean A, Chartier-­Kastler E, et al. Adrenal tumours are more predominant in females regardless of their histological subtype: a review. World J Urol. 2013;31:1037–1043. 252. Arlt W, Allolio B. Adrenal insufficiency. Lancet. 2003;361:1881–1893. 253. Broersen LHA, Pereira AM, Jørgensen JOL, et al. Adrenal insufficiency in corticosteroids use: systematic review and meta-­analysis. J Clin Endocrinol Metab. 2015;100:2171–2180. 254. Dörner J, et al. GLI1(+) progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-­like tissue. Mol Cell Endocrinol. 2017;441:164–175. 255. Looyenga BD, Hammer GD. Origin and identity of adrenocortical tumors in inhibin knockout mice: implications for cellular plasticity in the adrenal cortex. Mol Endocrinol. 2006;20:2848–2863. 256. Looyenga BD, Wiater E, Vale W, et al. Inhibin-­A antagonizes TGFbeta2 signaling by down-­regulating cell surface expression of the TGFbeta coreceptor betaglycan. Mol Endocrinol. 2010;24:608–620. 257. Röhrig T, et al. Toying with fate: Redirecting the differentiation of adrenocortical progenitor cells into gonadal-­like tissue. Mol Cell Endocrinol. 2015;408:165–177. 258. Nishimoto K, et al. Aldosterone-­stimulating somatic gene mutations are common in normal adrenal glands. Proc Natl Acad Sci U S A. 2015;112:E4591–E4599. 259. Lim JS, Rainey WE. The potential role of aldosterone-­producing cell clusters in adrenal disease. Horm Metab Res. 2020;52:427–434. 260. Nishimoto K, et al. Adrenocortical zonation in humans under normal and pathological conditions. J Clin Endocrinol Metab. 2010;95:2296– 2305. 261. Hadjidemetriou I, et al. DLK1/PREF1 marks a novel cell population in the human adrenal cortex. J Steroid Biochem Mol Biol. 2019;193:105422. 262. Wils J, et al. The neuropeptide substance P regulates aldosterone secretion in human adrenals. Nat Commun. 2020;11:2673.

86 Genetic Disorders of the Adrenal Cortex Christopher J. Smith, Avinaash Maharaj, Louise A. Metherell, and Leonardo Guasti

OUTLINE Primary Adrenal Insufficiency: An Overview, 1430 Genetic Disorders of the Adrenal Cortex, 1431 Steroidogenesis, 1431 Hypoplasia, 1437

Familial Glucocorticoid Deficiency-­Like (FGD-­Like), 1439 Other Causes Of Syndromic Adrenal Insufficiency, 1441 Conclusion, 1443 Further Resources, 1443

 

KEY POINTS  • Primary adrenal insufficiency is a result of adrenal dysfunction and is characterized by low cortisol and aldosterone levels, as well as high adrenocorticotropic hormone. • Conversely, secondary and tertiary adrenal insufficiency are results of disordered pituitary or hypothalamic function. • Glucocorticoids, mineralocorticoids, and androgens are produced from cholesterol by enzyme cascades in the adrenal cortex.

PRIMARY ADRENAL INSUFFICIENCY: AN OVERVIEW Primary adrenal insufficiency (PAI) is the result of disordered adrenal function and is characterized by low cortisol and aldosterone levels, as well as high plasma adrenocorticotropic hormone (ACTH). This differs from secondary and tertiary adrenal insufficiency (SAI), resulting from disordered pituitary or hypothalamic function, which presents with low cortisol levels and ACTH deficiency. While cortisol and aldosterone are both deficient in PAI, in SAI, where the adrenals remain functional, it is only cortisol levels that are reduced. This is due to aldosterone being regulated through the renin-­angiotensin system (RAS) and explains the relatively different clinical presentation of PAI and SAI. Glucocorticoid production is discussed in detail elsewhere in this text, but in brief, ACTH, produced in the pituitary gland under the control of the hypothalamus–pituitary–adrenal axis, induces cortisol production through both transcription-­independent stimulation and increased gene expression of steroidogenic enzymes via melanocortin 2 (MC2R) receptors in the adrenocortical cells of the zona fasciculata.1 Upon ACTH activation, cholesterol is transported to the inner mitochondrial membrane (IMM) by steroidogenic acute regulatory protein (STAR) and converted to pregnenolone by cytochrome P450 side chain cleavage enzyme (CYP11A1) (Fig. 86.1). Pregnenolone is subsequently transported out of the mitochondria and hydroxylated to 17-­OH-­pregnenolone by P450c17 (CYP17A1) before being converted to 17-­OH-­progesterone (17-­OHP) through the action of 3β-­ hydroxysteroid dehydrogenase (HSD3B2) at the endoplasmic reticulum. 17-­OHP is next hydroxylated by P450c21 (CYP21A2) to

1430

produce 11-­deoxycortisol, which is then returned to the mitochondria, and P450c11β (CYP11B1) completes the cortisol synthesis pathway.2 Glucocorticoids regulate ACTH secretion by a negative feedback loop,3,4 maintain cardiac function,5-­7 and are essential for hepatic glycogen deposition.4,8 Consequently, glucocorticoid deficiency typically presents with hypoglycemia, hypotension, reduced stroke volume, and ACTH-­mediated hyperpigmentation (via activation of MC1R in melanocytes). Aldosterone production takes place in the zona glomerulosa, the outermost layer of the adrenal cortex, where its synthesis is controlled by the RAS. Briefly, a drop in blood pressure results in the release of renin from juxtaglomerular cells in the kidneys, which is then proteolytically cleaved by angiotensinogen from the liver to produce angiotensin I. Angiotensin-­converting enzyme produced by the lungs converts angiotensin I to angiotensin II, which stimulates the release of aldosterone from the adrenal cortex via binding to angiotensin II receptor type I.9 Upon angiotensin II activation, cholesterol is processed in the same manner as cortisol synthesis to produce pregnenolone. With the absence of P450c17 in the zona glomerulosa, pregnenolone is solely converted to progesterone through HSDB2. Progesterone is subsequently oxidised by P450c21 to 11-­deoxycorticosterone (DOC), substrate for zona glomerulosa-specific P450c11AS (CYP11B2), which produces corticosterone, a glucocorticoid. Corticosterone is then further processed through the action of P450c11AS to produce 18-­hydroxycorticosterone and finally aldosterone.2 Mineralocorticoids regulate sodium, potassium and hydrogen ion levels in the kidneys, and therefore a deficiency of these can result in hypotension, hyponatremia, hyperkalemia, and metabolic acidosis.10 The functions of corticosteroids and the effects of their absence due to autoimmune and infectious adrenal insufficiency are explored extensively elsewhere in the text. This chapter will provide an overview of the clinical phenotype, comorbidities, and mechanisms of action (where known) for genetic disorders of the adrenal cortex resulting in PAI. KEY POINTS  • Primary adrenal insufficiency can be caused by hereditary mutations that affect steroidogenic enzymes, development and growth of the adrenal gland, resistance to adrenocorticotropic hormone action or adrenal destruction.

CHAPTER 86  Genetic Disorders of the Adrenal Cortex

Lipid droplet 1.

2. Cholesterol

Pregnenolone 3. 17-OH-pregnenolone 4. 17-OH-progesterone

Cortisol

6.

5. 11-deoxycortisol

Figure 86.1  Cortisol production pathway in the zona fasciculata. CYP11A1 conversion of cholesterol to pregnenolone is the rate-­ limiting step of steroidogenesis, performed at the inner mitochondrial membrane. Pregnenolone is transported to the endoplasmic reticulum, where a cascade of enzyme activity results in the production of 11-­deoxycortisol, which is subsequently returned to the mitochondria and converted to cortisol. The genes encoding the enzymes involved at each step of the pathway are as follows: 1. STAR, 2. CYP11A1, 3. CYP17A1, 4. HSD3B2, 5. CYP21A2 6. CYP11B1.

GENETIC DISORDERS OF THE ADRENAL CORTEX PAI encompasses a heterogeneous group of diseases distinguished by decreased production of cortisol and steroid hormones by the adrenal cortex and arises from anomalies in the development of the adrenal gland, impaired steroidogenesis, resistance to ACTH action (familial glucocorticoid deficiency [FGD]) or adrenal destruction. In adults, most cases are caused by adrenal destruction due to autoimmune Addison disease, whereas in childhood the cause is usually hereditary and monogenic in origin and may be related to considerable morbidity and mortality. The International Classification of Pediatric Endocrine Diagnoses Consortium (http://www.icped.org/) classifies them into congenital adrenal hyperplasias (CAH), adrenal hypoplasia congenita (AHC), and FGD-­ like (http://www.icped.org/revisions/0/2015/ diagnoses/#!/8), with CAH and AHC leading to adrenal insufficiencies of both gluco-­and mineralocorticoids, whereas in FGD-­like disease patients chiefly lack glucocorticoids (Table 86.1). CAH accounts for most of PAI cases in childhood, with the most common type due to mutations in CYP21A2, but defects in CYP11B1, CYP17A1, CYP11A1, and HSD3B2 can also result in CAH.11 This results in a compensatory increase in ACTH that drives the adrenal hyperplastic response due to its trophic action on adrenocortical cells.12,13 Congenital adrenal hypoplasia is characterized by underdevelopment of the adrenal glands and can be caused by defects in nuclear receptor subfamily 5, group A, member 1 (NR5A1) or the nuclear receptor subfamily 0, group B, member 1 (NR0B1) but is also seen as a constituent part of complex multisystem growth disorders caused by mutations in genes such as cyclin-­dependent kinase inhibitor 1C (CDKN1C) (Table 86.1). Finally, FGD-­like or ACTH resistance syndromes14 are characterized by isolated glucocorticoid deficiency in a patient who retains normal mineralocorticoid production. FGD-­ like syndromes have considerable phenotypic variation, dependent on the gene defect, but

1431

normally present at an early age with neonatal hypoglycemia, seizures, hyperpigmentation, and increased susceptibility to infection. The first mutations were identified in MC2R and its accessory protein MRAP; however, with advances in next-­generation sequencing and genetic diagnosis tools, further gene mutations have been discovered that give rise to disorders with isolated cortisol deficiency through defects in cell proliferation (mini-­chromosome maintenance complex component 4 [MCM4]) and cellular redox balance (nicotinamide nucleotide transhydrogenase [NNT] and thioredoxin reductase 2 [TXNRD2]). KEY POINTS  • Mutations in genes that encode adrenal steroidogenic enzymes are common causes of congenital adrenal hyperplasia. • Congenital adrenal hyperplasia can present differently depending on the dysfunctional enzyme, and patients can have varied glucocorticoid, mineralocorticoid, and androgen production. • Mutations in CYP11A1 and STAR can also cause congenital lipoid adrenal hyperplasia, a disease where lack of hormone production results in increased accumulation of cholesterol, and consequently mitochondrial oxidative damage.

Steroidogenesis Defects in enzymes within the adrenal steroidogenic cascade (Fig. 86.2) are common causes of CAH. As a complex network of enzymes, mutations in different genes can cause variable disease phenotypes due to deficiency of glucocorticoid, mineralocorticoid, or both, as well as possible gonadal insufficiency.

Cytochrome P450 Family 21 Subfamily A Member 2 (CYP21A2). CYP21A2 encodes steroid 21-­hydroxylase, the steroidogenic enzyme that converts progesterone to deoxycortisone and 17-­OHP to deoxycortisol (Fig. 86.2). Mutations in CYP21A2 cause 90% of CAH and result in reduced mineralocorticoid and glucocorticoid production, as well as androgen excess, leading to hypergonadism. CAH due to steroid 21-­hydroxylase deficiency presents with three major phenotypes: classical salt-­wasting, classical simple virilizing, and nonclassical. The absence of aldosterone results in the inability to retain sodium, which is consequently lost in urine, a process known as salt wasting.15 Classical salt-­wasting is the more severe of the two classical forms and constitutes 75% of cases.16 Due to mutations resulting in less than 2% enzyme activity, undiagnosed cases can lead to life-­threatening adrenal crisis in the first 2 weeks of life.17,18 In addition, 46, XY patients are born with normal genitalia, putting them most at risk, while 46, XX patients are commonly born with genital ambiguity, prompting further evaluation and treatment.18 For classical salt-­wasting cases, both mineralocorticoid and glucocorticoid therapy are required. In the simple virilizing cases, which possess greater than 2% enzyme activity, sufficient aldosterone is produced to prevent salt wasting. For both disease variants, patients undergoing postnatal development may develop signs of androgen excess, including precocious pubarche, adrenarche, acne, and advanced bone age leading to reduced final height.19,20 Furthermore, 46, XY patients are likely to develop testicular adrenal rest tumors, benign testicular tumors that morphologically and functionally resemble adrenal tissue.21 Patients with the nonclassical form of the disease retain up to 50% of enzyme activity. This allows sufficient hormone production to prevent adrenal insufficiency and genital ambiguity in 46, XX patients, resulting in many patients going undiagnosed.18,22 The primary method to diagnose steroid 21-­hydroxylase deficiency is to measure serum levels of 17-­OHP, the substrate for steroid 21-­hydroxylase, during newborn screening.18

Nuclear recep- NR0B1 tor subfamily 0 group B member 1

300200

613571

609300 Congenital adrenal 10q24.32 hyperplasia, ambiguous genitalia, hypokalemic hypertension, delayed puberty

202110

CYP17A1

POR

613890 Congenital adrenal hyperplasia, male pseudohermaphroditism

201810

HSD3B2

Cytochrome P450 Oxidoreductase

118485 Adrenal insufficiency, 46,XY sex reversal

613743

CYP11A1

300473 Adrenal hypoplasia congenital, Hypogonadotrophic hypogonadism

Xp21.2

124015 Congenital adrenal 7q11.23 hyperplasia, virilization in females, Antley– Bixler syndrome

1p12

15q24.1

8p11.23

600617 Lipoid congenital adrenal hyperplasia

201710

STAR

6p21.33

 Steroidogenic acute regulatory protein Cytochrome P450, family 11, subfamily A member 1 Hydroxy-­delta-­ 5-­steroid dehydrogenase, 3 beta-­ and steroid delta-­ isomerase 2 Cytochrome P450 family 17, subfamily A, polypeptide 1

613815 Congenital adrenal hyperplasia, ambiguous genitalia, virilisation

201910

PHENOTYPE

X:30,304, 205-­30, 309,389

7:75,915,154-­ 75,986,854

Nucleus

ER, cytosol

XR

AR

AR

ER 10:102,830, 530-­102,837, 412

AR

Mitochondria

AR

AR

AR

Mitochondria

ER

1:119,414,930-­ Mitochondria, 119,423,033 ER

15:74,337, 761-­74,367, 645

8:38,142,699-­ 38,150,951

6:32,038, 414-­32,041, 643

59

Embryonic lethal 63 incomplete penetrance, hypomorph with no adrenal phenotype noted Yes, gonadal 68 Yes, No phenotype in adrenal males, adrenal phenotype, phenotype on abnormal aging bone mineralization

69

51

56 Yes, no adrenal phenotype, decreased bone mineral density No

Yes, embryonic lethal, no adrenal phenotype tested

45

34

27

11

N/A

41

31

N/A

HUMAN REF

No

No

Yes, Prewean- Yes, model for ing lethality p450scc complete penetrance

Yes (Cyp21a1) No preweaning lethality (in) complete penetrance No Yes, model for CLAH

GENOME SUBCELLULAR GENOMIC COORDINATES LOCATION OF MODE OF MOUSE MOUSE MODEL MOUSE PROTEIN LOCATION (GRCH38) INHERITANCE MODEL IMPC OTHER REF

Cytochrome CYP21A2 P450 family 21 subfamily A member 2

GENE NAME

OMIM OMIM GENE ID DISEASE GENE

TABLE 86.1  Monogenetic causes of primary adrenal insufficiency and associated phenotype(s) in humans.

1432

611812

Wnt family member 4

231550

617825

605378 Hypoadrenalism, alacrima, achalasia, neurological dysfunction

606448 Isolated GC deficiency (cardiac defects)

607878 Isolated GC deficiency (TART, DCM)

614736

NNT

TXNRD2

609196 Isolated GC deficiency

607398

MRAP

Aladin WD AAAS repeat nucleoporin

Thioredoxin reductase 2

607397 Isolated GC deficiency

202200

174762

N/A

MC2R

610456

617053

Sterile alpha SAMD9 motif domain containing 9 Polymerase POLE epsilon 1

600856 IMAGE Syndrome -­intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies

614732

Melanocortin 2 receptor Melanocortin 2 receptor accessory protein Nicotinamide nucleotide transhydrogenase

PHENOTYPE

12q13.13

22q11

5p12

21q22

18p11.21

12q24.33 

7q21.2 

11p15.4

Cytosol

AR

12:53,307,455-­ Cytoskeleton, 53,321,609 nucleus, cytosol

AR

AR

Mitochondria

AR

AR

AR

AD

AD

Mitochondria, cytosol

22:19,875, 521-­19, 941,817

5:43,601,091-­ 43,707,395

12:132,623, Nucleus 757-­132,687, 518 18:13,882,041-­ Membrane 13,915,706 21:32,291,812-­ Membrane 32,314,783

7:93,099,517-­ 93,117,978 

11:2,883,217-­ 2,885,774

AR

1:22,117,307-­ 22,143,980 

Extracellular, ER, membrane, Golgi Nucleus

AD

9:124,481,235-­ Nucleus 124,507,398

No

Yes, adrenal phenotype not noted, decreased bone mineral density No

No

No

No

No

Yes, mice die within 24 h of birth No

No

Yes, embryonic lethal, no adrenal phenotype tested Yes, no adrenal phenotype

145

136

Continued

140

136

126

Yes, 50% 126 reduction in corticosterone

107

101

116

113

104

98

93

124

Yes, recapitulates FGD Yes, recapitulates FGD

Yes, no adrenal phenotype

82

HUMAN REF

87,88,89, 86 90,91

78

97 No, models for Beckwith– Wiedemann and Silver– Russell syndromes but no adrenal phenotype No N/A

No

Yes, adrenal and gonadal phenotype

GENOME SUBCELLULAR GENOMIC COORDINATES LOCATION OF MODE OF MOUSE MOUSE MODEL MOUSE PROTEIN LOCATION (GRCH38) INHERITANCE MODEL IMPC OTHER REF

184757 46,XY sex reversal, par- 9q33.3 tial or complete with or without adrenal failure 603490 1p36.12

Cyclin-­ CDKN1C dependent kinase inhibitor 1C

WNT4

612965

Nuclear recep- NR5A1 tor subfamily 5A1

GENE NAME

OMIM OMIM GENE ID DISEASE GENE

TABLE 86.1—cont’d

602136

300371

613497

614862

214100

300100

278000

PEX6

PEX1

ABCD1

LIPA

10:89,213, 570-­89, 251,925 

Mitochondria, peroxisome, lysosome ER, cytosol Lysosome, nucleus

Peroxisome, cytosol

Peroxisome, cytosol

AR

XR

AR

AR, AD

AR, AD

AR

AR

N/A

Yes, model for adrenoleukodystrophy

201

179

Yes, mild Zellwe- 186 ger phenotype

No

No Yes, develop accumulations of triglycerides and cholesterol esters

Yes, no adrenal phenotype Yes, no adrenal phenotype

No

Yes, embryYes, good model 167 onic for APS1 lethality (complete?)

Yes, hypomorph 147,149 Yes, embryis small with onic/preadrenal pheweaning notype lethality complete penetrance Yes, prewean- Yes, recapitu155 ing lethality lates main complete characteristics penetrance of SGPL1 deficiency

203

202

185

184

165

155

147

HUMAN REF

IMPC, International Mouse Phenotyping Consortium; ER, endoplasmic reticulum; CLAH, congenital lipoid adrenal hyperplasia; AR, autosomal recessive; AD, autosomal dominant; XR, X-­linked recessive; OMIM, online Mendelian inheritance in man.

10q.23.31

Xq28

7q21.2

6:42,963, 864-­42,980, 223 7:92,487, 022-­92,528, 519 X:153,724, 850-­153, 744,754

Nucleus

21:44,285, 875-­44,298, 647

Peroxisomal Biogenesis Factor 6 Peroxisomal Biogenesis Factor 1 ATP Binding Cassette Subfamily D Member 1 Lysosomal Acid Lipase

240300

AIRE

Autoimmune regulator

ER

10:70,815, 919-­70,881, 183

10q22.1 603729 Primary adrenal insufficiency, Steroid resistant nephrotic syndrome, ichthyosis, primary hypothyroidism, primary gonadal failure, dysplidemia, neurological abnormalities, lymphopenia 21q22.3 607358 Autoimmune polyendocrine syndrome type I Hypoparathyroidism, immune deficiency, chronic candidiasis, autoimmune adrenal insufficiency 601498 6p21.1

617575

SGPL1

 Sphingosine-­1 phosphate lyase

Nucleus

8:47,960,940-­ 47,978,159

GENOME SUBCELLULAR GENOMIC COORDINATES LOCATION OF MODE OF MOUSE MOUSE MODEL MOUSE PROTEIN LOCATION (GRCH38) INHERITANCE MODEL IMPC OTHER REF 8q11.21

PHENOTYPE

602638 Adrenal failure, Natural Killer cell deficiency, short stature, chromosomal instability

607398

Mini-­ MCM4 chromosome maintenance complex component 4

GENE NAME

OMIM OMIM GENE ID DISEASE GENE

TABLE 86.1—cont’d

CHAPTER 86  Genetic Disorders of the Adrenal Cortex

1435

Cholesterol CYP11A1

CYP17A1

HSD3B2

CYP17A1

Progesterone CYP21A2

CYP17A1 17-hydroxy-pregnenolone

Pregnenolone

HSD3B2

CYP21A2

Deoxy-corticosterone

CYP17A1 HSD3B2

-hydroxy-progesterone

17

Deoxycortisol

DHEA HSD17B1 Testosterone

Androstenedione CYP19

HSD17B3

CYP19

Oestrone

Oestradiol

CYP11B1

CYP11B2 Corticosterone

Cortisol

CYP11B2 Aldosterone Zona glomerulosa mineralocorticoids

Zona fasciculata glucocorticoids

Zona reticularis androgens

Figure 86.2  Steroidogenic cascade of the adrenal gland. The adrenal gland produces mineralocorticoids, glucocorticoids, and androgens in different zones of the cortex. Mutations in several of the CYP genes result in congenital adrenal hyperplasia, leading to a compensatory increase in adrenocorticotropic hormone that drives the adrenal hyperplastic response due to its trophic action on adrenocortical cells. DHEA, dehydroepiandrosterone.

A naturally occurring mouse strain, H-­2aw18, with deletion of cyp21 and complement component c4 due to meiotic recombination, dies at an early postnatal stage.23 The loss of cyp21 leads to hyperproduction of ACTH with adrenocortical hyperplasia and accumulation of precursor steroids.24 Because mouse adrenals lack steroid 17-­alpha-­hydroxylase/17,20 lyase, the accumulated precursor is progesterone, and no androgenization occurs. The adrenals of these mice present with lack of zonation, hypertrophy, and hyperplasia of adrenocortical mitochondria, although adenovirus-­mediated transfer of human steroid 21-­hyrdoxylase into these mice has been shown to transiently restore adrenal steroidogenesis.24

Steroidogenic Acute Regulatory Protein (STAR). STAR is part of a complex responsible for the transportation of cholesterol from the outer mitochondrial membrane to the IMM, where it is processed to pregnenolone, reported as the rate-­limiting step of steroidogenesis (Fig. 86.1). Along with mitochondrial cholesterol side-­chain cleavage enzyme (CYP11A1), it is one of the key factors in hormone production in both the adrenals and the gonads.25 Mutations in STAR therefore inhibit the delivery of cholesterol to the steroidogenic enzyme cascade and prevent glucocorticoid and mineralocorticoid production.26 This shutdown of steroidogenesis due to mutations in STAR is known as congenital lipoid adrenal hyperplasia (CLAH), first characterized in 1996.27 Patients with this condition present in the first month of life with salt-­wasting adrenal and gonadal insufficiency.28 Due to dysfunctional gonadal steroidogenesis, CLAH infants with a 46, XY karyotype fail to produce testosterone in utero and have female genitalia, while infants with a 46, XX karyotype present with variable ovarian insufficiency at adolescence. A two-­hit model has been proposed to account for the pathogenesis of CLAH, with the first “hit” being lack of STAR and the second being cholesterol-­mediated oxidative damage.27 Upon ACTH stimulation, a steroidogenic cell gathers cholesterol from endogenous synthesis, stored lipid droplets or low-­density lipoprotein (LDL) receptor-mediated endocytosis, and STAR subsequently facilitates transport through the mitochondria. In cells with defective STAR transport, only minimal

residual STAR-­independent steroid biosynthesis occurs, resulting in low levels of steroidogenesis (first hit). Inadequate steroid levels drive an increase in steroidogenic stimuli and culminate in the accumulation of cholesterol within the cells. This progressive lipid accumulation leads to mitochondrial oxidative damage, which eventually culminates in reduced steroidogenic capacity and enlarged adrenal size (second hit).27 Beyond the “classic” disease of CLAH, partial defects in STAR can cause nonclassic CLAH (NCLAH) in 5% to 10% of patients.29,30 This disease predominantly affects glucocorticoid synthesis and masquerades as FGD, as patients have normal genitalia and may be fertile. Variants pR188C and pR192C reduce STAR’s interactions with the 3‐βOH group of cholesterol and cause partial loss of function (LOF).29,30 Long-­term reproductive follow-­up is required for NCLAH patients to ensure that correct pubertal development occurs, and appropriate counseling and sperm banking may be offered.28 Star knockout mice are born normally, indicating that STAR is not required for survival in utero; however, both genetic male and genetic female mice have female external genitalia.31 Most mice die within 1 to 2 days of birth, with none surviving beyond 16 days without treatment, most likely due to respiratory distress secondary to adrenal insufficiency.31 This was supported by the survival of pups that underwent corticosteroid replacement. Furthermore, levels of corticosterone and aldosterone were low compared to littermates, while levels of ACTH were elevated. Finally, although adrenal glands were smaller in knockout mice, they showed high quantities of lipid deposition, supporting the second hit of the two-­hit model of CLAH.31

Cytochrome P450, Family 11, Subfamily A Member 1 (CYP11A1). CYP11A1 encodes the mitochondrial cholesterol side-­ chain cleavage enzyme that performs the rate-­limiting step of steroidogenesis, cholesterol conversion to pregnenolone, at the beginning of the steroidogenic enzyme cascade (Fig. 86.2). Pregnenolone is subsequently processed to produce cortisol, aldosterone, and androgens in different pathways and, consequently, loss-­of-­function mutations can cause CAH associated with glucocorticoid and mineralocorticoid deficiency,

1436

PART 7  Adrenal

as well as disorders of sex development. Absence of the enzyme was previously believed to be incompatible with term gestation; however, several patients with side-­chain cleavage enzyme deficiency have been reported, initially in 2001.32-­34 Mutations in CYP11A1 classically result in mitochondrial cholesterol side-­chain cleavage enzyme syndrome characterized by primary adrenal failure in combination with 46, XY sex reversal, although, unlike STAR-­deficient subjects, adrenal size is unaffected. Similar to STAR, partial loss-­of-­function mutations (retention of up to 20% of wild-­type function) can produce a nonclassic form of the disease with a phenotype of predominant glucocorticoid deficiency with variable degrees of mineralocorticoid deficiency and sexual development, depending on the residual activity of the mutant proteins.35-­38 Patients can present at different ages during infancy, typically with hyperpigmentation, hypoglycemia, or prolonged illness with infections.28 Two recent studies have found that missplicing of CYP11A1 mRNA results in a truncated version of the protein that can cause adrenal insufficiency.39,40 Genetic analysis revealed that many families of European heritage with PAI are compound heterozygous for a c.940G>A variant (rs6161) on one allele of CYP11A1, with a second disruptive change on the other allele.39 The in vitro splicing assays showed that the predicted benign change of c.940G>A (p.E314K) resulted in missplicing and a truncated protein consistent with cleavage at the predicted amino acid change.39,40 This variant is carried by 1:140 people of European descent but has only been shown to cause PAI when inherited with a rare disruptive change on the other allele.28 A study investigating Cyp11a1 null mice showed that they do not survive weaning and present with corticosterone and aldosterone deficiency compared with wild-­type littermates. However, when treated with corticosteroids they survive to adulthood. Cyp11a1-­/-­ XY males exhibit sex reversal with feminization of external genitalia and disorganization of internal genitalia. The adrenals of the null mice are reduced in size, although they do show progressive lipid accumulation similar to Star knockout mice.41

Hydroxy-­Delta-­5-­Steroid Dehydrogenase, 3 Beta-­And Steroid Delta-­Isomerase 2 (HSD3B2). Mutations in HSD3B2 result in a very rare variant of CAH causing less than 0.5% of cases.42 HSD3B2 catalyzes the conversion of Δ5-­3β-­hydroxysteroids (pregnenolone, 17-­hydroxypregnenolone and dehydroepiandrosterone [DHEA]) to Δ4-­3-­ketosteroids (progesterone, 17-­OHP and androstenedione) (Fig. 86.2). HSD3B2 deficiency therefore impairs the synthesis of aldosterone, cortisol, and testosterone and results in high levels of renin, ACTH, and DHEA.43 DHEA can be converted to androstenedione by extraadrenal HSD3B1, resulting in high levels of testosterone in females, although this fails to compensate for absence of the hormone in males.43,44 Severe impairment of steroid synthesis was first mapped to HSD3B2 through identification of nonsense and frameshift mutations in 1992.45 In general, frameshift and nonsense mutations present with a salt-­wasting phenotype, while missense mutations are associated with residual enzyme activity and a non-salt-­wasting phenotype.15,46 Further presenting symptoms include incomplete masculinization in 46, XY patients, virilization in 46, XX patients, gynecomastia in males, hypospadias, and palpable testis.47-­49 This form of CAH is best diagnosed by high levels of 17-­hydroxypregnenolone in the blood plasma and is treated with glucocorticoid, mineralocorticoid, and possibly sex hormone replacement therapy.

Cytochrome P450 Family 17, Subfamily A, Polypeptide 1 (CYP17A1). Due to its location in the steroidogenic cascade (Fig. 86.2), steroid 17-­ alpha-­ hydroxylase/17,20 lyase (CYP17A1) acts as the

exclusive gateway to androgen production, with severe mutations resulting in impaired adrenal and gonadal sex steroid production, causing sexual infantilism and pubertal failure.44,50 Steroid 17-­ alpha-­ hydroxylase/17,20 lyase functions in the conversion of pregnenolone to 17-­hydroxypregnenolone and onwards to DHEA, as well as that of progesterone to 17-­OHP and onwards to androstenedione, and therefore deficiency of the enzyme results in low cortisol and androgens but high levels of DOC and corticosterone (Fig. 86.2). Pubertal failure is therefore a prominent feature of steroid 17-­alpha-­hydroxylase/17,20 lyase deficiency. Importantly, however, due to the high levels of DOC and corticosterone, which possess glucocorticoid properties, mutations in CYP17A1 do not result in glucocorticoid deficiency and adrenal crisis.44 High levels of DOC, however, cause sodium retention, leading to hypertension and consequently suppression of aldosterone production. CYP17A1 deficiency was first identified in 1988 in a patient with a four-­base duplication.51 46, XY patients with CYP17A1 mutations are usually diagnosed at birth due to ambiguous external genitalia.52 Conversely, as patients do not undergo adrenal crisis at a young age, many 46, XX patients are not diagnosed until adolescence. Although DOC accumulates, manifestations of mineralocorticoid excess tend not to occur until adolescence, where it presents with hypertension and hypokalemia.50 At puberty, lack of DHEA prevents adrenarche and development of pubic and axillary hair, and 46, XX patients can present with amenorrhea without hypertension and ovarian cysts.53-­55 At present there are no published examples of successful pregnancy in which either partner has 17-­hydroxylase deficiency. The deficiency is treated with the appropriate androgen replacement therapy and partial glucocorticoid replacement. While glucocorticoid therapy is not essential, due to corticosterone substitution, it functions to prevent adrenal axis suppression and normalize blood pressure, and therefore a partial replacement is used as a compromise.50 In one study, Cyp17a1-­/-­ mice died around embryonic day 7 and could not be rescued through maternal administration of DHEA, androstenedione, and 17-­OH pregnenolone.56 The International Mouse Phenotyping Consortium, however, presents data indicating that mice reach adulthood but display varying phenotypes, including increased circulating cholesterol level and absent male genitalia.

Cytochrome P450 Oxidoreductase (POR). Cytochrome P450 oxidoreductase (POR) is a flavoprotein that plays a key role in electron transport, donating electrons to mitochondrial P450 enzymes including steroid 17-­alpha-­hydroxylase/17,20 lyase and steroid 21-­hydroxylase and aromatase (CYP19A1).44 Therefore, in POR deficiency (PORD), a reduction in steroidogenesis is due to impairment of key steroidogenic enzymes resulting in a lack of glucocorticoids and androgens. As POR acts an electron donor for all microsomal cytochrome P450 enzymes, there are multiple comorbidities associated with PORD. These can, but do not always, include changes in drug metabolism, pathogenesis of skeletal dysplasia, and other skeletal manifestations that resemble Antley–Bixler syndrome.57-­59 The first mutations in POR linked with disordered steroidogenesis were identified in 2004.59 Most POR mutations retain some enzymatic function, resulting in high levels of pregnenolone, progesterone, 17-­ OHP, DOC, and corticosterone, low levels of DHEA and androstenedione, and variable levels of cortisol and aldosterone.44,60 Affected individuals can be detected in utero, as fetoplacental deficiency of POR can lead to virilization of the mother due to defective conversion of androgen precursors to estrogen.59 Once born, affected 46, XX patients present with ambiguous genitalia, but circulating androgens remain at a low level, and virilization does not progress.61 Urinary steroid metabolite profiling also provides a further useful noninvasive method

CHAPTER 86  Genetic Disorders of the Adrenal Cortex for diagnosis.44 PORD presentation varies greatly, from amenorrhea and polycystic ovaries in 46, XX patients and androgen deficiency in 46, XY patients to severe hormone disturbances resulting in atypical genitalia. One study showed that more than 80% of 46, XX patients in their cohort presented with differences in sex development (DSD), whereas DSD was present in only half of 46, XY patients.62 Patients do not develop mineralocorticoid deficiency, but require permanent or stress-­dosed glucocorticoid replacement therapy. In one study, targeted deletion of the translation start site and membrane-­ binding domain in Por-­/-­ mice resulted in embryonic lethality by day 13.5 of gestation, with embryos possessing multiple defects including abnormalities in the neural tube, heart, eye, and limb or a gross retardation of development.63 A further study created a hypomorphic mouse with 5% to 26% of normal POR activity that grew to adulthood and presented with decreased weight and decreased heart, lung, and kidney size.64 Interestingly no adrenal phenotype was noted in these mice. KEY POINTS  • Mutations in certain genes can cause congenital adrenal hypoplasia, which is underdevelopment or hypotrophy of the adrenal cortex. • Mutations in the genes encoding the transcription factors NROB1 and NR5A1 affect the transcription of genes involved in steroidogenesis and the development of the adrenal glands themselves. • Mutations in genes such as CDKN1C, SAMD9, and POLE1 can cause complex multisystem growth disorders that contain adrenal hypoplasia as a hallmark of the disease.

Hypoplasia Adrenal hypoplasia is the underdevelopment or hypotrophy of the adrenal cortex leading to reduced steroid hormone production, and subsequently PAI. For more detail on adrenocortical developmental genetics, refer to Chapter 85.

Nuclear Receptor Subfamily 0 Group B Member 1 (NROB1). AHC is a condition that results from the underdevelopment of the adrenal gland. It is caused by LOF mutations to dosage-­sensitive sex reversal, adrenal hypoplasia critical region on chromosome X, DAX1, encoded by NROB1, and classically presents with PAI and associated salt-wasting, hypogonadotropic hypogonadism (HH), and infertility.28 DAX1, a transcription factor belonging to the nuclear receptor superfamily, acts as a transcriptional repressor of genes involved in the steroidogenic pathway and the development of the hypothalamus–pituitary–gonadal axis.65,66 DAX1, in part, serves as a transcriptional repressor of steroidogenic factor 1 (SF-­1, also known as NR5A1)-­mediated transcriptional activation. Furthermore, DAX1 downregulates anti-­Müllerian hormone (AMH), a hormone that, in 46, XY embryos, inhibits folliculogenesis and development of female gonads.67 Loss of DAX1-­dependent inhibition therefore results in abnormal gonad development in 46, XY patients. This is supported by DAX1-­deficient mice presenting with testicular abnormality and infertility.68 Pathogenic variants in NROB1 were first reported as a cause of X-­linked AHC in 1994.69 Its association with Duchenne muscular dystrophy and glycerol kinase deficiency allowed for an AHC-­critical region of 200 to 500 kb to be physically mapped to the short arm of the X chromosome.69 Most affected infants present within the first months of life with classic PAI symptoms: low cortisol, high ACTH, low aldosterone, and increased renin resulting in salt wasting.70 Affected individuals are therefore treated with replacement doses of hydrocortisone and salt supplements, as well as sex hormone replacement to induce

1437

puberty if necessary. Patients may also present with paradoxical features such as precocious puberty, making the clinical spectrum of the disease quite variable.71 Late-­onset X-­linked AHC has also been well documented. Patients can also present with partial HH and infertility. This is often due to a partial LOF variant in the ligand-­like binding domain or an early stop variant leading to translation of a truncated protein.72-­74 Moreover, late-­ onset adrenal failure in patients with AHC has been preceded by cortisol excess in some patients.75 While it remains unclear how LOF NROB1 mutations could lead to these disparate adrenal phenotypes,65,66 data in mice with genetic LOF mutations in NROB1 are consistent with premature adrenocortical progenitor cell differentiation at the expense of ultimate adrenal failure.76

Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1). SF-­1 is a transcription factor belonging to the nuclear receptor superfamily and acts as a master regulator of both adrenal and gonadal development.77 Expressed in the adrenal cortex, testicular Leydig cells, and ovarian theca cells, SF-­1 regulates the transcription of genes encoding key steroidogenic enzymes such as CYP11A1, CYP11B1, CYP21, and STAR, as well as AMH, gonadotrophins, and aromatase.78 Indeed, SF-­1 acts as an active determinant of gender differentiation through upregulation of AMH.79 As demonstrated in mice, loss of Sf-­1 can therefore cause adrenal agenesis, gonadal dysgenesis with a subsequent female phenotype, and variable degrees of HH.78 Mutations in NR5A1 were first associated with adrenal insufficiency and XY sex reversal in 1999.80 Here a 46, XY patient presented with primary adrenal failure as well as phenotypically female genitalia as a result of failure of Müllerian regression due to loss of SF-­1– directed AMH production. In 46, XY patients, pathogenic variants are associated with a range of phenotypes, including testicular dysgenesis/ dysfunction, severe hypospadias, and infertility.81 Primarily causing defects in reproductive development, NR5A1 mutations have rarely been associated with PAI, with only six patients documented to date.28 Affected individuals with PAI can present with hyperpigmentation, hypotonia, jaundice, weight loss, and failure to thrive.82

Wnt Family Member 4 (WNT4). WNT4 belongs to the large WNT family of secreted glycoproteins that play a key role in the development of multicellular animals. WNT4 itself is considered the most important molecule for the correct differentiation of female gonadal tissues and is also known to play a role in the development of the kidneys, adrenals, pituitary gland, and mammary tissues.83,84 Considering that the gonads, kidneys, and adrenals all originate from the urogenital ridge, it can be assumed that WNT4 functions in early embryological development. Mutations in WNT4 can therefore disrupt the normal development of these organs and cause adrenal insufficiency. WNT4 is also known to repress the steroidogenic enzymes CYP17A1 and HSD3B2, and consequently loss of WNT4 leads to androgen excess and sex reversal.85 Adrenal insufficiency due to WNT4 mutations is rare, and the autosomal recessive syndrome it causes, SEx Reversion, Kidneys, Adrenal and Lung dysgenesis (SERKAL) syndrome, has been identified in three fetuses.86 This study used a candidate gene approach to identify a homozygous missense mutation in WNT4 (p.A114V) in three terminated fetuses. The parents of two of these fetuses were second-­degree cousins, with the third fetus also belonging to the wider kindred, and the pregnancies were terminated due to renal agenesis determined through ultrasonic scanning. This mutation markedly reduced WNT4 mRNA levels in transfected ovarian epithelium cells compared with wild-­type WNT4, resulting in a downregulation of WNT4-­dependent inhibition of β-­catenin degradation.86 This change in β-­catenin-induced gene expression could result in the developmental defects witnessed

1438

PART 7  Adrenal

in SERKAL syndrome. Wnt4-­deficient mice die within 24 h of birth, showing signs of kidney dysfunction and improper development of the pituitary gland, as well as partial sex reversal in females.87-­89 In the adrenal cortex, Wnt4-­deficient mice showed reduced expression of Cyp11b2 in the zona glomerulosa, resulting in significantly lowered aldosterone secretion in newborn mice, suggesting a role of Wnt4 in the development and maintenance of the zona glomerulosa.90 A mouse model in which Wnt4 was depleted in the adrenal cortex corroborated these results, while also uncovering crucial crosstalk between PKA and Wnt-­beta catenin signaling in determining the differentiation and fate of zona glomerulosa cells.91

Cyclin Dependent Kinase Inhibitor 1c (CDKN1C). A complex multisystem growth disorder that involves PAI is IMAGe syndrome, characterized by Intrauterine growth restriction, Metaphysical dysplasia, Adrenal hypoplasia, and Genitourinary anomalies (in males), and was first described in 1999.92 The syndrome is normally caused by heterozygous missense variants in cyclin-­dependent kinase inhibitor 1C (CDKN1C), a negative regulator of cell proliferation. All identified mutations that cause IMAGe syndrome are clustered in the proliferating cell nuclear antigen (PCNA) binding domain and cause a gain-­of-­function resulting in repressed growth.93 The mechanism is still unclear but may involve reduced degradation of CDKN1C and subsequently prolonged cell cycle repression by impairing entry into S phase.28,94 Conversely, LOF mutations outside of the PCNA binding domain of the CDKN1C gene contribute to the overgrowth phenotype of Beckwith–Wiedemann syndrome (including adrenal cytomegaly) due to an imprinting defect of the IGF2 locus at 11p15 (containing CDKN1C).93 IMAGe syndrome is clinically variable, with some patients presenting without adrenal insufficiency and with diabetes mellitus.95 This diabetes phenotype is most likely due to CDKN1C being involved in insulin-­producing β cells. CDKN1C is paternally imprinted on chromosome 11 and is consequently only expressed from the maternal allele; while this inheritance can mimic an X-­linked disease, IMAGe syndrome patients can be male or female.96 Expression of IMAGe-­causing mutations (mutations in the PCNA binding domain) in a human embryonic kidney (HEK) cell line demonstrated that, in IMAGe syndrome CDKN1C becomes more stable and does not undergo normal cell cycle– related oscillation, inhibiting progression from the G1 phase.94 Overexpression of wild-­type CDKN1C and Beckwith–Wiedemann syndrome mutant CDKN1C both inhibited cell proliferation, but not to the same extent as the IMAGe mutant. No mouse line has been created with an IMAGe-­mutant CDKN1C, but a mouse containing a Beckwith–Wiedemann mutant CDKN1C presented with no adrenal phenotype.97

Sterile Alpha Motif Domain Containing 9 (SAMD9). A further complex multisystem growth restriction disorder that causes adrenal hypoplasia is MIRAGE syndrome, named according to it symptoms: Myelodysplasia, Infections, Restricted growth, Adrenal hypoplasia, Gonadal anomalies, and Enteropathy.98 The syndrome is a result of gain-­of-­function mutations in the growth suppressor gene SAMD9, located on the long arm of chromosome 7 (7q21.2).98,99 Conversely, LOF mutations have been reported in tumors, including normophosphatemic familial tumoral calcinosis.100 Patients with MIRAGE syndrome commonly die at a young age (G) as part of a common haplotype, in combination with different LOF mutations in trans (11 different mutations in the 15-­patient cohort). Experiments in patient-­derived fibroblasts demonstrated that the intronic variant alters splicing, and part of intron 15 is retained. This inclusion of an extra 47 bp causes a frameshift that results in an early stop codon (p.Asn563Valfs*16). This mutation, along with a LOF mutation on the other allele, significantly reduces the protein levels of POLE1 in patient fibroblasts, leading to delayed S-­phase progression and, consequently, restricted cell growth.101 A single homozygous intronic variant (c.4444+3A>G) in POLE1 has been associated with immunodeficiency, lymphopenia, and short stature.102,103 Immunodeficiency was also identified in seven of the 15 patients from the discussed study, and therefore the spectrum of ­biallelic POLE mutations is greater than that of IMAGe syndrome caused by CDKN1C mutations.101 A further study investigated the effect of a knock-in mutation in mice to selectively inactivate the proofreading exonuclease activity of the enzyme while preserving its normal polymerase activity.104 This study demonstrated that the

CHAPTER 86  Genetic Disorders of the Adrenal Cortex ACTH MC2R

N C

MRAP

C N Adenylate  cyclase ATP cAMP

PKA

P CREB

Nuclear gene transcription Figure 86.3  Adrenocorticotropic hormone (ACTH) signaling in adrenocortical cells. Ligand binding results in generation of increased levels of cAMP, which activates protein kinase A (PKA) and subsequently phosphorylates cAMP response element binding protein (CREB). CREB is directly involved in the transcriptional regulation of steroidogenic enzymes.

impaired enzyme resulted in a strong mutator phenotype with a high incidence of neoplasms. KEY POINTS  • Familial glucocorticoid deficiency (FGD) is characterized by the lack of response to adrenocorticotropic hormone (ACTH) and results in glucocorticoid deficiency but unaffected plasma renin and aldosterone levels. • Mutations in MC2R and MRAP cause FGD through the disruption of ACTH signaling and consequential inactivation of the steroidogenic cascade. • NNT and TXNRD2 mutations cause FGD through uncontrolled oxidative stress in the cells of the zona fasciculata, which disrupt steroidogenesis.

Familial Glucocorticoid Deficiency-­Like (FGD-­Like) FGD, a hereditary unresponsiveness to ACTH, is characterized by glucocorticoid deficiency in a patient who retains normal plasma renin and aldosterone levels. It can be caused by several genes, and disease phenotypes are therefore often variable.

Melanocortin Type 2 Receptor (MC2R). MC2R encodes a G protein–coupled receptor that belongs to the melanocortin receptor family. Binding of its ligand ACTH triggers the activation of adenylate cyclase, which increases intracellular cAMP, subsequently activating PKA and the glucocorticoid steroidogenic cascade (Fig. 86.3). Mutations in MC2R account for the largest number of FGD cases, and the majority are nonconservative missense mutations that culminate

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in a failure to traffic the receptor to the cell surface, resulting in 20% to 100% reduced membrane expression in an in vitro study.105,106 MC2R mutations were first associated with FGD via a candidate gene sequencing approach of a family with the condition in 1993.107 More than 50 have now been described, and, interestingly, regional variants exist in MC2R; for example, p.S74I is the most common in the United Kingdom, while c.560delT is a recurrent mutation in Turkey.108 Mutations in MC2R therefore inhibit the initiation of steroidogenesis and disrupt the negative feedback loop, resulting in high ACTH plasma levels. It is important to note that, while MC2R mutations most typically cause FGD, in severe LOF cases due to homozygous frameshift mutations, minor disturbances to the renin–angiotensin–aldosterone axis have been documented, therefore resulting in a form of PAI with transient mineralocorticoid deficiency.109,110 Patients with MC2R mutations typically present with symptoms of glucocorticoid deficiency, including jaundice, hypoglycemia, failure to thrive, sepsis, and hyperpigmentation.111 Hyperpigmentation of the skin is due to supraphysiologic levels of ACTH causing overactivation of melanocortin 1 receptor (MC1R), culminating in the production of excess melanin. The age of onset is highly variable (range 0–16 years; median 2 years), with some patients presenting in later childhood with mild disease.106 Patients present with a mean height standard deviation score of +1.76 ± 1.52,106 believed to be due to increased ACTH-­level activation of melanocortin receptors expressed in bone and cartilaginous growth plates.112 This is supported by evidence from adrenalectomized mice showing that the melanocortin system plays a role in linear growth.112 Interestingly, however, Mc2r knockout mice do not exhibit any significant difference in body length compared with wild-­ type mice.113 Clinical observations indicate that the advanced growth rate in humans can be normalized with glucocorticoid replacement therapy.114,115

Mc2r Accessory Protein (MRAP). MRAP encodes a single transmembrane protein that acts as an essential cofactor in the trafficking of MC2R from the endoplasmic reticulum to the cell membrane to enable its functional expression.116 It exists as a homodimer with a unique antiparallel dual topology conformation essential for its function.117,118 Mutations in MRAP therefore prevent trafficking of MC2R to the plasma membrane and consequently inhibit the initiation of steroidogenesis through ACTH signaling. Mutations in the gene were first associated with FGD following linkage studies and homozygosity mapping using single nucleotide polymorphism array genotyping of a consanguineous family.116 Unlike the majority of MC2R mutations, which are missense and may result in some residual function, MRAP mutations are typically splice-­site or nonsense mutations.106 These are predicted to result in a truncated protein that lacks the highly conserved transmembrane domain. This therefore inhibits its function of trafficking MC2R from the endoplasmic reticulum and results in degradation of the receptor.105,116,119 Patients with MRAP mutations present with the same symptoms as those with MC2R mutations. However, due to a complete breakdown of ACTH signaling, the disease can often present with earlier onset. One study showed that the median age of onset for MRAP-­induced FGD was 0.08 years (compared with 2 years for MC2R mutations), and the range was significantly reduced, at 0 to 1.6 years.106 Considering the earlier onset and consequential earlier glucocorticoid replacement in these patients, the taller stature seen in MC2R patients is largely absent in the MRAP patient cohort.120 There are, however, exceptions to the rule, and sequencing of two affected families revealed homozygous missense mutations c.175T>G (pY59D) and c.76T>C (p.V26A), both of which presented with a later onset and subtler phenotype.121 Beyond the trafficking of MC2R, MRAP has been shown to interact

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PART 7  Adrenal H+

Inner mitochondrial membrane

NNT

H+ NADPH

Thioredoxin system

GSR

TXNRD2

Glutathione system

GSH/GSSG

TXN2

GLRX2

PRDX3

ROS H2O2

H2O + 1/2O2

Figure 86.4  The thioredoxin and glutathione redox homeostatic pathways. NADPH is the electron donor that drives the thioredoxin and glutathione pathways, with NNT being the primary regenerator of NADPH levels. TXNRD2 reduces both TXN2 and GLRX2 (glutathione reductase and GSH play a further role in reduction of GLRX2), which subsequently reduces PRDX3. PRDX3, a highly conserved thioredoxin-­dependent peroxide reductase, protects the cellular environment from oxidative stress due to its antioxidant properties.

with each of the melanocortin receptors, and it is postulated that its facilitation of MC4R expression could account for the obesity observed in one patient.122,123 This remains speculative, however, and a study comparing the phenotypic differences between MC2R and MRAP patients concluded that there was no evidence that MRAP deficiency impacted any other physiological function beyond that seen with a defective MC2R.106 A recent study showed that Mrap-­/-­ mice have almost complete penetrance of neonatal lethality (322 out of 325 mice) due to incomplete lung development, although this phenotype was rescuable by administration of corticosterone to pregnant dams. Mice that survived weaning showed the characteristic symptoms of MRAP deficiency: isolated glucocorticoid deficiency with normal mineralocorticoid function. Analysis of adrenal histology of knockout mice showed altered zonation, a thicker capsule, and lower mRNA expression of steroidogenic genes.124

Nicotinamide Nucleotide Transhydrogenase (NNT). NNT is the major enzymatic source of NADPH in the mitochondria, contributing 45% of the total supply.125 Existing as a dimer, it is expressed ubiquitously throughout the body, where it spans the IMM orchestrating H+ movement and supplying NADPH for the detoxification of reactive oxygen species (ROS) through the glutathione and thioredoxin pathways (Fig. 86.4). This role in redox homeostasis and removal of ROS contributes to limiting oxidative stress. NNT was initially linked to PAI through single nucleotide polymorphism genotyping of consanguineous families with cases of isolated glucocorticoid deficiency, followed by targeted exome sequencing that identified many causal variants in the gene in different families.126 This study documented 15 mutations in different kindreds that presented with a classical FGD phenotype, indistinguishable from mutations in MC2R and MRAP, with patients presenting with hypocortisolemia and

elevated ACTH with normal renin and aldosterone levels. Furthermore, this study demonstrated that NNT mutations cause adrenal dysfunction in humans, primarily affecting cells of the zona fasciculata responsible for cortisol production, and when NNT expression was stably knocked down in adrenocortical carcinoma cells in vitro, increased mitochondrial ROS generation with lowered reduced glutathione to oxidized glutathione was observed.126 A second study reported that FGD patients with NNT mutations present at a median age of 12 months, with a range of 3 days to 39 months, and 53% display hyperpigmentation.127 In a cohort of 23 patients, four presented with coexisting mineralocorticoid deficiency and showed evidence of hyponatremia, hyperkalemia, and Addisonian crises. Further studies have added comorbidities to the classical FGD symptoms, with a cardiac phenotype being linked to NNT mutations. Heterozygous LOF NNT mutation patients have been linked to left ventricular noncompaction,128 while a homozygous mutation (p.R379*) identified in a single patient resulted in features of glucocorticoid insufficiency with superimposed progressive left ventricular hypertrophy.129 Furthermore, testicular adrenal rest tumors have also been documented with NNT mutations, similar to CAH patients.129,130 These extra comorbidities make early screening in affected families essential. The mouse strain C57BL/6J carries an intrinsic inactivating mutation in Nnt that results in an untranslated protein. Analysis of the adrenal glands of 3-­month-­old mice of this strain shows slightly disorganized zona fasciculata with higher levels of apoptosis than the C57BL/6N strain without this mutation.126 A further study reported reduced steroidogenic capacity (50% reduction in corticosterone levels, perhaps due to reduction in Cyp11a1/Cyp11b1 expression) of the adrenals due to the inability of other antioxidant enzymes to compensate for the loss of NNT, leading to oxidative stress.131 Interestingly,

CHAPTER 86  Genetic Disorders of the Adrenal Cortex C57BL/6J mice do not exhibit a cardiac phenotype, despite lacking NNT.132

Thioredoxin Reductase 2 (TXNRD2). TXNRD2 is a dimeric NADPH-­dependent flavin adenine dinucleotide-­containing enzyme that reduces thioredoxin 2 and other substrates.133 It achieves this by utilizing electrons supplied by NNT (Fig. 86.4). The role of this protein, along with the glutathione system, is to eliminate ROS, which are generated in high quantities by the steroidogenic pathway and impede adrenocortical cellular function. The final step of cortisol production, performed by CYP11B1, accounts for approximately 40% of total electron flow from NADPH to ROS during steroidogenesis.134 This may explain the susceptibility of the zona fasciculata to oxidative stress and why NNT and TXNRD2 patients develop primarily glucocorticoid deficiency. Knockdown of TXNRD2 in adrenocortical carcinoma cells results in an increase in mitochondrial ROS levels, as it is unable to keep PRDX3 in its reduced form. This is only partially compensated for by the glutathione system.135 Mutations in TXNRD2 therefore can lead to oxidative stress-­induced damage to mitochondria and inhibit steroidogenesis. Thus far only one mutation of TXNRD2 has been linked to PAI, a stop-­gain mutation (p.Y447X) discovered in a Kashmiri kindred with seven affected individuals.135 These patients presented with a wide spectrum of disease with later onset in a number of family members. All clinically affected family members demonstrated a poor cortisol response to ACTH with normal mineralocorticoid levels, and only one patient presented with a comorbidity (cardiac failure). Txnrd2-­/-­ mice exhibit embryonic lethality due to severe growth restriction, cardiac anomalies, and hematopoietic dysregulation; however, no adrenal phenotype was noted.136 Electron microscopy of neonatal hearts showed severe swelling and destruction of mitochondrial cristae in cardiomyocytes, highlighting the damage that can be caused to mitochondria by loss of Txnrd2. This is supported by a further study that showed mitochondria isolated from Txnrd2 knockout cardiomyocytes were unable to utilize oxygen efficiently, and consequently generated high levels of ROS.137 Interestingly, two separate heterozygous mutations (p.G375R and p.A59T) have been identified in individuals with dilated cardiomyopathy, supporting a link between TXNRD2 and heart failure in humans.138

Aladin Wd Repeat Nucleoporin (AAAS). Triple A syndrome (Allgrove syndrome) is a rare autosomal recessive disease caused by mutations in AAAS, characterized by alacrima, achalasia, and adrenal failure. It is associated with a range of progressive motor, sensory, and autonomic neurological defects.139,140 AAAS encodes the nuclear pore complex protein alacrima-­achalasia-­adrenal insufficiency neurological disorder (ALADIN), which is expressed ubiquitously with predominance in the adrenal and central nervous system and was discovered to cause triple A syndrome in 2001.140,141 The clinical phenotype and age of onset of the disease are highly variable, even with members of the same family, with associated features including pupil and cranial nerve abnormalities, optic atrophy, and hypoglycemic seizures.142 Knockdown of AAAS in adrenocortical carcinoma cells results in increased susceptibility to oxidative stress, along with alteration of redox homeostasis leading to apoptosis.143,144 Furthermore, loss of AAAS has been shown to cause reduced expression of key steroidogenic enzymes STAR, mitochondrial cytochrome P450 11B1, steroid 17-­alpha-­hydroxylase/17,20 lyase, and steroid 21-­hydroxylase, which results in a significant reduction in cortisol production in human cell lines.143,144 Improvements in both cell viability and cortisol production were demonstrated by treatment with N-­acetylcysteine,

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which replenishes stores of reduced glutathione and acts as a direct scavenger of free radicals.143 This highlights the importance of the glutathione antioxidant pathway and promotes N-­acetylcysteine as a possible therapeutic adjunct for triple A syndrome. Interestingly, while Aaas knockout mice show female infertility, they fail to develop a phenotype similar to human triple A syndrome.145

Mini-­Chromosome Maintenance Complex Component 4 (MCM4). MCM4 is a protein essential for the initiation of eukaryotic genome replication. It possesses intrinsic DNA helicase activity and forms part of a hexameric complex with other MCMs. PAI appears at a higher prevalence in the Irish Traveller community, a genetically isolated population with higher levels of consanguinity than in the wider population (1 in 2506 compared with 1 in 201,898).146 Homozygosity mapping of a cohort of Irish Travellers who were MC2R-­, MRAP-­, and STAR-­ mutation negative highlighted a single variant in MCM4 (c.71-­1insG) that is predicted to produce a truncated protein (p.Pro24Argfs*4).147 This variant is unique to the Irish Traveller community and is associated with adrenal failure, growth retardation, and natural killer cell deficiency (also known as immunodeficiency 54).147,148 MCM4 patients present with a milder adrenal phenotype than other FGD syndromes, with onset of hypocortisolemia in childhood, sometimes following a period of normal adrenal function.147 This is possibly explained by the presence of a shorter isoform of the protein containing all necessary functional domains and partially compensating for the loss of the full-­length protein.148 Due to its role in genome replication, MCM4 patients suffer from chromosome fragility, increased susceptibility to viral infections, and an increased risk of developing neoplasms.148 Mice carrying a hypomorphic allele of Mcm4 present with capsular/subcapsular hyperplasia with nonsteroidogenic (Cyp11b1-­ and Cyp11b2-­negative) spindle-­like cells invading the cortex. This consequently reduces the number of steroidogenic cells in the zona fasciculata and is likely to reduce glucocorticoid output of the gland.147,149 KEY POINTS  • Primary adrenal insufficiency can also be caused by genes that do not fall into any of the above categories. • Mechanisms include accumulation of antisteroidogenic sphingolipids (SGPL1), autoantibodies against steroidogenic enzymes (AIRE), and accumulation of toxic very long chain fatty acids (ABCD1, PEX1, and PEX6).

Other Causes Of Syndromic Adrenal Insufficiency

Sphingosine-­1-­Phosphate Lyase 1 (SGPL1). SGPL1 performs the final degradative step in sphingolipid metabolism. A 5’-­phosphate-­dependent aldehyde-­lyase, it is localized to the endoplasmic reticulum, where it cleaves sphingosine 1-­phosphate (S1P) and degrades it to phosphoethanolamine and hexadecanal. Loss of the enzyme can result in an accumulation of sphingolipids and proapoptotic ceramides, a feature of sphingolipidoses (Gaucher disease and Niemann–Pick disease). Conversely, in healthy conditions, its degradation of S1P makes it the major modulator of S1P signaling, which has been linked to pathways involved in angiogenesis, immune cell trafficking, stem cell differentiation, and programmed cell death.150 Sphingolipid intermediates including ceramide, sphingosine, and S1P are known modulators of the steroidogenic pathway.151 In SGPL1 patient-derived fibroblasts, elevated levels of sphingosine and ceramides result in a reduced level of cortisol production.152 Sphingosine achieves this by acting as an endogenous antagonist of SF1, maintaining it in an inactive conformation.153 Conversely, S1P increases cortisol secretion through promoting the expression of multiple genes

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PART 7  Adrenal

involved in steroidogenesis, such as CYP17A1 and STAR.151,154 Mass spectroscopic analysis of sphingolipid intermediates in the plasma of an SGPL1 patient and heterozygous parents revealed elevated levels of ceramide and sphingosine, and to a lesser extent S1P.155 A further study observed a similar increase in ceramide, compared with S1P, in conditioned media from patient fibroblasts.156 This accumulation of antisteroidogenic intermediates could explain the reduced cortisol expression seen in SGPL1 patients. Patients with SGPL1 mutations are very rare, with most cases being lethal in utero. Those who do survive, present in infancy with multiple comorbidities, often including isolated glucocorticoid deficiency, (although mineralocorticoid deficiency has also been reported), and steroid-resistant nephrotic syndrome.155,156 Other comorbidities include primary hypothyroidism, ichthyosis, neurodevelopmental delay, primary gonadal failure, lymphopenia, dyslipidemia, and axonal degeneration.155-­157 Cytosolic accumulations of sphingolipid intermediates, along with mitochondrial dysfunction, could be the underlying pathology behind these manifestations.152,155 Sgpl1-­/-­ mice are born at normal Mendelian ratios, but most die within the first 2 weeks of life.155 In mice that survive weaning, cortical zonation is compromised, with less definition between the zona glomerulosa and zona fasciculata, and with cells of the zona fasciculata appearing smaller, with fewer lipid droplets. The adrenals of the knockout mice showed lower expression of Cyp11a1, which supported the disruption of steroidogenesis that was observed.155

Autoimmune Regulator (AIRE). AIRE is a nuclear protein that regulates self-­tolerance of T-­cells, with highest expression in the thymus, and is also expressed to a lesser extent in peripheral lymphoid tissues.158,159 Mutations in the gene cause autoimmune-­ poly-­ endocrinopathy-­ candidiasis–ectodermal-­dystrophy syndrome (APECED) (also known as autoimmune polyendocrine syndrome), a rare monogenic autosomal recessive disease that is most common in Finnish and Iranian Jewish populations.160 The classic criteria for diagnosis of APECED are the presence of any two of the following: chronic mucocutaneous candidiasis, chronic hypoparathyroidism, or PAI. However, the latest criteria are based on genetic and immunological features of the syndrome.160 Dysfunctional AIRE results in the production of autoantibodies against self-­antigens, including mitochondrial cholesterol side-­chain cleavage enzyme, steroid 17-­alpha-­hydroxylase/17,20 lyase, and steroid 21-­hydroxylase inhibiting steroidogenesis in the adrenals and gonads.161 Further studies have found that almost all patients possess antibodies to interferons and interleukins, which lead to a severe autoimmune disease affecting multiple endocrine organs and other tissues.162,163 Initially identified in 1997 to cause APECED, to date, approximately 115 mutations have been found to affect AIRE, with most of these variants being either nonsense or frameshift mutations resulting in a truncated protein, or a single amino acid missense variant.160,164,165 Due to the nature of autoimmune disease, AIRE deficiency presents with many different manifestations in a highly patient-­variable manner, making the disease unpredictable. These manifestations can include mucocutaneous candidiasis, chronic hypoparathyroidism, and PAI, as well as ectodermal dystrophy, hypogonadism, vitiligo, alopecia, malabsorption, and cataracts. The most common causes of death from APECED include hypocalcemia and adrenal crisis, oral and esophageal squamous cell carcinoma, and acute hepatitis.166 Aire-­/-­ mice develop normally in terms of weight, size, and maturation but present with autoimmune features such as exaggerated immune response, multiorgan lymphocytic infiltration, circulating autoantibodies, and infertility, as well as undetectable adrenal glands in some mice.167

ATP Binding Cassette Subfamily D Member 1 (ABCD1). ABCD1, located on chromosome Xq28, encodes a protein that transports very long chain fatty acids (VLCFAs) into the peroxisome, a small membrane-­enclosed organelle that contains enzyme pathways for lipid metabolism and detoxification processes.168 Mutations in ABCD1, commonly missense, nonsense, or splicing-­defect, result in failure to traffic VLCFAs into the peroxisome and their subsequent accumulation in plasma and tissues. VLCFAs are toxic to adrenocortical cells and also alter the structure of the cell membrane, suppressing the availability of MC2R and consequently inhibiting steroidogenesis.169,170 Furthermore, VLCFAs are also toxic to myelin-­producing oligodendrocytes, where they cause a disturbance in calcium homeostasis and mitochondrial dysfunction.171 This toxicity caused by VLCFAs through ABCD1 dysfunction results in the genetic metabolic disorder adrenoleukodystrophy (ALD).172 ALD is an X-­linked inheritance peroxisomal disease with two main phenotypes: cerebral demyelinating form and adrenomyeloneuropathy (AMN).173 Sufferers of the cerebral demyelinating form of the disease appear asymptomatic at birth but start to present with cognitive dysfunction, through declining school performance and behavioral problems, between 4 and 8 years of age. The disease then progresses rapidly, with central demyelination resulting in seizures, dementia, cortical blindness, coma, and death, most frequently within 2 years of presentation and before the age of puberty.174 The other form of the disease, AMN, presents in men in their 20s or above and is characterized by slow progressive mixed motor and sensory peripheral neuropathy, with 10% of cases developing cerebral demyelination.175 Although all 46, XY patients with ALD are eventually affected by AMN, the age of onset and rate of progression are highly variable.173 Virtually all 46, XY patients will develop adrenocortical insufficiency, with approximately 80% doing so before adulthood.176 Although ALD is deemed an X-­linked disease, adrenal insufficiency can be present in 46, XX heterozygous carriers of ABCD1 mutations.177 In these carriers, 63% have myelopathy, whereas 57% have peripheral neuropathy, and this increases to 80% for patients over the age of 60 years.178 This could be due to X-­inactivation of the nonmutant allele, but this explanation remains contested and is not supported by this particular study.178 One study found that Abcd1-­/-­ mice appear normal for the first 12 months of life and show an increase in VLCFA but no demyelination in the brain, spinal cord, or peripheral nerves, indicating that VLCFAs alone may not be sufficient to demyelinate the nervous system.179 A further study showed no statistical difference between wildtype and Abcd1-­/-­ mice in corticosterone production or oxidative stress, while knockout of Abcd2 resulted in the acceleration of ceroid production (lipogenic pigmentation) in the adrenal gland, the end product of oxidative damage.180 Peroxisomal Biogenesis Factors (PEX Genes). Peroxisomal diseases are divided into two main groups: disorders with a deficient enzyme or transporter (ALD), and peroxisome biogenesis disorders with multiple metabolic abnormalities, such as Zellweger spectrum disorders (ZSDs). ZSDs are autosomal recessive diseases with a range of severity due to mutations in PEX genes resulting in the reduction or absence of functional peroxisomes.181 The phenotype of ZSDs is variable, depending on the PEX gene mutated, but the most common features are liver dysfunction, developmental delay, adrenal insufficiency, and hearing/vision impairment.182 Adrenal insufficiency is caused by mutations in PEX1 and PEX6 and could be due to accumulation of VLCFAs; these are toxic to adrenocortical cells and suppress steroidogenesis, causing adrenal insufficiency in approximately 30% of patients.169,170,183,184 Furthermore, VLCFAs are also toxic to myelin-­ producing oligodendrocytes, resulting in hypomyelination and neurological defects.171

CHAPTER 86  Genetic Disorders of the Adrenal Cortex Of the 15 PEX genes, mutations in PEX1 are the most common cause of Zellweger syndrome, the most severe form of ZSDs.185 Variants within the gene can dictate the severity of the disease. p.G843D leads to a less severe form with long survival, whereas p.Pro970* results in a nonfunctional protein, a more severe form of the disease, and shorter survival.186-­188 Indeed, nonsense mutations have been shown to result in more severe cases than missense variants.189 Further symptoms can include epileptic seizures, craniofacial dysmorphism, leukodystrophy, and cerebellar ataxia.190 Zellweger disease, the most severe form, can itself be divided into three categories–– neonatal-­infantile, childhood, and adolescent-­adult––with severity of disease decreasing as age of presentation increases. There is no curative therapy for patients, and therefore treatment is supportive and symptomatic. Hypomorphic Pex1-­G843D mice recapitulate many of the human traits, such as adrenal insufficiency, growth retardation, and fatty liver with cholestasis.186

Lysosomal Acid Lipase (LIPA). LIPA encodes an enzyme that hydrolyses cholesteryl esters (CE) and triglycerides (TG) in lysosomes, producing free cholesterol and free fatty acids (FFA).191 Free cholesterol and FFAs function as key structural components of cell membranes, energy sources, and signaling molecules. Mutations in LIPA result in accumulation of CE and TG all over the body, particularly the liver, spleen, lymph nodes, bone marrow, and macrophages.191 This manifests as an autosomal recessive lysosomal storage disease with two phenotypes, presentation of which is dependent on the quantity of functional enzyme the patient possesses. Wolman disease is the early-­ onset form of LIPA deficiency; it presents in newborns who have no or almost no enzyme activity.192 Sufferers of this disease rarely survive beyond infancy and present with hepatomegaly, calcification of the adrenal glands, malabsorption, and hepatic failure.191,192 Patients who retain partial LIPA function (up to 12%) are diagnosed with CE storage disease (CESD), present later in life, if at all, and have variable clinical phenotypes.193 Patients can present with hepatomegaly, abdominal distension, and dyslipidemia, as well as, in severe cases, multiorgan failure and death .191 Interestingly, however, large adrenal calcium deposits are rare, although fine punctate calcifications are often detected. CESD is rare and is often misdiagnosed as nonalcoholic fatty liver disease or other forms of hypercholesterolemia.194 Mutations in LIPA have also been linked to coronary heart disease through genome-­wide association studies.195,196 This could be due to accumulation of TG and CE leading to foam cell formation and consequently atherosclerosis.197 High levels of oxidised LDL, a result of LIPA deficiency, have been shown to result in increased levels of intracellular calcium in cultured adrenal cells, which could explain the adrenal calcification observed in Wolman disease.198 This calcification, combined with the reduced abundance of free cholesterol, could inhibit steroidogenesis and result in a form of adrenal insufficiency in these patients.193 LIPA deficiency can be treated by bone marrow transplant, to normalize cholesterol levels, and enzyme replacement therapy.199,200 Lipa-­/-­ mice are born at Mendelian ratios and appear normal at birth. However, within 21 days, TG and CE accumulations appear in several organs at levels up to 30-­fold higher than those found in normal mice, and the liver appears one and a half to two times larger. Adrenal glands present with an expanded adrenal cortex due to lipid accumulations. Heterozygotes have approximately 50% enzyme functionality and do not show lipid accumulation.201

CONCLUSION The number of genes identified to cause PAI continues to increase, both those involved in isolated glucocorticoid deficiency and those

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involved in syndromic forms. With many dysfunctional genes causing similar and overlapping phenotypes, it is of vital importance to attain a genetic diagnosis for patient treatment. This process has been eased significantly with the dawn of next-­generation sequencing, which allows sequencing of all genes concomitantly rather than taking a candidate gene approach. Many PAI patients remain without a genetic diagnosis, and researchers are working to identify these causative genes.

FURTHER RESOURCES The following resources contain more information about the genes, mutations, and mouse models discussed in this chapter. ClinVar: Information about genomic variation and its relationship to human health; https://www.ncbi.nlm.nih.gov/clinvar/ Human Gene Mutation Database: A database for identifying disease-­causing mutations; https://mastermind.genomenon.com/ International Mouse Phenotyping Consortium: An international scientific endeavor to create and characterize the phenotype of 20,000 knockout mouse strains; https://www.mousephenotype.org/ Leiden Open Variation Database: An open-­source DNA variation database; www.lovd.nl Mouse Genome Informatics: International database resource for the laboratory mouse; http://www.informatics.jax.org/ Online Mendelian Inheritance in Man: An online catalogue of human genes and genetic disorders; https://omim.org/

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103. Thiffault I, Saunders C, Jenkins J, et al. A patient with polymerase E1 deficiency (POLE1): clinical features and overlap with DNA breakage/ instability syndromes. BMC Med Genet. 2015;16:31. 104. Albertson TM, Ogawa M, Bugni JM, et al. DNA polymerase epsilon and delta proofreading suppress discrete mutator and cancer phenotypes in mice. Proc Natl Acad Sci U S A. 2009;106:17101–17104. 105. Chung TT, Webb TR, Chan LF, et al. The majority of adrenocorticotropin receptor (melanocortin 2 receptor) mutations found in familial glucocorticoid deficiency type 1 lead to defective trafficking of the receptor to the cell surface. J Clin Endocrinol Metab. 2008;93:4948–4954. 106. Chung T-­TLL, Chan LF, Metherell LA, et al. Phenotypic characteristics of familial glucocorticoid deficiency (FGD) type 1 and 2. Clin Endocrinol. 2010;72:589–594. 107. Clark AJ, McLoughlin L, Grossman A. Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor. Lancet. 1993;341:461–462. 108. Guran T, Buonocore F, Saka N, et al. Rare causes of primary adrenal insufficiency: genetic and clinical characterization of a large nationwide cohort. J Clin Endocrinol Metab. 2016;101:284–292. 109. Chan LF, Metherell LA, Krude H, et al. Homozygous nonsense and frameshift mutations of the ACTH receptor in children with familial glucocorticoid deficiency (FGD) are not associated with long-­term mineralocorticoid deficiency. Clin Endocrinol (Oxf). 2009;71:171–175. 110. Lin L, Hindmarsh PC, Metherell LA, et al. Severe loss-­of-­function mutations in the adrenocorticotropin receptor (ACTHR, MC2R) can be found in patients diagnosed with salt-­losing adrenal hypoplasia. Clin Endocrinol (Oxf). 2007;66:205–210. 111. Clark AJ, Metherell LA, Cheetham ME, et al. Inherited ACTH insensitivity illuminates the mechanisms of ACTH action. Trends Endocrinol Metab. 2005;16:451–457. 112. Yeh JK, Evans JF, Niu QT, et al. A possible role for melanocortin peptides in longitudinal growth. J Endocrinol. 2006;191:677–686. 113. Chida D, Nakagawa S, Nagai S, et al. Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis. Proc Natl Acad Sci USA. 2007;104:18205–18210. 114. Imamine H, Mizuno H, Sugiyama Y, et al. Possible relationship between elevated plasma ACTH and tall stature in familial glucocorticoid deficiency. Tohoku J Exp Med. 2005;205:123–131. 115. Elias LL, Huebner A, Metherell LA, et al. Tall stature in familial glucocorticoid deficiency. Clin Endocrinol (Oxf). 2000;53:423–430. 116. Metherell LA, Chapple JP, Cooray S, et al. Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nat Genet. 2005;37:166–170. 117. Cooray SN, Almiro Do Vale I, Leung KY, et al. The melanocortin 2 receptor accessory protein exists as a homodimer and is essential for the function of the melanocortin 2 receptor in the mouse y1 cell line. Endocrinology. 2008;149:1935–1941. 118. Sebag JA, Hinkle PM. Melanocortin-­2 receptor accessory protein MRAP forms antiparallel homodimers. Proc Natl Acad Sci USA. 2007;104:20244–20249. 119. Janovick JA, Maya-­Nunez G, Conn PM. Rescue of hypogonadotropic hypogonadism-­causing and manufactured GnRH receptor mutants by a specific protein-­folding template: misrouted proteins as a novel disease etiology and therapeutic target. J Clin Endocrinol Metab. 2002;87:3255– 3262. 120. Novoselova TV, Chan LF, Clark AJL. Pathophysiology of melanocortin receptors and their accessory proteins. Best Pract Res Clin Endocrinol Metab. 2018;32:93–106. 121. Hughes CR, Chung TT, Habeb AM, et al. Missense mutations in the melanocortin 2 receptor accessory protein that lead to late onset familial glucocorticoid deficiency type 2. J Clin Endocrinol Metab. 2010;95:3497– 3501. 122. Rumié H, Metherell LA, Clark AJ, et al. Clinical and biological phenotype of a patient with familial glucocorticoid deficiency type 2 caused by a mutation of melanocortin 2 receptor accessory protein. Eur J Endocrinol. 2007;157:539–542. 123. Chan LF, Webb TR, Chung TT, et al. MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family. Proc Natl Acad Sci USA. 2009;106:6146–6151.

124. Novoselova TV, Hussain M, King PJ, et al. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation. Faseb J. 2018;32. fj201701274RR. 125. Nickel AG, von Hardenberg A, Hohl M, et al. Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure. Cell Metab. 2015;22:472–484. 126. Meimaridou E, Kowalczyk J, Guasti L, et al. Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency. Nat Genet. 2012;44:740–742. 127. Jazayeri O, Liu X, van Diemen CC, et al. A novel homozygous insertion and review of published mutations in the NNT gene causing familial glucocorticoid deficiency (FGD). Eur J Med Genet. 2015;58:642–649. 128. Bainbridge MN, Davis EE, Choi WY, et al. Loss of function mutations in NNT are associated with left ventricular noncompaction. Circ Cardiovasc Genet. 2015;8:544–552. 129. Roucher-­Boulez F, Mallet-­Motak D, Samara-­Boustani D, et al. NNT mutations: a cause of primary adrenal insufficiency, oxidative stress and extra-­adrenal defects. Eur J Endocrinol. 2016;175:73–84. 130. Hershkovitz E, Arafat M, Loewenthal N, et al. Combined adrenal failure and testicular adrenal rest tumor in a patient with nicotinamide nucleotide transhydrogenase deficiency. J Pediatr Endocrinol Metab. 2015;28:1187. 131. Meimaridou E, Goldsworthy M, Chortis V, et al. NNT is a key regulator of adrenal redox homeostasis and steroidogenesis in male mice. J Endocrinol. 2018;236:13–28. 132. Williams JL, Paudyal A, Awad S, et al. Mylk3 null C57BL/6N mice develop cardiomyopathy, whereas Nnt null C57BL/6J mice do not. Life Sci Alliance. 2020;3:e201900593. 133. Biterova EI, Turanov AA, Gladyshev VN, et al. Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism. Proc Natl Acad Sci USA. 2005;102:15018–15023. 134. Hanukoglu I. Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. Drug Metab Rev. 2006;38:171–196. 135. Prasad R, Chan LF, Hughes CR, et al. Thioredoxin reductase 2 (TXNRD2) mutation associated with familial glucocorticoid deficiency (FGD). J Clin Endocrinol Metab. 2014;99:E1556–E1563. 136. Conrad M, Jakupoglu C, Moreno SG, et al. Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function. Mol Cell Biol. 2004;24:9414–9423. 137. Kiermayer C, Northrup E, Schrewe A, et al. Heart-­specific knockout of the mitochondrial thioredoxin reductase (Txnrd2) induces metabolic and contractile dysfunction in the aging myocardium. J Am Heart Assoc. 2015;4:e002153. 138. Sibbing D, Pfeufer A, Perisic T, et al. Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 cause dilated cardiomyopathy. Eur Heart J. 2011;32:1121–1133. 139. Allgrove J, Clayden GS, Grant DB, et al. Familial glucocorticoid deficiency with achalasia of the cardia and deficient tear production. Lancet. 1978;1:1284–1286. 140. Handschug K, Sperling S, Yoon SJ, et al. Triple A syndrome is caused by mutations in AAAS, a new WD-­repeat protein gene. Human Mole Genet. 2001;10:283–290. 141. Cho AR, Yang KJ, Bae Y, et al. Tissue-­specific expression and subcellular localization of ALADIN, the absence of which causes human triple A syndrome. Exp Mol Med. 2009;41:381–386. 142. Houlden H, Smith S, De Carvalho M, et al. Clinical and genetic characterization of families with triple A (Allgrove) syndrome. Brain. 2002;125:2681–2690. 143. Prasad R, Metherell LA, Clark AJ, et al. Deficiency of ALADIN impairs redox homeostasis in human adrenal cells and inhibits steroidogenesis. Endocrinology. 2013;154:3209–3218. 144. Jühlen R, Idkowiak J, Taylor AE, et al. Role of ALADIN in human adrenocortical cells for oxidative stress response and steroidogenesis. PLOS ONE. 2015;10:e0124582. 145. Huebner A, Mann P, Rohde E, et al. Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to devel-

CHAPTER 86  Genetic Disorders of the Adrenal Cortex op a phenotype resembling human triple A syndrome. Mol Cell Biol. 2006;26:1879–1887. 146. O’Riordan SM, Lynch SA, Hindmarsh PC, et al. A novel variant of familial glucocorticoid deficiency prevalent among the Irish traveler population. J Clin Endocrinol Metab. 2008;93:2896–2899. 147. Hughes CR, Guasti L, Meimaridou E, et al. MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans. J Clin Invest. 2012;122:814–820. 148. Gineau L, Cognet C, Kara N, et al. Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency. J Clin Invest. 2012;122:821–832. 149. Chuang CH, Wallace MD, Abratte C, et al. Incremental genetic perturbations to MCM2-­7 expression and subcellular distribution reveal exquisite sensitivity of mice to DNA replication stress. PLoS Genet. 2010;6:e1001110. 150. Maceyka M, Harikumar KB, Milstien S, et al. Sphingosine-­1-­phosphate signaling and its role in disease. Trends Cell Biol. 2012;22:50–60. 151. Lucki NC, Li D, Sewer MB. Sphingosine-­1-­phosphate rapidly increases cortisol biosynthesis and the expression of genes involved in cholesterol uptake and transport in H295R adrenocortical cells. Mol Cell Endocrinol. 2012;348:165–175. 152. Maharaj A, Williams J, Bradshaw T, et al. Sphingosine-­1-­phosphate lyase (SGPL1) deficiency is associated with mitochondrial dysfunction. J Steroid Biochem Mol Biol. 2020;202:105730. 153. Lucki NC, Sewer MB. Nuclear sphingolipid metabolism. Ann Rev Physiol. 2012;74:131–151. 154. Ozbay T, Rowan A, Leon A, et al. Cyclic adenosine 5’-­monophosphate-­dependent sphingosine-­1-­phosphate biosynthesis induces human CYP17 gene transcription by activating cleavage of sterol regulatory element binding protein 1. Endocrinology. 2006;147:1427– 1437. 155. Prasad R, Hadjidemetriou I, Maharaj A, et al. Sphingosine-­1-­phosphate lyase mutations cause primary adrenal insufficiency and steroid-­resistant nephrotic syndrome. J Clin Invest. 2017;127:942–953. 156. Lovric S, Goncalves S, Gee HY, et al. Mutations in sphingosine-­1-­ phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency. J Clin Invest. 2017;127:912–928. 157. Janecke AR, Xu R, Steichen-­Gersdorf E, et al. Deficiency of the sphingosine-­1-­phosphate lyase SGPL1 is associated with congenital nephrotic syndrome and congenital adrenal calcifications. Hum Mutat. 2017;38:365–372. 158. Poliani PL, Kisand K, Marrella V, et al. Human peripheral lymphoid tissues contain autoimmune regulator-­expressing dendritic cells. Am J Pathol. 2010;176:1104–1112. 159. Suzuki E, Kobayashi Y, Kawano O, et al. Expression of AIRE in thymocytes and peripheral lymphocytes. Autoimmunity. 2008;41:133–139. 160. Pellegrino M, Bellacchio E, Dhamo R, et al. A novel homozygous mutation of the AIRE gene in an APECED patient from Pakistan: case report and review of the literature. Front Immunol. 2018;9:1835. 161. Söderbergh A, Myhre AG, Ekwall O, et al. Prevalence and clinical associations of 10 defined autoantibodies in autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab. 2004;89:557–562. 162. Meager A, Visvalingam K, Peterson P, et al. Anti-­interferon autoantibodies in autoimmune polyendocrinopathy syndrome type 1. PLoS Med. 2006;3:e289. 163. Puel A, Döffinger R, Natividad A, et al. Autoantibodies against IL-­17A, IL-­17F, and IL-­22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I. J Exp Med. 2010;207:291–297. 164. Bruserud Ø, Oftedal BE, Landegren N, et al. A longitudinal follow-­up of autoimmune polyendocrine syndrome type 1. J Clin Endocrinol Metab. 2016;101:2975–2983. 165. Finnish-German APECED Consortium. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-­type zinc-­finger domains. Nat Genet. 1997;17:399–403. 166. Borchers J, Pukkala E, Mäkitie O, et al. Patients with APECED have increased early mortality due to endocrine causes, malignancies and infections. J Clin Endocrinol Metab. 2020;105:e2207–e2213.

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167. Ramsey C, Winqvist O, Puhakka L, et al. Aire deficient mice develop multiple features of APECED phenotype and show altered immune response. Hum Mol Genet. 2002;11:397–409. 168. Wiesinger C, Kunze M, Regelsberger G, et al. Impaired very long-­chain acyl-­CoA β-­oxidation in human X-­linked adrenoleukodystrophy fibroblasts is a direct consequence of ABCD1 transporter dysfunction. J Biol Chem. 2013;288:19269–19279. 169. Whitcomb RW, Linehan WM, Knazek RA. Effects of long-­chain, saturated fatty acids on membrane microviscosity and adrenocorticotropin responsiveness of human adrenocortical cells in vitro. J Clin Invest. 1988;81:185–188. 170. Powers JM, Schaumburg HH, Johnson AB, et al. A correlative study of the adrenal cortex in adreno-­leukodystrophy-­-e­ vidence for a fatal intoxication with very long chain saturated fatty acids. Invest Cell Pathol. 1980;3:353–376. 171. Hein S, Schönfeld P, Kahlert S, et al. Toxic effects of X-­linked adrenoleukodystrophy-­associated, very long chain fatty acids on glial cells and neurons from rat hippocampus in culture. Hum Mol Genet. 2008;17:1750–1761. 172. Mosser J, Douar AM, Sarde CO, et al. Putative X-­linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature. 1993;361:726–730. 173. Engelen M, Kemp S, Poll-­The BT. X-­linked adrenoleukodystrophy: pathogenesis and treatment. Curr Neurol Neurosci Rep. 2014;14:486. 174. Kemp S, Berger J, Aubourg P. X-­linked adrenoleukodystrophy: clinical, metabolic, genetic and pathophysiological aspects. Biochim Biophys Acta. 2012;1822:1465–1474. 175. van Geel BM, Bezman L, Loes DJ, et al. Evolution of phenotypes in adult male patients with X-­linked adrenoleukodystrophy. Ann Neurol. 2001;49:186–194. 176. Dubey P, Raymond GV, Moser AB, et al. Adrenal insufficiency in asymptomatic adrenoleukodystrophy patients identified by very long-­chain fatty acid screening. J Pediatr. 2005;146:528–532. 177. el-­Deiry SS, Naidu S, Blevins LS, et al. Assessment of adrenal function in women heterozygous for adrenoleukodystrophy. J Clin Endocrinol Metab. 1997;82:856–860. 178. Engelen M, Barbier M, Dijkstra IM, et al. X-­linked adrenoleukodystrophy in women: a cross-­sectional cohort study. Brain. 2014;137:693–706. 179. Kobayashi T, Shinnoh N, Kondo A, et al. Adrenoleukodystrophy protein-­deficient mice represent abnormality of very long chain fatty acid metabolism. Biochem Biophys Res Commun. 1997;232:631–636. 180. Lu J-­F, Barron-­Casella E, Deering R, et al. The role of peroxisomal ABC transporters in the mouse adrenal gland: the loss of Abcd2 (ALDR), Not Abcd1 (ALD), causes oxidative damage. Lab Invest. 2007;87:261–272. 181. Waterham HR, Ebberink MS. Genetics and molecular basis of human peroxisome biogenesis disorders. Biochim Biophys Acta. 2012;1822:1430– 1441. 182. Bowen P, Lee CS, Zellweger H, et al. A familial syndrome of multiple congenital defects. Bull Johns Hopkins Hosp. 1964;114:402–414. 183. Berendse K, Engelen M, Linthorst GE, et al. High prevalence of primary adrenal insufficiency in Zellweger spectrum disorders. Orphanet J Rare Dis. 2014;9:133. 184. Matsumoto N, Tamura S, Moser A, et al. The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6. J Hum Genet. 2001;46:273–277. 185. Reuber BE, Germain-­Lee E, Collins CS, et al. Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders. Nat Genet. 1997;17:445–448. 186. Hiebler S, Masuda T, Hacia JG, et al. The Pex1-­G844D mouse: a model for mild human Zellweger spectrum disorder. Mol Genet Metab. 2014;111:522–532. 187. Maxwell MA, Nelson PV, Chin SJ, et al. A common PEX1 frameshift mutation in patients with disorders of peroxisome biogenesis correlates with the severe Zellweger syndrome phenotype. Hum Genet. 1999;105:38–44. 188. Maxwell MA, Allen T, Solly PB, et al. Novel PEX1 mutations and genotype–phenotype correlations in Australasian peroxisome biogenesis disorder patients. Hum Mut. 2002;20:342–351.

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189. Preuss N, Brosius U, Biermanns M, et al. PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease. Pediatr Res. 2002;51:706–714. 190. Klouwer FC, Berendse K, Ferdinandusse S, et al. Zellweger spectrum disorders: clinical overview and management approach. Orphanet J Rare Dis. 2015;10:151. 191. Pericleous M, Kelly C, Wang T, et al. Wolman’s disease and cholesteryl ester storage disorder: the phenotypic spectrum of lysosomal acid lipase deficiency. Lancet Gastroenterol Hepatol. 2017;2:670–679. 192. Abramov A, Schorr S, Wolman M. Generalized xanthomatosis with calcified adrenals. AMA J Dis Child. 1956;91:282–286. 193. Aguisanda F, Thorne N, Zheng W. Targeting Wolman disease and cholesteryl ester storage disease: disease pathogenesis and therapeutic development. Curr Chem Genom Transl Med. 2017;11:1–18. 194. Zhang B, Porto AF. Cholesteryl ester storage disease: protean presentations of lysosomal acid lipase deficiency. J Pediatr Gastroenterol Nutr. 2013;56:682–685. 195. Coronary Artery Disease (C4D) Genetics Consortium. A genome-­wide association study in Europeans and South Asians identifies five new loci for coronary artery disease. Nat Genet. 2011;43:339–344. 196. Wild PS, Zeller T, Schillert A, et al. A genome-­wide association study identifies LIPA as a susceptibility gene for coronary artery disease. Circ Cardiovasc Genet. 2011;4:403–412.

197. Zschenker O, Illies T, Ameis D. Overexpression of lysosomal acid lipase and other proteins in atherosclerosis. J Biochem. 2006;140:23–38. 198. Nègre-­Salvayre A, Salvayre R. UV-­treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. 4. Calcium is involved in the cytotoxicity of UV-­treated LDL on lymphoid cell lines. Biochim Biophys Acta. 1992;1123:207–215. 199. Krivit W, Peters C, Dusenbery K, et al. Wolman disease successfully treated by bone marrow transplantation. Bone Marrow Transplant. 2000;26:567–570. 200. Valayannopoulos V, Plantaz D, Vara R, et al. Clinical effect of sebelipase alfa on survival and growth in infants with lysosomal acid lipase deficiency (Wolman disease). Mol Genet Metab. 2014;111:S108. 201. Du H, Duanmu M, Witte D, et al. Targeted disruption of the mouse lysosomal acid lipase gene: long-­term survival with massive cholesteryl ester and triglyceride storage. Hum Mol Genet. 1998;7:1347–1354. 202. Cartier N, Sarde CO, Douar AM, et al. Abnormal messenger RNA expression and a missense mutation in patients with X-­linked adrenoleukodystrophy. Hum Mol Genet. 1993;2:1949–1951. 203. Aslanidis C, Ries S, Fehringer P, et al. Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity. Genomics. 1996;33:85–93.

87 Enzymes and Pathways of Human Steroidogenesis Richard J. Auchus and John D.C. Newell-­Price

OUTLINE Overview of the Human Steroidogenic Enzymes and ­Steroidogenesis, 1449 Cytochrome P450 Enzymes, 1450 Hydroxysteroid Dehydrogenases and Reductases, 1450 Acute Regulation of Steroidogenesis, 1451 Chronic Maintenance of the Steroidogenic Machinery, 1452 Human Steroidogenic Cytochrome P450 Enzymes, 1454 P450scc (Encoded by CYP11A1), 1454 P450c17 (Encoded by CYP17A1), 1454 P450c21 (Encoded by CYP21A2), 1456 P450c11β and P450c11AS (Encoded by CYP11B1 and CYP11B2 in Humans), 1456 P450aro (Aromatase), 1458 Redox Partner Proteins, 1458 Ferredoxin, 1458 Ferredoxin Reductase, 1459

P450 Oxidoreductase, 1459 Cytochrome b5, 1460 Steroidogenic Dehydrogenases and Reductases, 1460 3β-­Hydroxysteroid Dehydrogenase/Δ5→Δ4-­Isomerases, 1460 17β-­Hydroxysteroid Dehydrogenases, 1461 Steroid 5α-­Reductases, 1461 3α-­Hydroxysteroid Dehydrogenases, 1462 11β-­Hydroxysteroid Dehydrogenases, 1462 Steroid Sulfonation, 1463 Pathways, 1464 Adrenal Steroidogenic Pathways, 1464 Gonadal Steroidogenic Pathways, 1464 The Backdoor Pathway to Dihydrotestosterone, 1464 Pathways to 11-­Oxygenated Androgens, 1464 Androgen Synthesis in Prostate Cancer, 1468

 

OVERVIEW OF THE HUMAN STEROIDOGENIC ENZYMES AND STEROIDOGENESIS All steroid hormones derive from cholesterol in a process that can be conceptualized as having six distinct components: 1. The conversion of cholesterol to pregnenolone. Although viewed superficially as a single chemical transformation, the mobilization of cholesterol into the steroidogenic pathways is a complex event that serves as a key locus of regulation and also conventionally defines a tissue as “steroidogenic.” In humans, only the cells of the adrenal cortex, testicular Leydig cells, ovarian granulosa cells, and trophoblast cells of the placenta possess high capacity to cleave cholesterol into pregnenolone (the C21 precursor of all active steroid hormones) and isocaproaldehyde, while skin and specific glial and neuronal cells of the brain have lower capacity for local synthesis. The differences in how this process is regulated and in how pregnenolone is subsequently metabolized define the roles of the various steroidogenic cells and tissues in human physiology. Unlike peptide-­secreting glands, steroidogenic cells do not store steroid hormones and intermediates; it is the activation of this first step that enables the rapid synthesis and release of steroids in response to hormonal and environmental stimuli. 2. The transformation of pregnenolone to active hormones, intermediates, and secreted steroid derivatives. The repertoire of enzymes and cofactor proteins present in a given steroidogenic cell is responsible for the characteristic steroid profile of that cell type, and the coordinate regulation of their expression promotes the completion of all steps of a given pathway. Thus, these enzymes determine

qualitatively what steroids are made, but since these steps are not kinetically limiting, it is step 1 that quantitatively regulates how much steroid is made at a given moment. 3. Peripheral metabolism of hormones and precursors. Although not “steroidogenic” as defined by being able to convert cholesterol to pregnenolone, some organs, such as the liver, possess tremendous capacity to transform various steroids. For example, 70% to 80% of circulating testosterone in normally cycling women derives from the conversion of adrenal dehydroepiandrosterone (DHEA). Steroids can be activated in target tissues, such as the conversion of testosterone to dihydrotestosterone (DHT) in the prostate. In contrast, active androgens and estrogens are inactivated in the uterus and in other peripheral tissues. 4. Multiple layers of regulation. The regulation of steroidogenesis by tropic hormones, such as adrenocorticotropic hormone (ACTH), angiotensin II, and luteinizing hormone (LH), is well known; less well appreciated are the multiple levels at which such regulation may occur. These include the transcriptional regulation of the genes encoding steroidogenic enzymes and cofactors; regulation of mitochondrial protein import; transfer of electrons from nicotinamide adenine dinucleotide phosphate (NADPH); posttranslational modification and proteosomal degradation of steroidogenic enzymes; and subcellular localization and/or targeting. 5. Catabolism and unproductive metabolism. A panopoly of steroids can be isolated from human plasma and tissues, many of which have negligible biological activity. Most inactive by-­ products derive from hepatic transformations (e.g., 6β-­hydroxylation and

1449

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PART 7  Adrenal 21

20

18

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11

D

C

2

α

10

A

3

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9

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16

15

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5 4

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β Figure 87.1  Cycloperhydropentanophenanthrene steroid nucleus. Steroid rings are identified with boxed capital letters, and carbon atoms are numbered. Substituents and hydrogens are labeled as α or β if they are positioned behind or in front of the plane of the page, respectively.

(P450scc); the two isozymes of 11-­hydroxylase (P450c11β and P450c11AS); and three of the principal enzymes in vitamin D metabolism (1α-­hydroxylase, 24-­hydroxylase, and 25-­hydroxylase). Type 1 enzymes receive electrons from the reduced form of NADPH via ferredoxin (FDX1), a small, soluble, iron-­sulfur protein. FDX1 does not oxidize NADPH directly, however, but receives the two electrons from NADPH via the flavoprotein ferredoxin reductase (FDXR, Fig. 87.3). Type 2 enzymes, in contrast, receive electrons from NADPH via the flavin adenine dinucleotide (FAD)-­flavin mononucleotide (FMN) two-­ flavin protein, P450 oxidoreductase (POR). Type 2 enzymes are exclusively located in the smooth endoplasmic reticulum and constitute 50 of the 57 human P450 enzymes. P450 enzymes activate molecular oxygen using their heme center and electrons from NADPH. Substrate binding is required prior to heme reduction with one electron, which enables oxygen binding, the second one-­electron transfer, and formation of the iron-­oxygen complex, which then oxygenates the substrate. Thus, P450 reactions on steroids are limited to oxygen insertion (hydroxylation) reactions and, in a few notable cases, oxidative carbon–carbon bond cleavage reactions (Table 87.2).

Hydroxysteroid Dehydrogenases and Reductases 5β-­reduction of C21 and C19 steroids, and 4-­hydroxylation of estrogens), which promote renal excretion of these steroids. 6. More than one pathway to specific steroids. Particularly for the terminal steps of long pathways, such as the synthesis of DHT or estradiol, two or more routes may yield the same final product. Often the different routes utilize different enzymes, and these enzymes may be found in different tissues under different regulatory mechanisms. The relative importance of these pathways differ with age, sex, and physiologic state. Steroids are molecules derived from the cyclopentanoperhydrophenanthrene four-­ring hydrocarbon nucleus (Fig. 87.1). Most enzymes involved in steroid biosynthesis are either cytochrome P450 enzymes or hydroxysteroid dehydrogenases (Fig. 87.2).1 All P450-­mediated hydroxylations and carbon–carbon bond cleavage reactions are mechanistically and physiologically irreversible. Hydroxysteroid dehydrogenase reactions are mechanistically reversible and can run in either direction under certain conditions in vitro, but each hydroxysteroid dehydrogenase drives steroid flux predominantly in either the oxidative or reductive mode in vivo.2 However, two or more hydroxysteroid dehydrogenases drive the flux of a steroid pair in opposite directions, some favoring ketosteroid reduction and others favoring hydroxysteroid oxidation. KEY POINTS  • The conversion of cholesterol to pregnenolone is the quantitative, rate-­ limiting step of steroidogenesis and the site of acute regulation. • Steroidogenesis follows a specific sequence with some branch points and redundancies, and each step is either irreversible or has a strong directional preference. • Steroid formation features multiple layers of regulation, redundancy for some pathways, and multiple activities for some key enzymes.

Cytochrome P450 Enzymes Mammalian cytochrome P450 enzymes fall into two broad classes: type 1 and type 2.1 Type 1 enzymes and their electron transfer proteins reside in the mitochondria (Table 87.1) of eukaryotes; almost all bacterial P450s are also type 1 enzymes. Human type 1 P450 enzymes include the cholesterol side-­chain cleavage enzyme

All hydroxysteroid dehydrogenases (HSDs) and related enzymes use nicotinamide cofactors either to reduce or to oxidize the steroid by two electrons through a hydride transfer mechanism.1 Most examples involve the conversion of a secondary alcohol to a ketone or vice versa, and in the case of the 3β-­hydroxysteroid dehydrogenase/Δ5→Δ4-­ isomerases, the dehydrogenation is accompanied by the isomerization of the adjacent carbon–­carbon double bond from the Δ5 (between carbons 5 and 6) to the Δ4 positions (see Figs. 87.1 and 87.2). The human steroid 5α-­reductases types 1 and 2, which are included with the HSDs for convenience, reduce olefinic carbon–carbon double bonds to the saturated state rather than acting on carbon centers bonded to oxygen. The HSDs can be categorized according to either structural or functional classification schemes. Structurally, HSDs are members of either the short-­chain dehydrogenase reductase (SDR) or aldo-­ keto reductase (AKR) families.2 The SDR enzymes are β-­α-­β proteins, where up to seven parallel β-­strands fan across the center of the molecule, forming the “Rossman fold” characteristic of oxidation/reduction enzymes that use nicotinamide cofactors. The AKR enzymes are soluble proteins that contain a beta-­barrel or triosephosphate isomerase (TIM-­barrel) motif in which eight parallel β-­ strands lie in a slanted circular distribution like the staves of a barrel. In both cases, the active site contains a critical tyrosine and lysine pair of residues involved in proton transfer from or to the steroid alcohol during catalysis. Functionally, HSDs act either as true dehydrogenases, using NAD+ as a cofactor to convert hydroxysteroids to ketosteroids; or as ketosteroid reductases, utilizing predominantly NADPH to reduce ketosteroids. Many HSDs catalyze either oxidation or reduction in vitro based on the pH and cofactor concentrations, but these enzymes, when expressed in intact mammalian cells, drive steroid flux primarily in one direction.2 These directional preferences derive primarily from the relative abundance of the oxidized and reduced form of cofactors and the relative affinity of each enzyme for NAD(H) versus NADP(H) because cofactor concentrations exceed steroid concentrations by many orders of magnitude.2 Consequently, the directional preference of some “reductive” enzymes can be reduced or reversed by depleting cells of NADPH or by mutations that attenuate the preferential binding of NADPH over NAD(H).3,4

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis

Cholesterol

Aldosterone P450c11AS FDX1/FDXR

P450scc/FDX1/FDXR

StAR

11-dehydrocorticosterone

P450c21

3βHSD1 & 2 Pregnenolone

11-deoxycorticosterone

Progesterone

11βHSD1

POR P450c17 POR

P450c17 POR

Corticosterone P450c11β

P450c21

17OH-pregnenolone

17OH-progesterone

P450c17 POR+b5

11-deoxycortisol POR

P450c17 POR+b5

Cortisol FDX1/FDXR 11βHSD1

11-oxygenated androgens

3βHSD

11βHSD2

P450aro

DHEA

Estrone

Androstenedione 17βHSD3

17βHSD2

11βHSD2

Backdoor pathway

3βHSD

17βHSD1

1451

17βHSD1 17βHSD2

3βHSD Androstenediol

Cortisone

POR 17βHSD2

P450aro Testosterone

Estradiol POR

5α-Reductase1 & 2

Androstanediol Androsterone Androstanedione

Dihydrotestosterone 3αHSDs 17βHSDs

Figure 87.2  Major human steroidogenic pathways. Key enzymes and cofactor proteins are shown near arrows indicating chemical reactions. The steroidogenic acute regulatory protein (StAR) protein (oval) mobilizes cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, where P450scc cleaves cholesterol to pregnenolone, the first committed intermediate in steroid biosynthesis. The steroids in the first column are Δ5-­steroids, which constitute the preferred pathway to C19 steroids in humans, and the dashed arrow indicates poor flux from 17α-­hydroxyprogesterone to androstenedione. Steroids in the second column and farther right are Δ4-­steroids, except the C18 estrogens (estrone and estradiol) and 5α-­reduced steroids, including the potent androgen dihydrotestosterone and other androstanes (bottom row). Diagonal arrows indicate additional pathways from 17α-­hydroxyprogesterone (backdoor pathway) and androstenedione (11-­oxygenated androgens) illustrated in Fig. 87.7. Not all intermediate steroids, pathways, and enzymes are shown.

KEY POINTS  • The two major classes of steroid biosynthetic enzymes are cytochrome P450 enzymes and the hydroxysteroid dehydrogenases/reductases. • Of the steroidogenic cytochrome P450 enzymes, P450scc, P450c11β, and P450c11AS are mitochondrial and use ferredoxin/ferredoxin reductase as electron transfer proteins; P450c17, P450c21, and P450aro reside in the endoplasmic reticulum and use P450-­oxidoreductase as their electron transfer protein. • The hydroxysteroid dehydrogenases catalyze mechanistically reversible oxidation/reduction reactions using NAD[P][H] cofactors; some enzymes strongly favor hydroxysteroid oxidation, and others strongly favor ketosteroid reduction in intact cells.

TABLE 87.1  Intracellular Location of

Steroidogenic Proteins Mitochondria P450scc P450c11β P450c11AS Ferredoxin reductase Ferredoxin StAR 3βHSD1 and 2

Cytoplasm

Endoplasmic Reticulum

17βHSD1 StAR 3βHSD1 and 2 AKR1C1-­4

P450c17 P450c21 P450aro P450-­oxidoreductase 17βHSD1, 2, 3, 6* Cytochrome b5 3βHSD1 and 2 11βHSD1 and 2

Acute Regulation of Steroidogenesis

HSD, Hydroxysteroid dehydrogenase. *17βHSD4 is located in peroxisomes.

Every time a pulse of ACTH reaches the adrenal cortex, or a pulse of LH reaches the gonad, a subsequent pulse of steroid hormone production is observed within minutes. Although it has long been known that the loss of trophic hormones from the pituitary gland leads to adrenal and gonadal atrophy, the action of ACTH and LH to promote organ survival and to maintain steroidogenic capacity occurs at three distinct

levels. First, as seen in long-­term exposure to ACTH (e.g., in Cushing disease), ACTH promotes adrenal growth. This growth occurs primarily by ACTH stimulating the production of cyclic adenosine monophosphate (cAMP), which, in turn, promotes the synthesis of insulin-­like growth factor 2 (IGF-­2), basic fibroblast growth factor,

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PART 7  Adrenal

A

NADPH NADP+

P450

+– +–

–+ –+

FAD +– +–

Fedx

–+ –+

Fe

FeRed

B

NADPH P450

+

NADP+

FAD POR

FMN

– + – – + – + +

Fe CYTO ER

Figure 87.3  Electron transfer pathways for steroidogenic cytochrome P450 enzymes. A, In type I (mitochondrial) enzymes, the two electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) pass from the flavin (FAD) of ferredoxin reductase (FeRed) to the iron-­sulfur (Fe2S2, diamond with dots) cluster of ferredoxin (Fedx) and then to the heme of the P450 (square with Fe). Negatively charged residues in Fedx (–) guide docking and electron transfer with positively charged residues (+) in both Fedx and the P450. B, In type II (microsomal) enzymes, the flavoprotein P450-­oxidoreductase (POR) receives electrons from NADPH to its FAD moiety, transfers electrons to its FMN moiety, and after a conformational rearrangement, directly transfers electrons from the FMN to the P450. Negative charges on POR (–) and positive charges (+) on the P450 guide the interaction as with the type I P450; with some P450 enzymes, phosphorylation and cytochrome b5 also regulate electron transfer and catalysis. Heme of P450 is indicated by a square with iron atom (Fe).

and epidermal growth factor. Together, these growth factors stimulate adrenal cellular hypertrophy and hyperplasia. Second, ACTH acts long-­term through cAMP, and angiotensin II acts through the calcium/calmodulin pathway to promote the transcription of genes encoding various steroidogenic enzymes and electron-­donating cofactor proteins. Third, ACTH fosters the increased flow of cholesterol into the mitochondria, where it becomes substrate for the first and rate-­ limiting enzyme, P450scc. This acute response occurs within minutes and is inhibited by inhibitors of protein synthesis (e.g., puromycin or cycloheximide), indicating that a short-­lived protein species mediates this process. Although other proteins are involved in the chronic replenishment of mitochondrial cholesterol, abundant biochemical, clinical, and genetic evidence implicates the steroidogenic acute regulatory protein (StAR) as this labile protein mediator.5 StAR is a 37-­kD phosphoprotein that is cleaved to a 30-­kD form when it enters the mitochondrion. Overexpression of mouse StAR in mouse Leydig MA-­10 cells increased their basal steroidogenic rate,5 and cotransfection of expression vectors for both StAR and the P450scc system in nonsteroidogenic COS-­1 cells augmented pregnenolone synthesis above that obtained with the P450scc system alone.6 Mutations in StAR cause congenital lipoid adrenal hyperplasia,6,7 in which very

little steroid is made; and targeted disruption of the Star gene in the mouse causes a similar phenotype.8 The mechanism of StAR’s action is not completely understood. After other proteins transport cholesterol to the mitochondria, StAR acts on the outer mitochondrial membrane (OMM),9 and its activity in promoting steroidogenesis is proportional to its residency time on the OMM.10 When expressed in cytoplasm or added to mitochondria in vitro, both the 37-­kD “precursor” and the 30-­kD “mature form” of StAR are equally active, but StAR is inactive in the mitochondrial intramembranous space or matrix.10 Thus, it is StAR’s cellular localization, not its cleavage, that determines whether or not it is active. StAR has a sterol-­binding pocket that accommodates a single molecule of cholesterol.11 The interaction of StAR with the OMM involves conformational changes12 that are necessary for StAR to accept and discharge cholesterol molecules. Although StAR can transfer cholesterol between synthetic membranes in vitro, suggesting that other protein molecules are not needed for its action, this activity can also be seen with the inactive mutant R182L, which causes lipoid congenital adrenal hyperplasia.13 Thus StAR’s action to promote steroidogenesis can be dissociated from its cholesterol-­transfer activity. StAR appears to interact with a complex of OMM proteins that includes the peripheral benzodiazepine receptor (PBR, also termed the translocator protein [TSPO]), voltage-­dependent anion channel 1 (VDAC1), the acyl-­CoA-­binding-­ domain proteins ACBD-­1 and ACBD-­3, and possibly others. Each molecule of StAR appears to be recycled, moving hundreds of molecules of cholesterol before the cleavage/inactivation event.14 Although StAR is required for the acute steroidogenic response, steroidogenesis will persist in the absence of StAR at approximately 14% of the StAR-­ induced rate, accounting for the steroidogenic capacity of tissues that lack StAR (e.g., the placenta and the brain). TSPO ligands stimulate testosterone production in rats,15 yet mice with targeted deletion of the Tspo gene in Leydig cells have normal stimulated testosterone production and are fertile.16 These paradoxical findings suggest that more than one protein complex or cholesterol transfer mechanism can contribute to steroidogenesis in specific cells.

Chronic Maintenance of the Steroidogenic Machinery The acute regulation of steroidogenesis is determined by access of cholesterol to the P450scc enzyme, and P450scc is the rate-­limiting enzymatic step in steroidogenesis. Thus, the chronic regulation of steroidogenesis is quantitatively (how much) determined by CYP11A1 (encoding P450scc) expression17 and qualitatively (which steroids) determined by the expression and activities of downstream enzymes. The episodic bursts of cAMP resulting from the binding of ACTH and LH to their respective receptors are necessary but not sufficient for the continued expression of the steroidogenic enzymes and the production of steroids. Patients with inactivating mutations in the ACTH receptor (melanocortin type 2 receptor, MC2R)18 or LH receptor19 make negligible amounts of steroids from the affected glands. Conversely, activating mutations of the Gsα protein, which couples receptor binding to cAMP generation, and activating mutations of the LH receptor cause hypersecretion of steroids.20 Indeed, cAMP-­responsive elements have been identified in the genes for most of the human steroidogenic P450 enzymes, but this mechanism alone does not account for the diversity of steroid production observed in the various zones of the adrenal cortex, the gonads of both sexes, the placenta, and the brain. Other transcription factors (e.g., AP-­2, SP-­1, SP-­3, NF1C, NR4A1, NR4A2, GATA4, and GATA6) aid in defining the basal-­and cAMP-­ stimulated transcription of each gene, which is also regulated in a tissue-­specific manner by the regulatory elements unique to each gene. Among these factors, steroidogenic factor-­1 (SF-­1, NR5A1), an orphan nuclear receptor, coordinates the expression of steroidogenic enzymes

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CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis

TABLE 87.2  Key Human Steroidogenic Enzymes and Cofactor Proteins Protein

Gene

Gene Size (kb)

Chromosomal Locus

P450scc

CYP11A1

>20

15q23-­q24

P450c17

CYP17A1

6.6

10q24.3

P450c21

CYP21A2

3.4

6p21.1

ZG/ZF/ZR

Prog, 17OH-­Prog

P450c11β

CYP11B1

9.5

8q21-­q22

ZF/ZR, brain

P450c11AS

CYP11B2

9.5

8q21-­q22

ZG, brain, heart

P450aro

CYP19A1

>52

15q21.1

11-­Deoxycortisol 11-­DOC [cortisol] Corticosterone 11-­DOC [cortisol] 18OH-­corticosterone Androstenedione Testosterone

3βHSD1

HSD3B1

7.8

1p13.1

3βHSD2

HSD3B2

7.8

1p13.1

17βHSD1

HSD17B1

3.3

17q11-­q21

17βHSD2

HSD17B2

>40

16q24.1-­q24.2

17βHSD3

HSD17B3

>60

9q22

Gonads (L,G), placenta, brain, bone, fat Placenta, liver, brain Preg, 17OH-­Preg DHEA, Δ5-­A ZG/ZF>ZR Preg, 17OH-­Preg Gonad (L,T) DHEA, Δ5-­A Gonad (G), placenta, Estrone, [DHEA] breast Endometrium, Testosterone, estradiol, broadly DHT Gonad (L) Androstenedione, 5α-­A, 5α/3α-­A [DHEA]

Reductive 3αHSDs

AKR1C1-­4

13-­25 each

10p14-­p15

Liver, broadly

Oxidative 3α-­HSD

HSD17B6

>23

12q13

Liver, prostate, broadly

5α-­Reductase 1

SRD5A1

>35

5p15

Liver, brain, skin

5α-­Reductase 2

SRD5A2

>35

2p23

11βHSD1

HSD11B1

9

1q32-­q41

11βHSD2

HSD11B2

6.2

16q22

Ferredoxin Ferredoxin Reductase StAR

FDX1 FDXR

>30 11

11q22 17q24-­q25

Ubiquitous Ubiquitous

Mitochondrial P450s Ferredoxin

11β-­Hydroxysteroid Syndrome of dehydrogenase apparent mineralocorticoid excess Electron transfer Not described Electron transfer Not described

STAR

8

8p11.2

P450-­Oxido-­ Reductase

POR

73

7q11.2

ZF/ZG/ZR, gonad (L, T) Ubiquitous

Cholesterol flux within mitochondria Microsomal P450s

Sterol delivery to P450scc Electron transfer

Location ZG/ZF/ZR, gonads (L,G), placenta, brain ZF/ZR, gonads (L,T), brain

Principal Substrates

Major Activities

Deficiency Syndromes

Cholesterol Hydroxysterols

22R-­Hydroxylase 20R-­Hydroxylase 20,22-­Lyase 17α-­Hydroxylase 17,20-­Lyase [16α-­ Hydroxylase] [Δ16-­Synthase] 21-­Hydroxylase

Deficiency of all steroids

Preg, 17OH-­Preg, Prog [17OH-­Prog]* 5α-­reduced C21-­steroids

DHT, 5α-­A 5α-­reduced C21-­steroids [17β-­HSD: DHEA Androstenedione, 5α/3α-­A] Adiol, 5α/3α-­A, 5α/3α-­reduced C21 steroids, (products)

Testosterone, Δ4/C21-­ steroids Prostate, genital Testosterone, Δ4/C21-­ skin steroids Liver, brain, plaCortisone, 11-­Dehydrocenta, fat, broadly ­corticosterone [products] Kidney, gut, plaCortisol, corticosterone centa

11β-­Hydroxylase [18-­Hydroxylase] 11β-­Hydroxylase 18-­Hydroxylase 18-­Oxidase 19-­Hydroxylase 19-­Oxidase aromatization 3β-­Dehydrogenase Δ5/4-­Isomerase 3β-­Dehydrogenase Δ5/4-­Isomerase 17β-­Ketosteroid reductase 17β-­Hydroxysteroid dehydrogenase 17β-­Ketosteroid reductase 3α-­Ketosteroid reductase 17β-­Ketosteroid reductase

17-­Hydroxylase deficiency Isolated 17,20-­ lyase deficiency 21-­Hydroxylase deficiency 11-­Hydroxylase deficiency Aldosterone synthase deficiencies Aromatase deficiency Not described 3βHSD deficiency Not described Not described 17-­Ketosteroid Reductase deficiency DSD resembling isolated 17,20-­ lyase deficiency

3α-­Hydroxysteroid dehydrogenase (3α-­, 3β-­, and 17β-­ ketosteroid reductase) 5α-­Reductase

Not described

5α-­Reductase

5α-­Reductase deficiency Cortisone reductase deficiency

11β-­Ketosteroid reductase

Not described

Lipoid CAH Multiple steroidogenic defects ± ABS Continued

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PART 7  Adrenal

TABLE 87.2  Key Human Steroidogenic Enzymes and Cofactor Proteins—cont’d Protein

Gene

Gene Size (kb)

Chromosomal Locus

Location

Principal Substrates

Major Activities

Deficiency Syndromes

Cytochrome b5

CYB5A

32

18q23

ZR>ZG/ZF, gonads, liver, erythrocytes

Allosteric factor with P450c17

Augments 17,20-­ lyase activity

36.5

1p36

Fat, liver

NADP+ reduction

17 85

19q13.3 10q24

ZR ZR, cartilage liver

Glucose-­6-­phosphate + NADP+ DHEA, other Δ5-­steroids ATP + sulfate APS + ATP

Isolated 17,20-­ lyase deficiency ± methemoglobinemia Cortisone reductase deficiency Not described Premature adrenarche with skeletal abnormalities

H6PDH

H6PD

SULT2A1 PAPSS2

SULT2A1 PAPSS2

Sulfonation ATP sulfurylase APS kinase

ZG/ZF/ZR, Adrenal zona glomerulosa/fasciculata/reticularis, respectively; L, Leydig cells; T, theca cells; G, granulosa cells; 17OH-­Preg, 17α-­ hydroxypregnenolone; 17OH-­Prog, 17α-­hydroxyprogesterone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; DOC, deoxycorticosterone; Adiol, 5α-­androstane-­3α,17β-­diol; Δ5-­A, androsta-­5-­ene-­3β,17β-­diol; 5α-­A, 5α-­androstane-­3,20-­dione; 5α/3α-­A, androsterone; DSD, disorder of sex development; CAH, congenital adrenal hyperplasia; ABS, Antley–Bixler syndrome, APS, adenosine phosphosulfate. *Steroids in brackets are poor substrates.

in adrenal and gonadal cells.21 By contrast, steroidogenesis in the brain and placenta22 is independent of SF-­1. Targeted disruption of SF-­1 in the mouse not only disrupts steroid biosynthesis but also blocks the development of the adrenal glands, gonads, and the ventromedial hypothalamus in homozygous animals.23 Furthermore, SF-­1 does not act in isolation, but its action is modified by other transcription factors (e.g., WT-­1 and DAX-­1)24 or by phosphorylation.25 Thus, the development of steroidogenic organs is intimately related to the capacity to produce steroids, and multiple factors acting on the genes for steroidogenic enzymes yield both common features and diversity among the steroidogenic tissues. Most steroidogenic enzymes derive from a single mRNA species. The most prominent exception to this paradigm is aromatase, whose gene has four different promoters that enable vastly different regulation of expression of the same aromatase protein in many different tissues.26 KEY POINTS  • The acute regulation of steroidogenesis is determined by access of cholesterol to the P450scc enzyme, and P450scc is the rate-­limiting enzymatic step in steroidogenesis. • Adrenocorticotropic hormone in the adrenal cortex, and luteinizing hormone in the gonad, stimulate this first step by the influx of cholesterol, mediated by steroidogenic acute regulatory protein.

HUMAN STEROIDOGENIC CYTOCHROME P450 ENZYMES P450scc (Encoded by CYP11A1) P450scc consumes three equivalents of NADPH and molecular oxygen during the conversion of cholesterol to pregnenolone. Although the enzyme is named for the cleavage of the cholesterol side chain, this process consists of three discrete steps: 1) the 22-­hydroxylation of cholesterol; 2) the 20-­hydroxylation of 22(R)-­hydroxycholesterol; and 3) the oxidative scission of the C20–C22 bond of 20(R), 22(R)-­ dihydroxycholesterol—the side-­ chain cleavage event. The enzyme will utilize free hydroxysterol intermediates as substrates for the side-­ chain cleavage reaction, a tool that is used experimentally because the

hydroxysterols are much more soluble than cholesterol and because their access to P450scc is independent of StAR.6 In vivo, however, little of these free intermediates probably accumulate, because their kcat/Km ratios are much higher than for cholesterol,27 and the high KD for pregnenolone (approximately 3000 nM) drives product dissociation. This complex process is the rate-­limiting step in steroidogenesis, with turnover numbers of only approximately 20 molecules of cholesterol per molecule P450scc per minute,27 and all three reactions utilize the iron oxene (compound I) species without incorporation of dioxygen atoms into pregnenolone or isocaproaldehyde during the third reaction.28 The x-­ray crystal structure of the P450scc–ferredoxin complex shows that cholesterol substrate binds with the steroid nucleus angled to approximately 45 degrees with the heme ring, with the side chain extended across the heme,29 which is consistent with the known order of reactions. P450scc will also cleave the side chain of other hydroxysterols (e.g., 7-­dehydrocholesterol), and 20-­and 22-­hydroxylates vitamin D. The CYP11A1 gene (Table 87.2) encodes an mRNA of 2 kb. Following translation, a 39-amino acid mitochondrial leader peptide that targets P450scc to the mitochondria is then proteolytically removed to yield a 482-amino acid protein. Forms of P450scc engineered to lack the mitochondrial leader are inactive,30 demonstrating that the mitochondrial environment is required for activity. Expression of P450scc is induced in the adrenal zonae fasciculata and reticularis,31 testis, and ovary by cAMP; and in the zona glomerulosa by intracellular calcium/protein kinase C.32 In contrast, placental P450scc expression is constitutive and is caused at least in part by the long terminal repeat-­ binding protein family of transcription factors.22 Side-­chain cleavage activity and pregnenolone biosynthesis have been demonstrated in the rat and human brain33; and abundant P450scc expression is found in the rodent brain, especially in fetal life. Deletion of the gene for P450scc has been described in rabbits and mice,34 abrogating all steroidogenesis and thus proving that P450scc is the only enzyme that can convert cholesterol to pregnenolone. While homozygous mutations in P450scc might be expected to be embryonic lethal by eliminating placental progesterone synthesis, patients have been described having P450scc mutations that typically retain partial enzymatic activity.35

P450c17 (Encoded by CYP17A1) P450c17 catalyzes both the 17α-­hydroxylase and 17,20-­lyase reactions,36 with additional regulatory mechanisms for the 17,20-­lyase reaction.

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis The CYP17A1 gene (Table 87.2) encoding P450c17 is expressed in the adrenals and gonads,37 and leads to a single 2.1-­kb mRNA species yielding a 57-­kD protein in these tissues; mutations in this gene produce a spectrum of deficiencies in 17-­hydroxysteroids and C19 steroids.38 Human P450c17 17-­hydroxylates both pregnenolone and progesterone with approximately equal efficiency, but all other reactions show prominent differences among substrates. The 17,20-­ lyase activity is approximately 50 times more efficient for the 17α-­hydroxypregnenolone-­ to-­ DHEA reaction than for the 17α-­hydroxyprogesterone-­to-­ androstenedione reaction.39 Although the rate of the lyase reaction can be increased more than 10-­fold by the addition of a molar equivalent of cytochrome b5,39 the Δ5 preference persists, and the lyase rate never quite reaches the rate of the hydroxylase reactions. Residue N202 of P450c17 serves as a hydrogen bond donor (NH) to the 3-­keto oxygen of Δ4 steroids or acceptor (CO) for the 3-­hydroxyl hydrogen of Δ5 steroids,40 and mutation N202S eliminates the Δ5 preference for the 17,20-­lyase reaction.41 In addition, human P450c17 16α-­hydroxylates progesterone but not pregnenolone,39 and, in the presence of cytochrome b5, diverts approximately 10% of pregnenolone metabolism to a Δ16 andiene product that is also formed by this pathway in pigs and that acts as a porcine pheromone. Although experiments to study the chemistry of human P450c17 often require manipulations that could be considered nonphysiologic, the remarkable consistency for substrate preferences and kinetic constants observed for the modified, solubilized P450c17 expressed in Escherichia coli and native P450c17 expressed in yeast microsomes, or intact COS-­1 cells, or that obtained from human tissues and cells, serve to verify these conclusions. In addition, the 5α-­ reduced C21 steroids dihydroprogesterone (5α-­pregnane-­3,20-­dione) and allopregnanolone (5α-­pregnan-­3α-­ ol-­20-­one) are excellent substrates for the 17α-­hydroxylase activity of P450c17.42 Furthermore, 17α-­hydroxylated allopregnanolone (5α-­ pregnane-­3α,17α-­diol-­20-­one) is the most efficient substrate yet identified for the 17,20-­lyase activity of human P450c17, and its cleavage to androsterone is minimally dependent on cytochrome b5,42 unlike 17α-­hydroxypregnenolone metabolism to DHEA.39 The conversion of 5α-­pregnane-­3α,17α-­diol-­20-­one to androsterone by the 17,20-­lyase activity of P450c17, first described in the testes of tammar wallaby pouch young,43 provides an alternative or “backdoor” pathway to DHT (discussed later), by which DHT is produced without utilizing DHEA, androstenedione, and testosterone as intermediates. Consequently, the presence of 5α-­reductases in steroidogenic cells does not preclude the production of C19 steroids, but rather paradoxically enhances the production of DHT by directing flux to 5α-­reduced precursors of DHT. Human enzymes catalyze all of the other reactions required to complete this alternate route to DHT,44 and the fetal adrenal gland produces 5α-­reduced androgens, at least in some pathologic states. The backdoor pathway provides a major route to DHT in pathologic states in which 17α-­hydroxyprogesterone accumulates, including 21-­hydroxylase deficiency and P450 oxidoreductase deficiency.45 Androgen production by the backdoor pathway probably contributes to the severe virilization in newborn girls with 21-­and 11-­hydroxlase deficiencies, while those with 3β-­ HSD2 deficiency, whose adrenal glands cannot make 17α-­hydroxyprogesterone, are minimally virilized.46 Furthermore, 11-­oxygenated pregnanes are also substrates for human P450c17, which can generate 11-­oxygenated androgens.47 The chemistry of P450c17-­mediated hydroxylations is believed to proceed via the common iron oxene species and “oxygen rebound” mechanism proposed for prototypical P450 hydroxylations.48 The mechanism(s) of the 17,20-­lyase reaction involving a carbon–carbon bond cleavage, however, is not resolved, despite considerable study. The capacity of chemically formed iron oxene,48 but not hydrogen peroxide alone, to support catalysis suggest that the same heme-­oxygen

1455

complex might participate in both hydroxylations and the 17,20-­ lyase reaction, but spectroscopic studies suggest the participation of a peroxy-­anion intermediate.49 The sequential reactions follow distributive rather than processive kinetics,50 meaning that the intermediate 17-­hydroxysteroids are released from the active site prior to the 17,20-­ lyase reaction. One consequence of the Δ5 preference of the human enzyme for the 17,20-­lyase reaction is that most human C19-­and C18-­steroids derive from DHEA as an intermediate. This Δ5 preference allows for the phenomenon of adrenarche to occur in humans, an event that only takes place in large primates.51 However, Δ5-­lyase activity is not sufficient for adrenarche to occur, because some monkeys (e.g., rhesus macaques) produce high amounts of DHEA throughout life, but most mammals (e.g. cattle, dogs, cats) never produce much DHEA.51 The biochemistry of P450c17, with its differential regulation of 17α-­hydroxylase and 17,20-­lyase activities, provides clues to the genesis of this enigmatic process of adrenarche. P450c17 is a phosphoprotein, and phosphorylation selectively enhances the 17,20-­lyase activity.52 Mitogen-­associated kinase p38α (MAPK14), can phosphorylate P450c17 and confer 17,20-­ lyase activity to recombinant P450c17 in vitro and in immortalized cell systems,52 and this kinase activity is counterbalanced by protein phosphatase 2A, which in turn is regulated by cAMP via phosphoprotein SET.53 Cytochrome b5 also augments 17,20-­lyase activity, and high expression of b5 in the zona reticularis of monkeys and humans54 suggests that the developmentally regulated expression of cytochrome b5 might be a key event. The transcriptional regulation of cytochrome b5 in the adrenal gland is similar to that of P450c17,55 and the mechanisms enabling zone-­specific expression have not been elucidated. Finally, limiting steroid flux to the Δ5 pathway by low 3β-­HSD activity in the zona reticularis (where most DHEA derives) potentiates the effect of increased 17,20-­lyase activity.54,56 The initial description of 17α-­hydroxylase deficiency was a case in which both 17α-­hydroxylase and 17,20-­lyase products were absent.57 When the gene for human P450c17 was cloned, patients with 17α-­ hydroxylase deficiency were found to harbor mutations in the CYP17A1 gene, and approximately 100 mutations scattered throughout the CYP17A1 gene have been characterized.38 Mutations in W406R and R362C accounted for the high prevalence of 17α-­hydroxylase deficiency in Brazil,58 and triple deletion of D487, S488, F489, and Y329fs accounted for most cases of 17α-­hydroxylase deficiency in China and Southeast Asia.38 Isolated 17,20-­lyase deficiency is extremely rare, mainly caused by mutations in arginines 347 and 358.59,60 Computer modeling studies demonstrate that R347H and R358Q neutralize positive charges in the redox-­ partner binding site.59 Biochemical studies confirm that mutations R347H and R358Q impair interactions of P450c17 with its electron donor POR and with cytochrome b5.61 Therefore, these cases of isolated 17,20-­lyase deficiency are not caused by an inability of the mutant enzymes to bind the intermediate 17α-­ hydroxypregnenolone, but rather the uncoupling of electron transfer with catalysis.59,61,62 In contrast, mutation E305G has been shown to cause 17,20-­lyase deficiency by selectively disrupting binding of 17α-­ hydroxypregnenolone and DHEA synthesis despite enhanced conversion of 17α-­hydroxyprogesterone to androstenedione.63 This unusual variant of isolated 17,20-­ lyase deficiency provides further genetic evidence that the flux of androgens derived from conversion of 17α-­ hydroxyprogesterone to androstenedione in the minor Δ4 pathway is not sufficient to form normal male external genitalia. Mutation V366M is thought to selectively impair 17,20-­lyase activity by interfering with substrate binding and egress.64 One of the first patients reported to have isolated 17,20-­lyase deficiency bears a homozygous mutation in P450 oxidoreductase (G539R), further emphasizing the crucial role of

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PART 7  Adrenal

efficient electron transfer in the 17,20-­lyase reaction.65 Apparent isolated 17,20-­lyase deficiency may also be caused by specific mutations in cytochrome b5.66,67 The P450c17 inhibitors ketoconazole and abiraterone are used to treat castration-­resistant prostate cancer (CRPC) by suppressing testosterone production beyond limits achieved with medical or surgical castration.68 The x-­ray crystal structures of modified human P450c17 with bound abiraterone or galeterone (TOK-­001) show the azole nitrogens bound to the heme iron with the α-­face of the steroid facing the heme and the A-­ring draped against the I-­helix.69 These structures explain the 17α-­hydroxylation chemistry of P450c17 and suggest that the 17,20-­lyase reaction involves oxidation from the α-­ face as well. KEY POINTS  • The 17-­hydroxylase and 17,20-­lyase activities of P450c17 qualitatively regulate the type of steroid hormones produced in a cell. • Cytochrome b5 augments the 17,20-­lyase activity of P450c17 10-­fold by increasing the coupling of electron transfer to substrate oxidation.

P450c21 (Encoded by CYP21A2) Microsomal P450c21 performs adrenal 21-­hydroxylation of the Δ4 steroids 17α-­hydroxyprogesterone and progesterone, an essential step in the biosynthesis of both mineralocorticoids and glucocorticoids (see Fig. 87.2). The human P450c21 protein is found only in the adrenal glands; the extra-­adrenal 21-­hydroxylase activity found in other organs such as the liver and the aorta is not catalyzed by P450c21 but appears to be catalyzed by CYP2C9, CYP3A4, and possibly CYP2C19 and other enzymes as well.70 The locus containing the CYP21 genes is among the most complex in the human genome and explains why 21-­hydroxylase deficiency (affecting approximately 1 in 14,000 live births) is one of the most common autosomal-­ recessive diseases. The CYP21A2 gene and the CYP21A1P pseudogene lie on chromosomal locus 6p21.1 in the midst of the human leukocyte antigen (HLA) locus. Because the HLA locus is highly recombinogenic, exchange between the CYP21A1P and CYP21A2 loci is common. Thus, approximately 95% of 21-­hydroxylase mutations derive from gene micro-­or macroconversion events where some or all of the CYP21A1P pseudogene replaces the corresponding area of the CYP21A2 gene, thus reducing the expression of the encoded P450c21 protein and/or impairing its activity.71 Most of the remaining mutations causing 21-­hydroxylase deficiency are CYP21A2 gene deletions, again caused by recombination events; only approximately 1% of CYP21A2 mutations do not occur via recombination. In addition, at least eight additional genes lie in this locus (Fig. 87.4), including the liver-­specific C4A and C4B genes and the ubiquitously expressed tenascin X or TNXB gene, the disruption of which is one cause of Ehlers–Danlos syndrome (EDS). Occasionally, a patient with 21-­hydroxylase deficiency and EDS will have a contiguous gene syndrome with tenascin-X deficiency as well.72 Heterozygosity for TNXB causes a mild form of EDS characterized by joint hypermobility, and up to 7% of patients with classic CAH also have this form of EDS.73 It is clear that genotype consistently predicts phenotype in very severe and very mild cases of 21-­hydroxylase deficiency. In contrast, patients with P450c21 variants (e.g., the common P30L and V281L mutations and the less common R339H and P453S mutations), which have 5% to 20% of wild-­type activity, can have various phenotypes, implying additional factors that can modify the clinical manifestations of 21-­hydroxylase deficiency.71

Unlike P450c17, P450c21 is not very sensitive to the abundance of POR or regulated via cytochrome b5. The x-­ray crystal structure of bovine P450c21 contains two molecules of bound steroid, one with the steroid nucleus perpendicular to the heme ring, so positioning the C-­21 hydrogen atoms directly above the heme iron for hydroxylation.74 The second steroid molecule lies toward the periphery of the enzyme and is unlikely to undergo hydroxylation, but it may contribute to enzyme structure or allostery. The structure of human P450c21 contains only the progesterone molecule in the active site with V359 holding the steroid in place.75 Mutation of V359 to the smaller residues alanine and glycine increases progesterone mobility and confers 16α-­hydroxylase activity to the enzyme.76 The 21-­hydroxylase reaction of human P450c21 shows large intra-­and intermolecular kinetic isotope effects,77,78 meaning that C-­H bond breaking is substantially rate-­limiting. Impairments of substrate binding, catalysis, heme incorporation, and thermal stability are observed for the common mutations causing varying degrees of 21-­hydroxylase deficiency.79 KEY POINTS  • The CYP21 gene locus lies within the is highly human leukocyte antigen complex, and recombination exchange between the CYP21A1P and CYP21A2 loci is common. • 21-­hydroxylase deficiency is one of the most common autosomal recessive disorders. • Genotype predicts phenotype in very severe and very mild cases of 21-­hydroxylase deficiency.

P450c11β and P450c11AS (Encoded by CYP11B1 and CYP11B2 in Humans) The classical descriptions of distinct deficiencies in 11β-­hydroxylase, 18-­hydroxylase (previously called corticosterone methyl oxidase I or CMOI), and 18-­oxidase (CMOII) suggested that three enzymes executed these three respective transformations. Analogous to the scenario for P450c17, a single enzyme80 and corresponding gene were found in bovine adrenal glands that possessed all three activities. In contrast, humans have two genes named CYP11B1 and CYP11B281 that encode the mitochondrial enzymes 11β-­hydroxylase (P450c11β) and aldosterone synthase (P450c11AS), respectively, and rats, but not mice, have three functional Cyp11b genes. Although P450c11β and P450c11AS both possess 11β-­hydroxylase activities, P450c11AS also performs the two oxygenations at C18 required for aldosterone biosynthesis.82 P450c11β catalyzes a small amount of 18-­hydroxylase but no 18-­oxidase activity.83 Mutations in CYP11B1 cause 11β-­hydroxylase deficiency,84 whereas defects in CYP11B2 cause variable aldosterone synthase deficiency, with salt wasting, failure to thrive, and hypotension.85 Severe defects can impair all P450c11AS activities, leading to the clinical phenotype of CMOI deficiency,82 while P450c11β provides 11β-­hydroxylase activity in the zona fasciculata. Fortuitously, mutations R181W plus V386A, which mainly impair 18-­oxidase activity, are found in patients and cause CMOII deficiency.86 Unlike the multistep distributive kinetics of P450scc and P450c17, P450c11AS uses a highly processive mechanism to synthesize aldosterone from deoxycorticosterone (DOC) without release of the intermediates.87 Consequently, more aldosterone is formed and at a faster rate using DOC as the substrate than corticosterone, even though one additional step is required. Furthermore, only a trace of aldosterone is formed from 18-­hydroxycorticosterone, presumably due to formation of a cyclic acetal in solution with unfavorable geometry, which may explain why a processive mechanism from DOC is essential for efficient aldosterone synthesis.87

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis Class I A

Class II C B

Short arm of chromosome 6

0

10

C2

20

30

Bf RD

G11/RP

40

50

1457

60

70

TNF 250Kb 390Kb

80

90

DR DQ >300Kb

DP

GLO

100 110 120 130 140 150 160 170 180 kb

G11/RP

XB

C4A

21A ZA

XA YA

C4B

21B XB-S ZB

XB

YB

Figure 87.4  Genetic map of the human leukocyte antigen (HLA) locus containing the genes for P450c21. The top line shows the chromosome 6p21.1 region, with the telomere to the left and the centromere to the right. Most HLA genes are found in the class I and class II regions; the class III region containing the CYP21 genes lies between these two. The second line shows the scale (in kb) for the diagram immediately below, showing (from left to right) the genes for complement factor C2, properdin factor Bf, and the RD and G11/RP genes of unknown function; arrows indicate transcriptional orientation. The bottom line shows the 21-­hydroxylase locus on an expanded scale, including the C4A and C4B genes for the fourth component of complement, the CYP21A1P pseudogene (21A), and the active CYP21A2 gene (21B) that encodes P450c21. XA, YA, and YB are adrenal-­specific transcripts that lack open reading frames. The TNXB gene (XB) encodes the extracellular matrix protein tenascin-­X; XB-­S encodes a truncated adrenal-­specific form of the tenascin-­X protein whose function is unknown. ZA and ZB are adrenal-­specific transcripts that arise within the C4 genes and have open reading frames, but it is not known if they are translated into protein; however, the promoter elements of these transcripts are essential components of the CYP21A1P and CYP21A2 promoters. The arrows indicate transcriptional orientation. The vertical dotted lines designate the boundaries of the genetic duplication event that led to the presence of A and B regions.

The coding regions of the CYP11B1 and CYP11B2 genes share 93% amino acid identity and the same exonic gene structure found in all mitochondrial P450 genes. Despite the sequence similarities of these tandem genes, located within 40 kb on chromosome 8q24.3, the expression of P450c11AS is restricted to the adrenal zona glomerulosa, whereas P450c11β is found in the zona fasciculata and zona reticularis, emphasizing activity of zona-­specific gene promoters. The regulation of P450c11β is driven mainly by cAMP in response to ACTH, whereas P450c11AS expression derives from potassium and angiotensin II activation of the protein kinase C pathway.88 Thus, under normal circumstances, 18-­hydroxylase and 18-­oxidase activities are restricted to the zona glomerulosa, where 17-­hydroxylase activity is low, limiting the repertoire of steroids that can undergo 18-­oxygenation. Although the organization of two highly homologous adjacent CYP11B1 and CYP11B2 genes on chromosome 8 is reminiscent of the CYP21A1P and CYP21A2 genes, gene conversion in the CYP11B locus occurs rarely. Glucocorticoid-­remediable aldosteronism (GRA) arises, however, when an unequal crossing over of the CYP11B1 and CYP11B2 genes creates a third, hybrid gene in which the ACTH-­regulated promoter of CYP11B1 drives expression of a chimeric protein with aldosterone synthase activity.89 As a result, 18-­hydroxylase and 18-­oxidase activities are ectopically expressed in the zona fasciculata, leading to elevated renin-­independent production of aldosterone, as well as 18-­oxygenated metabolites of cortisol. The expression of this gene is

suppressed by blunting ACTH production with glucocorticoids such as dexamethasone, which is used for diagnosis and treatment.90 The genetics of GRA has assisted in the precise identification of residues in P450c11AS that enable 18-­oxygenase activities: residues 288, 296, 301, 302, 325, and, perhaps most importantly, 320 are critical for 18-­oxygenase activities.91,92 Therefore, crossovers 3′ to codon 320 do not enable aldosterone synthase activity. These key residues lie in or near the I-­helix, which contains the catalytically important threonine residue implicated in oxygen activation for almost all P450s; thus, these mutations would be expected to alter active site geometry. The x-­ray crystal structure of modified human P450c11AS with bound DOC reveals the β-­face of the steroid nucleus facing the heme iron, which enables oxygenation at C-­11β, to form corticosterone, and subsequently at C-­18, to form 18-­hydroxycorticosterone and aldosterone.93 In the x-­ray crystal structure of P450c11AS with the inhibitor fadrozole bound to the active-­site heme, the cyanophenyl ring of the R-­ fadrozole enantiomer forms hydrophobic interactions with the I-­helix.93 In the corresponding structure of the P450c11β-­fadrozole complex, S-­fadrozole adopts an opposite orientation pointed away from the I-­helix.94 The marked and unexpected differences for these two enzymes may contribute to their catalytic differences and enable the design of selective inhibitors. Osilodrostat, which is structurally related to R-­fadrozole, potently inhibits both P450c11β and P450c11AS and is approved for the treatment of Cushing disease in the United States and Europe.95 The activity of osilodrostat inhibiting P450c11β, and so

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lowering circulating cortisol levels, precluded its clinical development as an antihypertensive agent acting to reduce aldosterone secretion by inhibiting P450c11AS. Compounds that selectively inhibit P450c11AS but not P450c11β have begun to appear96 and are being advanced into clinical trials. In addition to 21-­carbon steroids, both P450c11β and P450c11AS efficiently 11β-­hydroxylate 19-­carbon steroids, including androstenedione and testosterone.93 In adrenal vein effluent, 11β-­ hydroxyandrostenedione is the third most abundant steroid after DHEAS and cortisol and is stimulated by ACTH.97 In peripheral tissues, 11β-­ hydroxyandrostenedione is metabolized to 11-­ ketotestosterone, which is nearly as potent an agonist of the human androgen receptor as testosterone98 and a dominant androgen in teleost fish and some reptiles. Accumulating evidence has demonstrated important contributions of these 11-­oxygenated androgens to human physiology and disease99 (see Pathways to 11-­Oxygenated Androgens later). KEY POINTS  • Expression of P450c11AS is restricted to the adrenal zona glomerulosa, whereas P450c11β is found in the zona fasciculata and zona reticularis. • Regulation of P450c11β is driven mainly by cyclic adenosine monophosphate in response to adrenocorticotropic hormone (ACTH), whereas P450c11AS expression derives from potassium and angiotensin II activation of the protein kinase C pathway. • Glucocorticoid-­remediable aldosteronism arises from an unequal crossing over of the CYP11B1 and CYP11B2 genes to creates a hybrid gene in which the ACTH-­regulated promoter of CYP11B1 drives expression of a chimeric protein with aldosterone synthase activity.

P450aro (Aromatase) The oxidative demethylation of C19 steroids, mainly androstenedione and testosterone, consumes three equivalents of molecular oxygen and NADPH, yielding formic acid and C18 steroids with an aromatic A-­ring; hence the common name for this enzyme, aromatase. As is the case for P450scc, each subsequent oxygenation proceeds with greater efficiency with a distributive mechanism,100 aiding in the completion of this transformation that is essential for estrogen biosynthesis in all animals.26 The mechanism of this aromatization must account for the incorporation of the final oxygen atom from molecular oxygen into the formic acid by-­product. The data are consistent with hydroxylation at C2 of 19-­oxo-­androstenedione via the iron oxene,101 followed by an enzyme-­assisted rearrangement and tautomerization of the intermediate dienone to the phenolic A-­ring.101 The x-­ray structure of human aromatase with bound androstenedione supports this model, showing the steroid positioned with the β-­face, C-­19 methyl group, and A-­ring adjacent to the heme iron.102 Because all estrogen synthesis requires aromatase activity, the potent aromatase inhibitors letrozole, anastrozole, and exemestane are now first-­line treatment for metastatic estrogen receptor-positive breast cancer in postmenopausal women. P450aro is expressed in steroidogenic tissues (ovarian granuloma cells, placenta), in brain, and in nonsteroidogenic tissues, especially fat and bone.26 The CYP19A1 gene for P450aro spans over 75 kb (Table 87.2) and contains five different transcriptional start sites26 with individual promoters that permit the tissue-­specific regulation of expression in diverse tissues. P450aro is a glycoprotein, but glycosylation per se does not appear to affect activity. Studies of patients with aromatase deficiency confirm that biologically significant estrogen synthesis derives entirely from this enzyme.103 In fetuses homozygous for aromatase deficiency, the principal manifestation results from its deficiency in the placenta,103 because ovarian

steroidogenesis is quiescent during fetal life. Although huge amounts of estriol and estradiol are produced by the fetoplacental unit, estrogens are not needed for fetal development, the maintenance of pregnancy, or the onset of parturition; all of these processes proceed normally in fetuses lacking StAR, P450c17, or aromatase, or even in fetuses wholly lacking adrenal because of mutations in SF-­1 or DAX-­1. However, in the absence of placental aromatase activity, androgenic C19 steroids derived from the fetal adrenal are passed into the maternal circulation, causing marked virilization of the mother.103 Furthermore, in pregnancies in which the mother has poorly treated 21-­hydroxylase deficiency, maternal testosterone values can exceed 300 ng/dL (a midpubertal value for males), yet the fetus is not virilized,104 because the maternal testosterone is efficiently metabolized to estradiol by placental aromatase. Thus, placental aromatase is a key enzyme in protecting both the fetus and mother from unwanted androgen exposure. After birth, individuals with aromatase deficiency grow normally and continue linear growth after completion of puberty, with males producing normal amounts of testosterone. However, when treated with estrogens, aromatase-­deficient subjects fuse their epiphyses and cease linear growth.105 These observations provide powerful evidence that bony maturation and epiphyseal fusion in children are mediated by estrogens, not androgens, even in males. These observations have led to the experimental use of aromatase inhibitors in various disorders of accelerated bone maturation.

REDOX PARTNER PROTEINS The proteins collectively referred to as redox partners channel reducing equivalents from NADPH to the heme centers of P450 enzymes.1 Many studies, however, suggest that these proteins promote catalysis by more than just their electron transfer properties. Because of this, the precise nature of the interactions of the P450s with their redox partners is of considerable importance.

Ferredoxin Ferredoxin (FDX1) is encoded by a gene on chromosome 11q22 that spans over 30 kb. FDX1 is a small (14-­kD), soluble Fe2S2 electron shuttle protein that resides either free in the mitochondrial matrix or is loosely bound to the inner mitochondrial membrane.106 FDX1 is expressed in many tissues, and its expression in steroidogenic tissues is induced by cAMP in parallel with P450scc.107 Bovine FDX1 consists of two domains,108 a core region and an interaction domain. The core region contains residues 1 to 55 and 91 to end (bovine numbering), including the four cysteines whose sulfur atoms tether the Fe2S2 cluster to the protein. Residues 56 to 90 form the interaction domain, which is a hairpin containing a helix at its periphery that includes acidic residues critical for the interaction of FDX1 with P450scc,109 including aspartates 72, 76, and 79, plus glutamate 73). Similarly, the carboxylate group of D76 in FDX1 is cross-­ linked in vitro with lysine amino groups substituted for R370 or R366 of P450c11β and P450c11AS, respectively,110 which is consistent with direct electrostatic pairings. A hydrophobic interaction with the adjacent L80 of FDX1 is also critical for binding and catalysis.110 The Fe2S2 cluster lies in a protuberance in the molecule at the junction of its two domains. The charged residues of FDX1 cluster in the interaction domain, giving the molecule a highly negatively charged surface above the Fe2S2 cluster (see Fig. 87.3A). This description of the FDX1 molecule concurs with earlier studies that showed that overlapping sets of negative charges on FDX1 drive its interactions with positive charges on both P450scc and FDXR, which reduces oxidized FDX1. Because a preponderance of the evidence favors a model in which the same surface of FDX1 shuttles between FDXR and the P450 to transport

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis electrons,110,111 a model of how FDX1 interacts with FDXR should suggest how mitochondrial P450s interact with FDX1.

Ferredoxin Reductase Like FDX1, ferredoxin reductase is widely expressed in human tissues, but its expression is two orders of magnitude higher in steroidogenic tissues.112 The primary RNA transcript from the 11-­kb FDXR gene (Table 87.2) on chromosome 17q24-­q25 is alternatively spliced, generating two mRNA species that differ by only 18 bp, but only the protein encoded by the shorter mRNA is active in steroidogenesis.113 Unlike most steroidogenic genes, the promoter for FDXR contains six copies of GGGCGGG sequences, which is the canonical binding site for the transcription factor SP-­1 typically found in “housekeeping” genes. Accordingly, cAMP does not regulate transcription of the FDXR gene, as is the case for FDX1 and P450scc,112 implying that FDXR plays additional roles in human physiology beyond steroidogenesis. Given their essential roles in the conversion of cholesterol to pregnenolone, no null mutations in FDXR or FDX1 have been described in humans. Bovine FDXR also consists of two domains, each comprising a β-­ sheet core surrounded by α-­helices.114 The NADP(H)-­binding domain is a compact region composed of residues 106 to 331 (bovine numbering), whereas the more open FAD domain, formed by the remaining N-­and C-­terminal residues, binds the dinucleotide portion of FAD across a Rossman fold with the redox-­active flavin isoalloxazine ring abutting the NADP(H) domain. By analogy to related structures including glutathione and thioredoxin reductases, the nicotinamide ring of NADPH is modeled to lie adjacent to the flavin ring in position to transfer its two electrons to the FAD. Thus, intramolecular electron transfer occurs in the cleft formed by the angled apposition of these two domains. Within this cleft, basic residues abound, including arginines 240 and 244, which are important for interactions with FDX1.113 Hypothetical docking of the two structures suggests that the negative surface of FDX1 fits elegantly into the positive surface of FDXR, even with NADP(H) bound.114 These studies illustrate the similarities in the electrostatic interactions of FDX1 with both FDXR and the mitochondrial P450s.

P450 Oxidoreductase The flavoprotein POR is expressed widely in human tissues and serves as the sole electron-­transfer protein for all microsomal P450s, including xenobiotic-­ metabolizing hepatic P450s, steroidogenic P450s, and P450s found in other tissues such as the kidney and brain.115 Crystallographic studies show that POR contains two lobes, one binding FAD and the other binding FMN, and a flexible N-­terminus that tethers it to the endoplasmic reticulum.116 NADPH is bound to the cofactor-­binding domain above the FAD in a β-­sheet–rich FAD domain, and an α-­helical connecting domain joins the FAD and the FMN domains. A disordered “hinge” of approximately 25 residues lies between the FMN domain and the connecting domain, suggesting that the FMN and FAD domains can move substantially relative to each other. In the x-­ray structure of rat liver POR, the FMN and FAD lie at the base of a cleft formed by the butterfly-­shaped apposition of the FAD and FMN domains, reminiscent of the electron transfer surface of FDXR.114 Nuclear magnetic resonance (NMR) and small-­angle x-­ray scattering studies117 and ion mobility spectrometry confirm that, once the electrons reach the FMN domain, POR “opens up” via its hinge so that the FMN domain may dock with the redox-­partner binding site of the P450 by electrostatic interactions (see Fig. 87.3). The surface of the electron-­donating FMN domain is dominated by acidic residues, whereas the redox-­partner binding site

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of P450 enzymes contain numerous basic residues. Negative charges of the FMN domain guide interactions with positive charges on the P450. The FMN approaches no closer than 18 Å from the heme, similar to the 16-­Å distance of FAD from the Fe2S2 cluster in the modeled FDXR-­FDX1 complex, and presumably similar to the distance of the heme from the Fe2S2 cluster in the P450-­FDX1 complex.114 These distances are too far for electrons to “jump” directly to the heme; rather, electron transfer apparently uses the polypeptide chain as a conduit.118 Basic residues in the redox-­partner-binding surface are crucial for interactions with POR and for electron transfer, and these positive charges in human P450c17 are critical for maximal 17,20-­lyase activity.61 Thus, these structures demonstrate several key principles of the electron transfer proteins involved in human steroidogenesis: NADPH and prosthetic groups lie at the interfaces of protein domains in which electron transfer occurs; the electron transfer surfaces are negatively charged to pair with positive charges on the P450s; the terminal electron transfer moiety (FMN domain or FDX1) must be mobile or soluble to pass electrons on to the P450; and electrons flow from the FMN or Fe2S2 cluster along the adjacent polypeptide chain to the heme.

Cytochrome P450 Oxidoreductase Deficiency: A Disorder Affecting Multiple P450 Enzymes. Beginning with a clinical report in 1985,119 several patients have been described with clinical and hormonal findings, suggesting partial deficiencies of both P450c17 and P450c21. Some of these individuals were born to mothers who had become virilized during pregnancy, suggesting fetoplacental aromatase deficiency, and many also had the Antley–Bixler skeletal malformation syndrome characterized by craniosynostosis and radioulnar synostosis. Approximately half of patients with Antley–Bixler syndrome have normal steroidogenesis and normal genitalia; these subjects have dominant, gain-­of-­function mutations in the gene for fibroblast growth factor type 2 receptor (FGFR2); however, patients with Antley–Bixler syndrome who also have genital anomalies and disordered steroidogenesis do not have FGFR2 mutations. The initial report described three patients with Antley–Bixler syndrome, genital ambiguity, and hormonal findings, suggesting partial deficiencies of 17α-­hydroxylase and 21-­hydroxylase, as well as a fourth patient who was phenotypically normal but had a similar hormonal profile; all had recessive, loss-­of-­ function amino acid replacement mutations in POR.120 One of these patients was born to a woman who had become virilized during the pregnancy, suggesting partial fetoplacental aromatase deficiency. In vitro biochemical assays of the recombinant mutant POR proteins showed that the mutations in the Antley–Bixler subjects had severely impaired, but not totally absent, activity, whereas the mutations found in a phenotypically normal subject with amenorrhea were less severe.120 Examination of the POR and FGFR2 genes in a series of 32 patients established that the recessive POR mutations and the dominant FGFR2 mutations segregate completely.121 Approximately 100 POR-­deficient patients have been described.122 It appears unlikely that subjects will be found who are homozygous for null POR alleles, because knockouts of POR in mice cause embryonic lethality. POR is required for the activities of all hepatic drug-­metabolizing P450 enzymes, and liver-­specific ablation of the Por gene in mice results in phenotypically and reproductively normal animals with profoundly impaired drug metabolism.123 Furthermore, the human POR mutations identified in patients interfere with in vitro drug metabolism by most hepatic P450 enzymes to varying degrees, and at least one report describes impaired metabolism of test drugs by a POR-­deficient patient.124 Consequently, it seems likely that some patients with POR deficiency may also metabolize drugs poorly.

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The human POR gene, located on chromosome 7, consists of 16 exons (Table 87.2). The sequence of this gene in 842 normal persons from four ethnic groups revealed a high degree of polymorphism.125 The coding sequence variant A503V, found on approximately 28% of all alleles, reduced the 17α-­hydroxylase and 17,20-­lyase activities of P450c17 to approximately 60% of normal121,125 and some, but not all drug metabolism by CYP3A4 and CYP2D6, but had no measurable effect on the activities of P450c21 or hepatic CYP1A2 or CYP2C19. The POR promoter polymorphism –152C→A, found on 13% of Caucasian alleles, reduced POR gene transcription by about half in both adrenal and liver cells in culture, but the potential clinical significance of this is unknown.

Cytochrome b5 The small (12-­to 17-­kD) hemoprotein cytochrome b5 (CYB5A, b5) is found in two forms: the full-­length protein is membrane-­bound in the liver, and a soluble form lacking the C-­terminal membrane anchor is found in erythrocytes. Full-­length cytochrome b5 is expressed in both the adrenal and the gonads, where it can interact with P450c17; the adrenal expression is largely confined to the zona reticularis and may contribute to the genesis of adrenarche.54 While cytochrome b5 appears to transfer the second electron for some P450 reactions,126 apo-­b5, which does not transfer electrons, also stimulates the 17,20-­ lyase activity of human P450c17.39 These experiments suggest that cytochrome b5 does not act alone as an electron donor, but rather functions in concert with POR to somehow aid catalysis, yet b5 might substitute for POR in other P450 systems. The soluble form of bovine b5 was one of the first proteins studied by x-­ray crystallography, and a wealth of structural data for b5 have been acquired using molecular dynamics and NMR spectroscopy for both the holo-­and apo-­b5.127,128 Analogous to FDX1, b5 consists of two domains, a heme-­liganding core 1 domain (residues 40 to 65, bovine numbering); and a structural core 2 domain, from which the C-­terminal membrane-­anchoring helix extends. The heme extends more to the periphery of cytochrome b5 than does the Fe2S2 cluster of FDX1, and the entire surface is dominated by negatively charged residues rather than just one cluster of negative charges near the heme. In addition, the core 1 domain acquires considerable conformational flexibility in apo-­b5, whereas the core 2 domain remains folded as in holo-­b5.127 Finally, the C-­terminal membrane-­spanning helix (exiting the core 2 domain) is required to stimulate the 17,20-­lyase activity of human P450c17, but the signal peptide is not.129 Genetic and biochemical studies have implicated basic residues in P450c17, including R347, R358, R449, and K89, as important for its interaction with cytochrome b5,59,129,130 while E48 and E49 of cytochrome b5 are required for high 17,20-­lyase activity.131 The major effect of cytochrome b5 on the P450c17-­POR complex to augment 17,20-­lyase activity is to increase the coupling of electron transfer to product formation, from 5% to almost 50%.62 KEY POINTS  • Redox partner proteins play and essential role in transferring reducing equivalents from nicotinamide adenine dinucleotide phosphate to the heme centers of P450 enzymes. • Ferredoxin reductase contains one flavin prosthetic group and transfers electrons to the iron-­sulfur protein ferredoxin, the ultimate electron-­ transfer protein to the mitochondrial P450 enzymes. • P450-­oxidoreductase (POR) contains two flavins and is the sole electron-­ transfer protein for all microsomal P450 enzymes; mutations in POR impair multiple P450 activities. • Cytochrome b5 augments the 17,20-­lyase activity of the P450c17-­POR complex.

STEROIDOGENIC DEHYDROGENASES AND REDUCTASES 3β-­Hydroxysteroid Dehydrogenase/Δ5→Δ4-­Isomerases

Conversion of Δ5 steroids into their Δ4 congeners, a step required for the production of progestins, mineralocorticoids, glucocorticoids, and sex steroids, consists of two chemical transformations, both performed by the 3β-­hydroxysteroid dehydrogenase/Δ5→Δ4-­isomerase (3βHSD) enzymes. The first reaction is the oxidation of the 3β-­ hydroxyl group to the ketone, and during this process NAD+ is converted to NADH. The intermediate Δ5-­3-­ketosteroid remains tightly bound to the enzyme with nascent NADH, and the presence of NADH in the cofactor-­binding site activates the enzyme’s second activity, the Δ5→Δ4-­isomerase activity.132 Competition experiments have shown that the dehydrogenase and isomerase activities reside in a single active site,133 yet these enzymes are often referred to by their dehydrogenase activity alone. Although rodents contain multiple 3βHSD isoforms, human beings have only two active genes. The type 1 enzyme (3βHSD1) is expressed in the placenta, liver, brain, and some other tissues.134 This isoform is required for placental progesterone production during pregnancy, which may explain why a deficiency of 3βHSD1 has never been described. In contrast, the type 2 enzyme (3βHSD2) is by far the principal isoform in the adrenal glands and the gonads.135 Deficiency of 3βHSD2 causes the rare form of congenital adrenal hyperplasia known as 3βHSD deficiency.46 The presence of the type 1 isozyme in these patients helps to explain the paradox of why 46,XX individuals born with severe 3βHSD2 deficiency can virilize slightly in utero and can have elevated 17-­hydroxyprogesterone (17OHP) in newborn screening tests. The 3βHSD block in the adrenal gland diverts Δ5-­steroids away from cortisol and toward DHEA; extraadrenal 3βHSD1 enables 17OHP and testosterone synthesis despite absent 3βHSD2 in the adrenal. The N367T allele of 3βHSD1 is variably present in all populations, and this variant has equivalent catalytic activity as wild-­type 3βHSD1; however, the N-­to-­T substitution disrupts a ubiquitination site. In prostate cancer cells, the 367T allele is resistant to degradation and provides a selective advantage by maintaining the 3βHSD activity required to form active androgens from DHEA.136 The types 1 and 2 enzymes share 93.5% amino acid identity, and all biochemical studies comparing the two enzymes yield very similar results. The enzymes are strongly inhibited by Δ4 products137 and by synthetic Δ4 steroids such as medroxyprogesterone acetate and the 3-­keto-­Δ4-­metabolite of abiraterone.138 Compared to the type 1 enzyme, the type 2 enzyme is higher affinity/lower capacity (lower Km, and Vmax) and thus efficiently converts Δ5 steroids pregnenolone, 17α-­hydroxypregnenolone, DHEA, and androst-­5-­ene-­ 3β,17β-­diol to their Δ4 congeners in the adrenals and gonads.133 The enzymes are primarily membrane-­bound and are found both in the microsomal and mitochondrial fractions during subcellular fractionation.133 Considerable evidence suggests that 3βHSD activity is an important factor in regulating adrenal production of DHEAS. The human fetal adrenal, which produces vast amounts of DHEAS, contains little 3βHSD immunoreactivity.139 Furthermore, the expression of 3βHSD in the innermost regions of the adrenal cortex declines as the zona reticularis develops in childhood, and 3βHSD immunoreactivity is low in the zona reticularis of the adult rhesus macaque and of humans.54 Thus, the development of an adrenal cell type (reticularis) that is relatively deficient in 3βHSD activity is a necessary component of adrenarche, in which adrenal production of the Δ5 steroids DHEA and DHEAS rises exponentially.140

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis

17β-­Hydroxysteroid Dehydrogenases There are at least 14 human 17β-­hydroxysteroid dehydrogenase (17βHSD) isoforms; these isoforms vary widely in size, structure, substrate specificity, cofactor utilization, and physiologic functions.141 This section focuses on the human isoforms that possess significant, rather than gratuitous, 17βHSD activity.

17β-­Hydroxysteroid Dehydrogenase Type 1. In the late 1980s, three independent groups reported the cloning of 17βHSD1 cDNA, the first of any human HSD.142 Located on chromosome 17q25 adjacent to a pseudogene, the HSD17B1 gene (Table 87.2) encodes a 34-­kD protein subunit that is expressed primarily in the placenta and in ovarian granulosa cells of developing follicles.141 The enzyme, which is active only as a dimer, accepts mainly estrogens such as estrone, although it also has low catalytic activity for the conversion of androstenedione to testosterone and DHEA to androst-­5-­ene-­3β,17β-­diol.143 Although the enzyme can oxidize 17β-­hydroxysteroids in the presence of NAD+ in vitro at a high pH, the enzyme functions in vivo to reduce estrone to estradiol and 16α-­hydroxyestrone to estriol.143 Sequence alignments with other members of the SDR family identified a Y-­X-­X-­X-­K active-­site motif in residues 155 to 159, which was confirmed crystallographically.144 The structure demonstrates that cofactor lies across the β-­sheet core of the protein in a Rossman fold characteristic of all SDR enzymes. Steroid appears to dangle from the top of the enzyme almost perpendicular to the cofactor, with a hydrophobic pocket holding the body of the steroid in place while the 3-­hydroxyl forms hydrogen bonds with H221 and E282. At the place where the steroid and cofactor meet, S142, Y155, and K159 help to form a proton-­relay system that drives catalysis. Because steroid flux to estrogens preferentially occurs via the aromatization of androstenedione to estrone, 17βHSD1 appears to be required for the conversion of estrone to biologically active estradiol in the ovary and placenta. This role has not been proven unequivocally, because no cases of human 17βHSD1 deficiency have been reported, although female mice with targeted disruption of the Hsd17b1 gene show reduced fertility.145 Such a disease is theoretically compatible with life, because fetuses with aromatase deficiency103 and estrogen insensitivity (ERα mutations) are viable. Nevertheless, this enzyme is probably critical for ovulation and may be important in the pathogenesis and progression of estrogen-­dependent breast cancers.146

17β-­Hydroxysteroid Dehydrogenase Type 2. In contrast to the “activating” role of 17βHSD1 in the placenta and ovary, human endometrium inactivates estradiol by the conversion to estrone. The 17βHSD1 mRNA is not detected in the human uterus, but a related cDNA encoding microsomal HSD17B2 was cloned147 and found to be expressed in endometrium, placenta, and other tissues.148 17βHSD2 converts estradiol to estrone and oxidizes testosterone and DHT to their inactive 17-­ ketosteroid homologs androstenedione and 5α-­ androstanedione, respectively. The widespread tissue distribution and broad substrate specificity of 17βHSD2 suggests that its role in human physiology is to protect tissues from excessive exposure to active steroid hormones by oxidation to inactive 17-­ketosteroids.141 Human deficiency of this most active human inactivating (oxidizing) 17βHSD has not been described, but targeted disruption of the mouse Hsd17b2 gene results in placental defects and embryonic lethality,149 which suggests a broad and fundamental importance of this isoenzyme. The type 2 enzyme also oxidizes 20α-­dihydroprogesterone to progesterone, but this activity is low relative to its 17βHSD activity.147

17β-­Hydroxysteroid Dehydrogenase Type 3. A form of 46,XY disordered sex development (DSD) lacking the enzyme capable of

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reducing androstenedione to testosterone, was reported in the 1970s.150 When the large, complex HSD17B3 gene encoding 17βHSD3 was cloned, patients with the androgenic “17-­ketosteroid reductase deficiency” were found to harbor mutations in this gene,151,152 proving the central role of this enzyme in male sexual differentiation and marking 17βHSD3 as the only 17βHSD enzyme whose role in human physiology is genetically established by a deficiency syndrome. Nonetheless, patients with 17βHSD3 deficiency produce enough testosterone to virilize incompletely at puberty, suggesting that one or more additional human 17βHSD enzymes convert androstenedione to testosterone (see later). Similarly, the human ovary exports some testosterone despite an absence of 17βHSD3 expression, and women with 17βHSD3 deficiency produce normal amounts of androgens and estrogens.153 Unlike 17βHSD1, which has been the subject of intense biochemical study, relatively little is known about 17βHSD3 enzymology. This knowledge gap is at least in part caused by the very hydrophobic nature of the encoded 310-amino acid protein, hampering the expression of this enzyme in bacteria. From experiments in transiently transfected HEK-­293 cells, 17βHSD3 reduces all of the C19 17-­ketosteroids that serve as precursors of testosterone and DHT in human beings, including DHEA, 5α-­androstanedione, and androsterone.152 The conversion of DHEA to androst-­5-­ene-­3β,17β-­diol by 17βHSD3 probably contributes significantly to testicular testosterone synthesis. Estrogens such as estrone are poor substrates for human 17βHSD3.143

17β-­ Hydroxysteroid Dehydrogenase Type 4. Many additional HSD isoforms have been described in rodents and in humans, but the activities of these isoforms for steroids are generally poor. For example, the type 4 enzyme is a trifunctional protein located in peroxisomes, but its (oxidative) HSD activity toward estradiol is 106 times slower than its 3-­hydroxyacyl-­coenzyme A dehydrogenase activity.154 Deficiency of the type 4 enzyme causes Zellweger syndrome, in which bile acid synthesis is disturbed but steroidogenesis is not affected. Thus, this enzyme has 17βHSD activity as one of its repertoire of transformations, but steroidogenesis is not its principal physiologic function.

17β-­Hydroxysteroid Dehydrogenase Type 5 (Aldo-­Keto Reductase Family 1 Member C3). Unlike 17βHSD types 1 to 4, which are SDR enzymes, the type 5 enzyme (AKR1C3) is an AKR enzyme that is expressed both in steroidogenic and nonsteroidogenic tissues.155 Originally described as hepatic 3αHSD type 2 for its ability to reduce DHT to 5α-­androstane-­3α,17β-­diol, this protein was later found also to have 17βHSD activity.155 This enzyme, now known as AKR1C3 (see 3α-­HSDs), may account for much of the extratesticular androstenedione-­to-­testosterone conversion, although its catalytic efficiency as a 17βHSD is poor156 compared with its 20α-­HSD activity with progesterone and DOC or its prostaglandin dehydrogenase activity, reducing PGH2 to PGF2α. Nevertheless, AKR1C3 is more highly expressed in the human fetal adrenal during the time of sexual differentiation than 17βHSD344 and may participate in androgen production, particularly in virilizing congenital adrenal hyperplasias. The postnatal adrenal also expresses low levels of AKR1C3, accounting for the small amount of testosterone secreted directly by the adrenal.157

17β-­Hydroxysteroid Dehydrogenase Type 6. This enzyme is discussed under the 3α-­hydroxysteroid dehydrogenases below.

Steroid 5α-­Reductases The conversion of testosterone to DHT in target tissues was described in the 1960s,158 and studies using fibroblasts suggested that at least two human enzymes with different pH optima and genetics performed these transformations.159 These initial results were confirmed when the genes

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PART 7  Adrenal

encoding the type 1 and type 2 enzymes were cloned, and patients with clinical 5α-­reductase deficiency were found to have deletions or mutations in the SRD5A2 gene.160 The two isoforms are very hydrophobic 30-­kD microsomal proteins that share 50% identity. The type 1 enzyme is expressed in adult nongenital skin and liver and is not expressed significantly in fetal peripheral tissues, but the enzyme is expressed in the fetal testis and fetal adrenal very early in gestation.44 Consequently, deficiency of the type 2 enzyme is not compensated for by the type 1 enzyme in the fetus.161 The type 2 enzyme remains the predominant enzyme in genital skin, male accessory sex glands, and prostate, whereas the type 1 enzyme accounts for most of the hepatic 5α-­reduction. Although 5α-­reductase activity is generally discussed in the context of male genital differentiation and androgen action, both isozymes reduce a variety of steroids in what are believed to be degradative pathways in humans. Progesterone, 17OHP, and related C21 steroids are the best substrates for both 5α-­reductases, particularly the type 1, and cortisol, cortisone, corticosterone, and related compounds are also good substrates.162 The 5α-­(and 5β-­) reduced steroids may be metabolized further and conjugated for excretion in the urine. Given the importance of 5α-­reductase type 2 in prostate growth, inhibitors of the type 2 enzyme have been developed for the treatment of prostatic hyperplasia and the prevention of its recurrence after surgery. Finasteride selectively inhibits human 5α-­reductase type 2, whereas dutasteride inhibits both the type 1 and type 2 isoenzymes. Both drugs are approved for treatment of prostatic hyperplasia in the United States. Although the function of 5α-­reductase type 2 is firmly established from the studies of its deficiency, which is a form of 46,XY DSD, the role of the type 1 isoform in humans is less clear, but its expression in the fetal adrenal and testis and the now-­established role of the backdoor pathway in human male sexual development suggest that the type 1 enzyme might participate in normal and abnormal sexual development. Furthermore, the type 1 isoenzyme appears to be the dominant 5α-­reductase expressed in most prostate cancers and thus controls the pathways of precursor flux to DHT.163 Given the abundant expression of the type 1 isoform in liver, and its high activity with C21 steroids, this enzyme has been ascribed a role of degrading circulating C21 steroids in preparation for excretion in the urine. However, disruption of the Srd5a1 gene in mice results in delayed parturition, a defect that can be rescued with 5α-­androstane-­3α,17β-­diol. In immature mice, 5α-­reductase type 1 is expressed both in the ovary and in the Leydig cells, and this enzyme participates in the testicular synthesis of 5α-­ androstane-­3α,17β-­diol via two pathways.164 Whether 5α-­reductases are expressed in postnatal human adrenal or gonads in normal physiology or in pathologic states is not known.

3α-­Hydroxysteroid Dehydrogenases The four major human 3α-­hydroxysteroid dehydrogenases (3αHSDs) are AKR enzymes with reductive preferences that belong to the AKR1C family (AKR1C1, 2, 3, and 4). The 3αHSDs types 1, 2, 3, and 4 are trivial names for AKR1C4, 1C3, 1C2, and 1C1, respectively, which are clustered on chromosome 10p14-­p15. Each enzyme has its characteristic tissue distribution165 and repertoire of catalytic activities.2,156 AKR1C3 also performs the 17βHSD reaction with androstenedione and is known also as 17βHSD5, and all of these AKR1C isoforms catalyze additional reactions, such as the 20α-­reduction of pregnanes. In the brain, 3αHSDs reduce 5α-­dihydroprogesterone to tetrahydroprogesterone (allopregnanolone), which is an allosteric activator of the GABAA receptor-­chloride channel complex with a nanomolar affinity166 and is approved for the treatment of postpartum depression in the United States.167 AKR1C4 is abundant in liver but has been found in adrenal and gonad; AKR1C3 was cloned from liver, prostate, and brain; AKR1C2 is found in the prostate and brain;

and AKR1C1 is abundant in the uterus. The amino acid sequences of the type 2 and 3 isoenzymes differ by only a few residues and have common allelic variations. These minor differences in sequences, however, cannot be neglected, because these differences might alter substrate utilization. In the central nervous system, selective serotonin reuptake inhibitors (e.g., the antidepressant drugs fluoxetine and paroxetine) directly lower the Km of rat brain type 2 3αHSD for 5α-­dihydroprogesterone by almost 10-­fold,168 which explains why these drugs augment brain allopregnanolone concentrations, perhaps contributing to their antidepressant activity. In addition, x-­ray crystallography has shown that the β subunit of the mammalian voltage-­gated potassium channel is a tetrameric structure169 in which each subunit closely resembles the rat liver 3αHSD (AKR1C9)170 and even contains bound NADP+ with high occupancy. Although the broader implications of this work are not yet known, these studies suggest a role of HSDs in coupling intracellular redox state to membrane excitation. The 3αHSDs differ from the 11βHSDs, 3βHSDs, and 17βHSDs types 1 through 4 in several respects, because all reductive 3αHSDs are AKR enzymes rather than SDR enzymes.2 As AKR enzymes, they function as monomers with a TIM-­barrel structure; they bind cofactor with the nicotinamide ring draped across the mouth of the “barrel” rather than lying on a Rossman fold; and their kinetic mechanisms are highly ordered, with cofactor dissociation the final and rate-­limiting step.171 Tight NADP(H) binding derives from interaction of R276 with the 2′-­phosphate, and mutation of R276 eliminates a conformational change associated with tight binding172 and attenuates or reverses the preference for ketosteroid reduction in intact cells.3 As shown in the structure of AKR1C9,170 their active sites also contain tyrosine and lysine residues to facilitate proton transfer during catalysis, but these residues are distantly located in linear sequence rather than confined to the Y-­X-­X-­X-­K motif as in SDR enzymes. In contrast to the reductive 3αHSDs, the oxidative 3αHSDs belong to the SDR family and show greatest similarity to the retinol dehydrogenase or cis-­retinol/androgen dehydrogenase (RoDH/CRAD) subfamily.173 Although several of these RoDH/CRAD enzymes show some 3αHSD activity, the most active enzyme has been called RoDH, the microsomal 3αHSD, 3(α→β)-­hydroxysteroid epimerase, or formally 17βHSD6, whose HSD17B6 cDNA was first cloned from prostate.174 This enzyme converts the inactive C19 steroid 5α-­androstane-­3α,17β-­ diol to DHT, and thus likely catalyzes the final step in the backdoor pathway from 17OHP to DHT via androsterone.44 Prolonged incubation of 3α-­hydroxysteroids with cells transfected with the cDNA for 17βHSD6 or with microsomes containing the recombinant enzyme, however, yields subsequent 3-­ketosteroid metabolites, including both 3α-­and 3β-­hydroxysteroids and 17β-­hydroxysteroids.175 Hence, this enzyme has complex catalytic flexibility, and may serve a variety of biological functions.

11β-­Hydroxysteroid Dehydrogenases The 11β-­hydroxysteroid dehydrogenases (11βHSD) regulate the bioactivity of endogenous and synthetic glucocorticoids, and a comparison of the types 1 and 2 enzymes exemplifies some key principles of HSD enzymology (Table 87.3). Both enzymes are hydrophobic, membrane-­ bound proteins that bind cortisol/cortisone and corticosterone/11-­ dehydrocorticosterone, but otherwise their properties and physiologic roles differ substantially176 (see Table 87.3). The type 2 enzyme shares only 21% sequence identity with 11βHSD1, whereas 11βHSD2 and 17βHSD2 share 37% identity and favor steroid oxidation in vivo. Thus, 11βHSD1 and 11βHSD2 are only distantly related members of the SDR family, yet they perform opposite functions in specific tissues in human physiology and pharmacology.

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis

TABLE 87.3  Comparison of 11β-Hydroxy­

steroid Dehydrogenases Types 1 and 2 Property

Type 1

Type 2

Size Orientation in ER Expression

34 kD Luminal Liver, decidua, lung, gonad, pituitary, brain, fat, bone Reduction NADPH via H6PDH Low affinity (Km 0.2-­2 μM) Moderate

41 kD Cytoplasmic Kidney, placenta, colon, salivary gland Oxidation Cytoplasmic NAD+ High affinity (Km 0.01-­0.1 μM) Strong

CRD (H6PDH > HSD11B1)

AME

Principal reaction Cofactor preference Substrate binding Inhibition by carbenoxolone Deficiency state

ER, Endoplasmic reticulum; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; NAD+, nicotinamide adenine dinucleotide, oxidized form; H6PDH, hexose-­6-­phosphate dehydrogenase; CRD, cortisone reductase deficiency; AME, apparent mineralocorticoid excess.

The 34-­kD type 1 enzyme (11βHSD1) is expressed in the liver, testis, lung, fat, and proximal convoluted tubule.177 The type 1 enzyme catalyzes both the oxidation of cortisol using NADP+ as a cofactor (Km 1–2 μM) and the reduction of cortisone using NADPH as a cofactor (Km 0.1–0.3 μM), with cortisone reduction being the dominant reaction in transfected cells.176 Some synthetic glucocorticoids such as prednisone are 11-­ketosteroids that must be reduced to their 11β-­hydroxy derivatives to attain biologic activity, and these transformations are performed mainly in the liver by 11βHSD1. In contrast, when recombinant 11βHSD1 is studied in vitro, cortisol oxidation with NADP+ is most efficient, and cortisone reduction is only achieved if NADP+ is scrupulously removed with an enzymatic NADPH regeneration system.178 Thus, the net flux of steroid driven by 11βHSD1 depends on the relative concentrations of available NADPH and NADP+, which usually favors reduction in cells, especially given the high Km of the enzyme for cortisol.178 The mechanism for the discrepancy between the prominent oxidative preference in vitro and the reductive dominance in vivo, however, is more complex and derives from the localization of 11βHSD1 in the lumen of the endoplasmic reticulum.179 In this compartment, the ratio of NADPH to NADP+ is not maintained by the cytoplasmic NADP+-­coupled dehydrogenases (mainly glucose-­6-­phosphate dehydrogenase), but by hexose-­ 6-­ phosphate dehydrogenase (H6PDH). Indeed, most patients with apparent cortisone reductase deficiency (CRD), which is characterized by high ratios of cortisone to cortisol and of their respective metabolites in blood and urine,180 bear loss-­ of-­function mutations in H6PDH181 rather than HSD11B1. These mutations impair regeneration of the NADPH within the endoplasmic reticulum, which serves as cofactor for the 11βHSD1 reaction. The genetics and pathophysiology of CRD provide an excellent example of the critical role of nicotinamide cofactors in HSD function and biology and the molecular complexity of steroidogenesis. In contrast, the 41-­kD type 2 enzyme catalyzes the oxidation of cortisol and corticosterone using NAD+, and, although this enzyme has a high affinity for its steroid substrates (Km 0.01–0.1 μM),182 catalysis of the reductive reactions by 11βHSD2 has not been conclusively demonstrated. In addition, 11βHSD2 also oxidizes 11β-­ hydroxyandrostenedione and 11β-­testosterone to their 11-­ketosteroids, as described in the 11-­oxyandrogen pathway section later. Cortisol is a potent agonist at the mineralocorticoid (glucocorticoid type 2) receptor in the distal nephron, but its oxidized 11-­keto derivative, cortisone, is

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not a mineralocorticoid. The reason cortisol does not act as a mineralocorticoid in vivo, even though cortisol concentrations can exceed aldosterone concentrations by three orders of magnitude, is because cortisol is enzymatically converted to cortisone in the cells lining the cortical and medullary collecting ducts. Thus, the type 2 enzyme inactivates the mineralocorticoid activity of cortisol in the kidney tubule,183 and inactivating mutations in the type 2 enzyme cause a syndrome of apparent mineralocorticoid excess184 (see Chapter 88). This enzyme system may be overwhelmed in cases of prodigious excess cortisol production, as seen in the ectopic ACTH syndrome, where cortisol acting at the mineralocorticoid receptor causes hypokalemia and hypertension. The presence of the type 2 enzyme in the placenta182 also inactivates endogenous and synthetic corticosteroids such as prednisolone, allowing the use of these agents during pregnancy without affecting the fetus. In contrast, 9α-­fluorinated steroids such as dexamethasone are minimally inactivated by the type 2 enzyme, primarily because of a shift in the oxidation/reduction preference rather than a reduction in affinity for the enzyme. It is this resistance to inactivation by placental 11βHSD2 that is essential for synthetic glucocorticoids to “cross the placenta” and to exert a pharmacologic effect on the fetus. Furthermore, the relatively high placental concentrations of NADP+ may also favor the oxidative action of 11βHSD1, so that both placental enzymes protect the fetus from the high maternal concentrations of cortisol that occur during pregnancy.176

STEROID SULFONATION The steroid sulfotransferase (SULT) enzymes are cytosolic enzymes that use 3′-­phosphoadenine-­5′-­ phosphosulfate (PAPS) as a sulfate donor. The SULT1E1 isoenzyme sulfonates the phenolic 3-­hydroxyl group of estrogens, and the SULT2A1 isoenzyme sulfonates the Δ5 steroids pregnenolone, 17-­hydroxypregnenolone, androst-­5-­ene-­3β, 17β-­diol, and most importantly DHEA. SULT2A1 is abundant in the adrenal zona reticularis,185 which efficiently converts most of the nascent DHEA to DHEAS. The sulfate donor PAPS derives from two molecules of ATP and sulfate, through the ATP sulfurylase and adenosine phosphosulfate (APS) kinases, the PAPS synthases types 1 and 2 (PAPSS1, PAPSS2). Whereas PAPSS1 is ubiquitously expressed, PAPSS2 expression is high in cartilage, adrenal, and liver. Loss-­of-­ function mutations in PAPSS2 deplete the adrenal gland of PAPS and increase production of unconjugated DHEA. This circulating DHEA is a substrate for peripheral 3βHSD1, which yields adrenal-­derived androgens. Rare cases of incomplete PAPSS2 deficiency have been described in girls with premature pubarche, advanced bone age, acne, hirsutism, and subsequent secondary amenorrhea.186 These children have mild to moderate skeletal deformities, whereas complete PAPSS2 deficiency causes autosomal recessive brachylomia and severe bone disease, primarily affecting the spine. KEY POINTS  • Conversion of Δ5 steroids into their Δ4 congeners, a step required for the production of progestins, mineralocorticoids, glucocorticoids, and sex steroids, consists of two chemical transformations, both performed by the 3β-­ hydroxysteroid dehydrogenase/Δ5→Δ4-­isomerase enzymes. • 17β-­hydroxysteroid dehydrogenase isoforms play critical roles in the production and degradation of sex steroids. • 3α-­hydroxysteroid dehydrogenases are involved in the “backdoor pathway” to DHT. • 11β-­hydroxysteroid dehydrogenases catalyze the interconversion of cortisol/cortisone, and corticosterone/11-­dehydrocorticosterone; 11βHSD2 also oxidizes 11β-­hydroxyandrostenedione and 11β-­testosterone to their 11-­ketosteroids in the 11-oxyandrogen pathway.

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PART 7  Adrenal

PATHWAYS Adrenal Steroidogenic Pathways Diagrams of steroidogenic pathways, such as that shown in Fig. 87.2, typically combine the pathways from multiple cell types to give a comprehensive and inclusive illustration; however, such diagrams may be misleading, as dominant pathways by which specific steroids are synthesized differ in each steroidogenic cell type. The three major pathways of steroidogenesis in each zone of the human adrenal cortex are shown in Fig. 87.6. The adrenal zona glomerulosa is characterized by three features: its expression of angiotensin II receptors, its unique expression of P450c11AS, and its absence of P450c17 expression. This combination of factors permits the zona glomerulosa to produce aldosterone under regulation by the renin/angiotensin system. By contrast, the adrenal zona fasciculata does not express angiotensin II receptors or P450c11AS, but instead expresses the melanocortin type 2 receptor (MC2R, the receptor for ACTH) and P450c11β, which cannot convert 18-­hydroxycorticosterone to aldosterone and has minimal capacity to convert corticosterone to 18-­hydroxycorticosterone.187 Both the zona glomerulosa and zona fasciculata express P450c21, but the zona fasciculata also expresses P450c17, which allows cortisol synthesis. The zona fasciculata, however, expresses very little b554; consequently, P450c17 in the zona fasciculata catalyzes 17α-­hydroxylation but much less 17,20-­lyase activity. Thus, the zona fasciculata produces two glucocorticoids, mainly cortisol and smaller amounts of corticosterone, under the influence of ACTH. Patients with severe mutations in P450c17 cannot synthesize cortisol but instead increase corticosterone production (as do rodent adrenal glands, which normally lack P450c17), explaining why they are not glucocorticoid-­deficient, despite the lack of cortisol (see Fig. 87.5). The adrenal zona reticularis also expresses MC2R but very little P450c21, P450c11β, or 3βHSD2, and as a result, the zona reticularis produces minimal amounts of cortisol. By contrast, high b5 expression in the zona reticularis54 maximizes the 17,20-­ lyase activity of P450c17,39 so that DHEA is produced and sulfated by SULT2A1 to DHEAS. As DHEA accumulates, small amounts are converted to androstenedione, and very small amounts of this androstenedione are converted to testosterone, probably by AKR1C3/17βHSD5. Thus, the pattern of steroid products secreted by each adrenal zone is determined by the enzymes produced in that zone and may be logically deduced from an understanding of their specific enzymatic properties.

Gonadal Steroidogenic Pathways Testicular synthesis of testosterone follows a pathway that is similar to C19-­steroid production in the adrenal zona reticularis, with the notable exceptions that Leydig cells express 3βHSD2 and 17βHSD3 but no SULT2A1, and the stimulus for steroidogenesis is transduced by the LH receptor rather than MC2R. Consequently, DHEA produced under LH stimulation is not sulfated but is readily converted to androstenedione and then testosterone, or via androst-­5-­ene-­3β,17β-­diol as the intermediate (Fig. 87.6). As in the adrenal gland, the principal pathway to C19 steroids is via Δ5 steroids to DHEA; the Δ4 pathway from 17OHP to androstenedione makes a minimal contribution.39,63 By contrast, ovarian steroidogenesis is more complex, as the enzymatic steps are partitioned between the granulosa and theca cells, which surround the oocyte and form a follicle. In addition, the patterns of steroidogenesis vary during the cycle, directed mainly to estradiol in the follicular phase and to progesterone in the luteal phase (see Fig. 87.6). The key point in ovarian steroidogenesis is that granulosa cells do not express P450c17. Thus, in general, steroidogenesis is initiated in granulosa cells under the influence of LH, which, via cAMP, stimulates the expression of P450scc.17 Pregnenolone and progesterone from granulosa cells diffuse into adjacent theca cells, where P450c17 and 3βHSD2

can act upon them to produce androstenedione. Some androstenedione is secreted or converted to testosterone (via AKR1C3/17βHSD5), and a portion returns to the granulosa cells, where androstenedione is converted to estrone and then to estradiol by P450aro and 17βHSD1, respectively. Thus, as with the three zones of the adrenal gland, the patterns of gonadal steroidogenesis are dictated by the cell-­specific expression of specific steroidogenic enzymes.

The Backdoor Pathway to Dihydrotestosterone An alternative pathway from 17OHP affords dihydrotestosterone when 17OHP is sequentially 5α-­and 3α-­reduced to 5α-­pregnane-­3α,17α-­ diol-­20-­one in steroidogenic cells expressing P450c17 (Fig. 87.7A).43 Not only is 5α-­pregnane-­3α,17α-­diol-­20-­one the most efficient substrate known for the 17,20-­lyase activity of human P450c17, but unlike the conversion of 17α-­hydroxypregnenolone to DHEA, its conversion to androsterone occurs efficiently without b5.42 The resulting androsterone may be reduced to 5α-­androstane-­3α,17β-­diol and then 3α-­oxidized to dihydrotestosterone, probably by RoDH (17βHSD6). Thus this alternative pathway is a “backdoor pathway” to dihydrotestosterone, which is produced without the intermediacy of DHEA, androstenedione, and testosterone. The backdoor pathway enables production of potent androgens from 17OHP, despite the poor 17,20-­ lyase activity of human P450c17 for 17OHP, by using 5α-­pregnane-­ 3α,17α-­diol-­20-­one as the substrate for the 17,20-­lyase reaction. The human backdoor pathway is relevant to normal physiology and to pathologic states. The only enzymatic activities unique to the backdoor pathway that are not found in “conventional” steroidogenic pathways are the oxidative and reductive 3αHSD activities, but at least one isoenzyme capable of catalyzing each requisite reaction is expressed in appropriate tissues of the human fetus.44 Mass spectrometric analyses of urinary steroids in patients with POR deficiency confirm that all the steroidal intermediates in the backdoor pathway are produced in patients with POR deficiency.188 Similar studies have shown that infants, children, and adults with 21-­hydroxylase deficiency who overproduce 17OHP, the “gateway steroid” of the backdoor pathway, excrete all the expected urinary metabolites of the backdoor pathway steroids, including high amounts of androsterone (5α-­reduced C19 steroid) relative to etiocholanolone (5β-­reduced C19 steroid).45 This androgen production by the backdoor pathway helps to explain why newborn girls with 21-­and 11β-­hydroxylase deficiencies can be severely virilized, while those with 3βHSD2 deficiency, who cannot generate much intraadrenal 17OHP, are minimally virilized.46

Pathways to 11-­Oxygenated Androgens In the testes of teleost fish, nascent testosterone is subject to 11β-­ hydroxylase and oxidative 11β-­hydroxysteroid dehydrogenase activities, yielding 11-­ketotestosterone as the major androgen product. The human Leydig cell expresses neither P450c11β nor 11βHSD2, but the human adrenal cortex expresses P450c11β in the zona fasciculata and zona reticularis, where androstenedione can be produced and 11β-­ hydroxylated. In fact, the concentration of 11β-­hydroxyandrostenedione in adrenal vein effluent is higher than androstenedione,97 and circulating concentrations of 11β-­hydroxyandrostenedione exceed those of androstenedione by a factor of 1.5 to 3 in males and females throughout life.98,189 Circulating 11β-­hydroxyandrostenedione is efficiently oxidized via 11βHSD2 in the kidney,190 yet little 11-­ketoandrostenedione accumulates. Instead, 11-­ketoandrostenedione is a much better substrate for AKR1C3 than androstenedione by roughly a factor of eight,191 and thus generates 11-­ketotestosteorne via two conversions of the adrenal precursor in peripheral tissues (Fig. 87.7B). Furthermore, 11-­ketotestosterone is a potent agonist of the human androgen receptor, nearly as active as testosterone.98

A

Cholesterol StAR

P450scc/FDX1/FDXR 3βHSD2

Pregnenolone

Progesterone

P450c21 POR

11-Deoxycorticosterone P450c11AS FDX1/FDXR Corticosterone

Zona Glomerulosa

P450c11AS FDX1/FDXR 18-Hydroxycorticosterone P450c11AS FDX1/FDXR

Aldosterone

B

Cholesterol Zona Fasciculata StAR

P450scc/FDX1/FDXR 3βHSD2

Pregnenolone

P450c21 POR

11-Deoxycorticosterone

P450c11β FDX1/FDXR

Corticosterone

P450c17 POR

P450c17 POR

3βHSD2

17-Hydroxy pregnenolone

C

Progesterone

17-Hydroxy progesterone

P450c21 POR

11-Deoxycortisol

P450c11β FDX1/FDXR

Cortisol

Cholesterol P450scc FDX1/FDXR

StAR

Pregnenolone P450c17 POR 17-Hydroxypregnenolone

Zona Reticularis

P450c17 POR+b5 3βHSD2

DHEA SULT2A1

PAPS

PAPSS2

Androstenedione

AKR1C3

Testosterone

2 ATP + SO4

DHEAS Figure 87.5  Major steroidogenic pathways of the human adrenal cortex. The conversion of cholesterol to pregnenolone is common to all three zones. A, In the zona glomerulosa, 3βHSD2 converts pregnenolone to progesterone. P450c17 is absent, but P450c21 produces 11-­deoxycorticosterone, which is a substrate for P450c11AS (aldosterone synthase), the enzyme catalyzing 11β-­hydroxylation and two 18-­oxygenations to complete aldosterone synthesis. B, The zona fasciculata expresses P450c17 but little cytochrome b5, so pregnenolone is hydroxylated to 17α-­hydroxypregnenolone but not cleaved to DHEA. Instead, 3βHSD2 yields 17α-­hydroxyprogesterone, the preferred substrate for P450c21, which produces 11-­deoxycortisol. P450c11β, which is unique to the zona fasciculata, completes the synthesis of cortisol. Corticosterone is normally a minor product (dashed arrows) derived from a parallel pathway without the action of P450c17. C, The zona reticularis has high P450c17 and cytochrome b5 but low 3βHSD2, so pregnenolone is sequentially oxidized to 17α-­hydroxypregnenolone and then DHEA. SULT2A1 sulfates DHEA, and DHEAS is exported to the circulation.

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A

Cholesterol P450scc FDX1/FDXR

StAR

Pregnenolone P450c17 POR 17-Hydroxypregnenolone

Testis Leydig Cell

P450c17 POR+b5 3β HSD2

DHEA 17βHSD3

Androstenedione

P450aro POR

Estrone

17βHSD3 AKR1C3 3β HSD2

Androstenediol

Testosterone

P450aro POR

Estradiol

Ovary

B Cholesterol P450scc

StAR

FDX1/FDXR Granulosa Cell

Pregnenolone

P450c17 POR

17-Hydroxypregnenolone P450c17 POR+b5

DHEA

3βHSD2

Androstenedione

P450aro POR

17βHSD1

AKR1C3 Theca Cell

Testosterone

Estrone

P450aro POR

Estradiol

Figure 87.6  Major steroidogenic pathways of the gonads. Both the Leydig cell of the testis and the granulosa cell of the ovary convert cholesterol to pregnenolone, and the Leydig and theca cells have the capacity to synthesize androstenedione and testosterone. The major difference in their enzymatic machinery is abundant 17βHSD3 expression in Leydig cells (A), which efficiently completes testosterone synthesis in the testis, whereas androstenedione is the major C19 product of the theca cell (B). The granulosa cells of the ovary contain abundant aromatase and 17βHSD1, which complete the biosynthesis of estradiol. Minor pathways are shown with dashed arrows.

Cholesterol 5

∆

Pregnenolone P450c17 4

∆ 3 β HSD

17α-hydroxy pregnenolone

5α 5 α R1

17α-hydroxy progesterone

5α, 3α

5α-pregnan17α-ol-3,20-dione

Red. 3 α HSD

5α-pregnan-3α, 17α-diol-20-one P450c17 ± Cyt b5

P450c17 +Cyt b5

Androsterone

Androstenedione

DHEA 3 β HSD

17 β HSD3

17 β HSD3

5 α R2

3 β HSD

Androsta-5ene-3β,17β-diol

17 β HSD3 AKR1Cs

AKR1C3 Ox. 3 α HSD

Testosterone

Androstanediol

DHT

A Cholesterol

Pregnenolone P450c17 POR 17α-hydroxy pregnenolone

3 β HSD

P450c17 POR Cyt b5 DHEA 3 β HSD

17α-hydroxy progesterone P450c17 POR Cyt b5 Androstenedione AKR1C3

Testosterone 5 α R1/2

Dihydrotestosterone

B

P450c11 β FDX1/FDXR

11β-hydroxy androstenedione

11 β HSD2

AKR1C3

AKR1C3 P450c11 β FDX1/FDXR

11β-hydroxy testosterone

11-keto androstenedione

11 β HSD2

5 α R1/2 5α-dihydro-11β-hydroxy testosterone

11-keto testosterone 5 α R1/2 5α-dihydro-11-keto testosterone

Figure 87.7  Additional important pathways in human androgen biosynthesis. A, The two pathways to DHT using the different 17,20-­lyase activities of human P450c17. In the conventional or Δ5-­pathway (solid arrows), the 17,20-­lyase activity of P450c17 requires cytochrome b5 to efficiently convert 17α-­hydroxyprogesterone to DHEA, and testosterone is reduced in target tissues by 5α-­reductase 2 (5αR2) to DHT. In the “backdoor” or 5α,3α-­pathway (broken arrows), 5α-­reduction by 5αR1 and 3α-­reduction of C21 steroids occurs in the steroidogenic tissue prior to the 17,20-­ lyase reaction. In the best characterized pathway, 5α-­pregnane-­3α,17α-­diol-­20-­one is cleaved to androsterone without requiring cytochrome b5 and reduced to 5α-­androstane-­3α,17β-­diol. The 5α-­androstane-­3α,17β-­diol is exported from the testis and metabolized to DHT by oxidative 3αHSDs (Ox. 3αHSD, probably 17βHSD6). Note that testosterone is not an intermediate in the backdoor pathway to DHT, that different isoforms of 5α-­ reductase appear to be involved in the two pathways, and that both reductive and oxidative 3αHSDs are required for the backdoor pathway. B, The 11-­oxygenated androgen pathway. The major substrate is androstenedione, which is 11β-­hydroxylated via P450c11β to 11β-­hydroxyandrostenedione in the adrenal cortex. Primarily in peripheral tissues, the enzyme 11βHSD2 oxidizes this adrenal product to 11-­ketoandrostenedione, which is an excellent substrate for AKR1C3. The 17β-­reduction via AKR1C3 yields 11-­ketotestosterone, a potent androgen, which can be further 5α-­reduced to 5α-­dihydro-­11-­ketotestosterone. Additional minor pathways are also shown. Continued

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3 β HSD1 DHEA

Androstenedione

AKR1C2,3

5 α R1

Androsterone

5α-androstanedione

AKR1C3

AKR1C3

AKR1C2,3

5 α R2 Testosterone

17 β HSD6

5α-dihydrotestosterone

C

AKR1C3

5α-androstane3α,17β-diol

17 β HSD6

Figure 87.7, cont’d  C, DHEA metabolism in castration-­resistant prostate cancer cells. DHEA is converted to androstenedione via 3βHSD1, which is then 5α-­reduced by 5α-­reductase type 1 (5αR1) to 5α-­androstanedione. AKR1C3 completes the pathway to dihydrotestosterone, and AKR1C enzymes plus 17βHSD6 reversibly convert these 5α-­reduced steroids to other side products.

In children with premature adrenarche98 and premature pubarche,192 circulating 11-­ketotestosterone is elevated compared to age-­matched controls and 2-­to 3-­fold higher than testosterone, regardless if DHEAS is elevated. Thus, while DHEAS might be the traditional biomarker of adrenarche, it is 11-­ketotestosterone that is the dominant circulating and biologically active androgen that yields the physical manifestations of adrenarche. Circulating concentrations of both 11β-­hydroxyandrostenedione and 11-­ketotestosterone exceed those of androstenedione and testosterone, respectively, in conditions of adrenal-­derived androgen excess with preserved 11β-­ hydroxylase activity, most prominently undertreated 21-­hydroxylase deficiency.193,194 Note that 11-­oxygenated androgen production is not elevated in 11β-­hydroxylase deficiency or 3β-­hydroxysteroid dehydrogenase deficiency, because 11β-­ hydroxylase activity is low or androstenedione is generated outside the adrenal cortex, respectively. In addition, circulating concentrations of 11β-hydroxyandrostenedione exceed those of androstenedione by at least a factor of two in most men and women,189 whereas 11-­ketotestosterone is the dominant circulating androgen in prepubertal children98 and is slightly higher than testosterone most women.189 Furthermore, while circulating concentrations of DHEAS, androstenedione, and testosterone all decline with age in men and women after early adulthood, those of 11β-­hydroxyandrostenedione and 11-­ketotestosterone are preserved without much change in healthy adults until the ninth decade of life.189 These observations are paralleled during aging by changes in the different zones of the adrenal cortex, with an expansion of the zona fasciculata, despite reduction in zona reticularis and the zona glomerulosa (in men), so allowing preservation of 11β-­hydroxylase activity that may then account for the preservation of 11β-­hydroxyandrostenedione and 11-­ ketotestosterone levels.195 Additional studies demonstrate that the 11-­oxygenated androgens are substrates for the 5α-­reductases (Fig. 87.7B), which amplifies the activity of 11-­ketotestosterone.196 Thus, the 11-­oxyandrogens are not curiosities or minor adrenal side-­ products, but rather important contributors to human physiology in children and women and to disease states, including 21-­hydroxylase deficiency, polycystic ovary syndrome, prostate cancer, and possibly to other conditions related to advanced age.99

Androgen Synthesis in Prostate Cancer The classical paradigms of steroidogenesis reflect the enzymes and pathways present in endocrine glands, which generate enough steroids to maintain circulating concentrations high enough to activate their cognate receptors in various tissues. Much less flux is required for a cell to generate biologically significant amounts of hormones— either from cholesterol or from circulating intermediates—to

activate the receptors in the same tissues, in an autocrine or intracrine manner. Under these circumstances, minor reactions, different enzymes, and modified pathways may dominate. CRPC is an example of this scenario, in which minute amounts of testosterone or dihydrotestosterone are sufficient to fuel disease progression. Most of the cognate mRNAs for the enzymes and proteins necessary to synthesize dihydrotestosterone from cholesterol are upregulated in CRPC metastases relative to primary tumors, and androgen concentrations in the metastases are preserved.197 Given that the abundance of most of the enzymes in the early steps is still quite low, however, it is not clear whether the majority of androgens in CRPC cells derive entirely from de novo synthesis or from metabolism of circulating intermediates.68 Abundant circulating DHEA and DHEAS could serve as the precursor, requiring only two to four steps to testosterone and dihydrotestosterone. The conversion of DHEA to dihydrotestosterone has been well documented in CRPC samples and in prostate cancer cell lines derived from such tumors.198 In these cells, the dominant pathway is oxidation of DHEA to androstenedione via 3βHSD1 (not 3βHSD2), then 5α-­reduction of androstenedione to 5α-­androstanedione via 5α-­reductase type 1 (not type 2), followed by reduction of 5α-­androstanedione to dihydrotestosterone via one or more 17βHSDs (Fig. 87.8C).163 In addition, dihydrotestosterone production via the “backdoor” pathway appears to occur,68 and oxidation of 5α-­androstane-­3α,17β-­ diol to dihydrotestosterone has been demonstrated.199 To complicate matters, AKR1C3, which may catalyze the critical (activating) 17β-­reduction of 17-­ketosteroids to testosterone and dihydrotestosterone, also catalyzes the (inactivating) 3α-­reduction of dihydrotestosterone, as does AKR1C2.199 Consequently, it is difficult to predict how changes in enzyme expression translate to shifts in the delicate balance between intracellular androgen synthesis and degradation. The evolving biology of androgen synthesis in CRPC continues to revise our traditional concept of steroidogenic pathways and to identify additional potential therapeutic targets.

KEY POINTS  • In the adrenal, zone-­specific expression of differing steroidogenic enzymes and receptors to adrenocorticotropic hormone and angiotensin II determine the specific repertoire and regulation of the steroids synthesized. • The human backdoor pathway is relevant to normal physiology and to pathologic states. • 11-­Oxygenated androgens are important in disease states, including 21-­hydroxylase deficiency, polycystic ovary syndrome, prostate cancer, and possibly to other conditions related to advanced age.

CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis

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CHAPTER 87  Enzymes and Pathways of Human Steroidogenesis 170. Hoog SS, Pawlowski JE, Alzari PM, et al. Three-­dimensional structure of rat liver 3α-­hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-­keto reductase superfamily. Proc Natl Acad Sci USA. 1994;91:2517– 2521. 171. Cooper WC, Jin Y, Penning TM. Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate: the example of rat liver 3α-­HSD (AKR1C9). J Biol Chem. 2007;282:33484– 33493. 172. Ratnam K, Ma H, Penning TM. The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3α-­hydroxysteroid dehydrogenase, a representative aldo-­keto reductase. Biochemistry. 1999;38:7856– 7864. 173. Napoli JL. 17β-­Hydroxysteroid dehydrogenase type 9 and other short-­ chain dehydrogenases/reductases that catalyze retinoid, 17β-­and 3α-­ hydroxysteroid metabolism. Mol Cell Endocrinol. 2001;171:103–109. 174. Biswas MG, Russell DW. Expression cloning and characterization of oxidative 17β-­and 3α-­hydroxysteroid dehydrogenases from rat and human prostate. J Biol Chem. 1997;272:15959–15966. 175. Chetyrkin SV, Hu J, Gough WH, et al. Further characterization of human microsomal 3α-­hydroxysteroid dehydrogenase. Arch Biochem Biophys. 2001;386:1–10. 176. White PC, Mune T, Agarwal AK. 11β-­hydroxysteroid dehydrogenase and the syndrome of apparent mineralocorticoid excess. Endocr Rev. 1997;18:135–156. 177. Tannin GM, Agarwal AK, Monder C, et al. The human gene for 11β-­ hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J Biol Chem. 1991;266:16653–16658. 178. Walker EA, Clark AM, Hewison M, et al. Functional expression, characterization, and purification of the catalytic domain of human 11β-­hydroxysteroid dehydrogenase type 1. J Biol Chem. 2001;276:21343– 21350. 179. Odermatt A, Arnold P, Stauffer A, et al. The N-­terminal anchor sequences of 11β-­hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane. J Biol Chem. 1999;274:28762– 28770. 180. Jamieson A, Wallace AM, Andrew R, et al. Apparent cortisone reductase deficiency: a functional defect in 11β-­hydroxysteroid dehydrogenase type 1. J Clin Endocrinol Metab. 1999;84:3570–3574. 181. Lavery GG, Walker EA, Tiganescu A, et al. Steroid biomarkers and genetic studies reveal inactivating mutations in hexose-­6-­phosphate dehydrogenase in patients with cortisone reductase deficiency. J Clin Endocrinol Metab. 2008;93:3827–3832. 182. Brown RW, Chapman KE, Edwards CRW, et al. Human placental 11β-­ hydroxysteroid dehydrogenase: evidence for and partial purification of a distinct NAD+-­dependent isoform. Endocrinology. 1993;132:2614–2621. 183. Funder JW, Pearce PT, Smith R, et al. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science. 1988;242:583–585. 184. Mune T, Rogerson FM, Nikkila H, et al. Human hypertension caused by mutations in the kidney isozyme of 11β-­hydroxysteroid dehydrogenase. Nat Genet. 1995;10:394–399.

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185. Seely J, Amigh KS, Suzuki T, et al. Transcriptional regulation of dehydroepiandrosterone sulfotransferase (SULT2A1) by estrogen-­related receptor alpha. Endocrinology. 2005;146:3605–3613. 186. Noordam C, Dhir V, McNelis JC, et al. Inactivating PAPSS2 mutations in a patient with premature pubarche. N Engl J Med. 2009;360:2310–2318. 187. Mulatero P, Curnow KM, Aupetit-­Faisant B, et al. Recombinant CYP11B genes encode enzymes that can catalyze conversion of 11-­deoxycortisol to cortisol, 18-­hydroxycortisol, and 18-­oxocortisol. J Clin Endocrinol Metab. 1998;83:3996–4001. 188. Homma K, Hasegawa T, Nagai T, et al. Urine steroid hormone profile analysis in cytochrome P450 oxidoreductase deficiency: implication for the backdoor pathway to dihydrotestosterone. J Clin Endocrinol Metab. 2006;91:2643–2649. 189. Davio A, Woolcock H, Nanba AT, et al. Sex differences in 11-­oxygenated androgen patterns across adulthood. J Clin Endocrinol Metab. 2020;105:e2921–e2929. 190. Gent R, du Toit T, Bloem LM, et al. The 11β-­hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-­ oxy C19 steroids with 11βHSD2 favouring 11-­ketotestosterone, 11-­ketoandrostenedione and 11-­ketoprogesterone biosynthesis. J Steroid Biochem Mol Biol. 2019;189:116–126. 191. Barnard M, Quanson JL, Mostaghel E, et al. 11-­Oxygenated androgen precursors are the preferred substrates for aldo-­keto reductase 1C3 (AKR1C3): implications for castration resistant prostate cancer. J Steroid Biochem Mol Biol. 2018;183:192–201. 192. Wise-­Oringer BK, Burghard AC, O’Day P, et al. The unique role of 11-­oxygenated C19 steroids in both premature adrenarche and premature pubarche. Horm Res Paediatr. 2020;93:460–469. 193. Turcu AF, El-­Maouche D, Zhao L, et al. Androgen excess and diagnostic steroid biomarkers for nonclassic 21-­hydroxylase deficiency without cosyntropin stimulation. Eur J Endocrinol. 2020;183:63–71. 194. Turcu AF, Nanba AT, Chomic R, et al. Adrenal-­derived 11-­oxygenated 19-­carbon steroids are the dominant androgens in classic 21-­hydroxylase deficiency. Eur J Endocrinol. 2016;174:601–609. 195. Tezuka Y, Atsumi N, Blinder AR, et al. The age-­dependent changes of the human adrenal cortical zones are not congruent. J Clin Endocrinol Metab. 2021;106:1389–1397. 196. Storbeck KH, Bloem LM, Africander D, et al. 11β-­ Hydroxydihydrotestosterone and 11-­ketodihydrotestosterone, novel C19 steroids with androgenic activity: a putative role in castration resistant prostate cancer? Mol Cell Endocrinol. 2013;377:135–146. 197. Montgomery RB, Mostaghel EA, Vessella R, et al. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-­resistant tumor growth. Cancer Res. 2008;68:4447–4454. 198. Evaul K, Li R, Papari-­Zareei M, et al. 3β-­hydroxysteroid dehydrogenase is a possible pharmacological target in the treatment of castration-­ resistant prostate cancer. Endocrinology. 2010;151:3514–3520. 199. Bauman DR, Steckelbroeck S, Williams MV, et al. Identification of the major oxidative 3α-­hydroxysteroid dehydrogenase in human prostate that converts 5α-­androstane-­3α,17β-­diol to 5α-­dihydrotestosterone: a potential therapeutic target for androgen-­dependent disease. Mol Endocrinol. 2006;20:444–458.

88 Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance Morag J. Young and Peter J. Fuller

OUTLINE Feedback Control of Aldosterone Secretion, 1474 Sodium Homeostasis, 1475 Potassium Homeostasis, 1476 Specificity-­Conferring Enzymes, 1476 11β-­Hydroxysteroid Dehydrogenase Type 2, 1476 Mineralocorticoid Receptors, 1476 Evolution, 1476 Structure, 1477 Genomic Versus Nongenomic Aldosterone Actions, 1478 Cellular Mechanisms of Aldosterone Action, 1478

Sodium Transport, 1478 Epithelial Sodium Channels, 1478 Na+/K+-­ATPase, 1479 Potassium Transport, 1479 Hydrogen Ion Transport, 1480 Nonepithelial Tissues, 1480 Cardiovascular System, 1480 Central Nervous System, 1481 Summary and Future Directions, 1481

 

The steroid hormone aldosterone first appeared in vertebrate evolution with the emergence of terrestrial life and the consequent need to conserve sodium and water.1 The primary and best-­characterized actions of aldosterone are those that stimulate sodium retention in transporting epithelia, particularly the distal nephron, distal colon, and salivary glands.1 In these epithelia, the conservation of sodium is associated with an increased secretion of both potassium and hydrogen ions. Aldosterone also has actions in the cardiovascular system, in inflammatory cells, in adipose tissue, and in the central nervous system (CNS). The existence of an adrenocorticoid natriuretic factor, distinct from the other adrenal steroid hormones, had been suspected for several years before its isolation by Simpson and colleagues in 1953.2 Using the toad urinary bladder as a model system, Crabbé3 was the first to show that aldosterone increases sodium transport in vitro. Subsequent studies demonstrated the presence of binding sites for aldosterone in the toad bladder and in other target tissues, particularly the principal cells of the cortical collecting duct of the kidney.4

FEEDBACK CONTROL OF ALDOSTERONE SECRETION Both serum sodium concentration and total body sodium are maintained within a narrow range by a complex set of endocrine feedback loops (Fig. 88.1). The most important of these involves the renin-­ angiotensin-­aldosterone system (RAAS), which responds to volume status. The feedback loops involved with potassium homeostasis operate in parallel and overlap those for sodium. The renin-­secreting juxtaglomerular cells of the kidney sense volume status. When the sodium status (and hence, intravascular volume) is low, renin will be secreted. Renin is an aspartyl protease that is synthesized as inactive prorenin and subsequently activated by the action of a protease. Renin release from the juxtaglomerular cells is influenced by several factors (Table 88.1). Stimulatory

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regulators include renal perfusion pressure, sympathetic nervous system, and prostaglandins, while inhibitory factors include dopamine, atrial natriuretic peptide (ANP) and angiotensin II (AngII). Renin cleaves angiotensinogen to release the decapeptide angiotensin I, which in turn is subject to further proteolysis by angiotensin-­ converting enzyme 1, primarily in the pulmonary vascular bed, to yield the octapeptide AngII. Ang II acts via its specific G protein–coupled receptor, the type 1 AngII receptor (AT1R), in the vasculature as a potent vasoconstrictor (thereby defending plasma volume and blood pressure) and on the adrenal glomerulosa cells to stimulate aldosterone synthesis.5 The latter response promotes sodium retention, with a consequent increase in intravascular volume. Aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex is regulated by transcription of the aldosterone synthase gene (CYP11B2), which encodes the last and rate-­limiting step in aldosterone biosynthesis (see Chapter 87). Although AngII is important in the regulation of aldosterone synthase activity, a response to low-­salt or high-­potassium diets is also seen in mice in which the angiotensinogen gene has been deleted.6 In these mice, the regulation of aldosterone is primarily directed by serum potassium levels. Both AT1R and potassium interact with potassium channels in the glomerulosa cell membrane. These channels maintain the hyperpolarization of the cell membrane; depolarization of the membrane through changes in ion flux through these channels results in an increased calcium flux, with consequent stimulation of aldosterone secretion. Experimental and clinical studies have identified channels involved in this coupling, including TWIK-­related acid sensitive channels, GIRK4 (G protein–activated inward rectifier potassium channel 4), an inward-­rectifying potassium channel encoded by the KCNJ5 gene, the α1 subunit of Na+/K+-­ATPase encoded by the ATP1A1 gene, PMCA3 (plasma membrane Ca2+-­ ATPase type 3) encoded by the ATP2B3 gene, Cav1.3 (a voltage-­ dependent calcium channel) encoded by the CACNAID gene, and

CHAPTER 88  Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance

Glomerulus Angiotensinogen Renin A-I

Na reabsorption

Adrenal

Aldosterone K excretion K

Cortical collecting duct Fig. 88.1  Interacting feedback loops controlling aldosterone secretion. Volume is regulated through the renin-­angiotensin system, and potassium is regulated through direct feedback. A-­I, Angiotensin I; A-­II, angiotensin II; BP, blood pressure; Na, sodium; K, potassium.

TABLE 88.1  Factors Regulating Renin

Release

Stimulatory

Inhibitory

Decreased perfusion pressure PGl2 ACTH

Increased chloride delivery at the macula densa Angiotensin II Atrial natriuretic factor Vasopressin α-­adrenergic stimulation Dopamine

β-­adrenergic stimulation

TABLE 88.2  Factors Regulating

Aldosterone Secretion Factor

Stimulatory

Inhibitory

Peptides

Angiotensin II Angiotensin III ACTH Vasopressin Endothelin Plasma potassium Serotonin Substance P

Atrial natriuretic peptide Somatostatin

Converting enzyme A-II

Volume BP

ACTH, Adrenocorticotropic hormone; PGl2, prostacyclin.

ClC-­2 (chloride channel protein 2) encoded by the CLCN2 gene.7 Somatic mutations in the KCNJ5, ATP1A1, ATP2B3, or CACNAID genes are found in approximately 88% of aldosterone-­producing adenomas,7 as discussed in Chapter 96. The secretion of aldosterone stimulated by potassium forms one arm of a negative feedback loop for potassium and aldosterone that this independent of AngII levels. Small increases in extracellular potassium levels stimulate calcium influx via depolarization of the glomerulosa cell plasma membrane. Plasma potassium levels also alter the sensitivity of the adrenal to AngII. Aldosterone also affects acid–base balance by increasing the exchange of hydrogen ions for sodium. Therefore, the net effect of an increase in aldosterone, as may result from an aldosterone-­producing tumor or exogenous mineralocorticoid administration (e.g., 9α-­ fludrocortisone), is sodium resorption, with consequent volume expansion, hypertension, and suppression of plasma renin activity, hypokalemia, and metabolic alkalosis (Chapter 96). The third principal regulator of aldosterone synthesis is adrenocorticotrophic hormone (ACTH), whose primary role is the regulation of cortisol synthesis in the adrenal fasciculata in the feedback loop between the pituitary corticotropes and the adrenal cortex, the hypothalamo-­pituitary-­adrenal axis (Chapter 85). ACTH acutely increases aldosterone synthesis, but chronic ACTH exposure results in a decrease in the levels of CYP11B2, and hence aldosterone synthesis8;

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Ions Other

Dopamine Ouabain

ACTH, Adrenocorticotropic hormone.

however, regulation of aldosterone synthesis is normal in patients with hypopituitarism.9 Other substances that have been shown to regulate aldosterone secretion are shown in Table 88.2. For example, intraadrenal regulation of aldosterone secretion has been proposed for substance P– positive nerve fibers. In the adrenal zona glomerulosa, a stimulatory tone for aldosterone secretion maintained by substance P acting via the neurokinin type 1 receptor on zona glomerulosa cells.10 Serotonin is exclusively synthesized and produced by adrenal subcapsular mast cells; it stimulates aldosterone secretion through a paracrine mechanism involving the serotonin type 4 receptor.11 The physiological and pathophysiological significance of these local regulators of aldosterone secretion is the subject of ongoing investigation. Aldosterone secretion is also subject to negative regulation. ANP is a potent inhibitor of aldosterone secretion, consistent with its role in promoting natriuresis. Dopamine is also a well-­characterized inhibitor of aldosterone secretion. Other inhibitors have been described, but their physiologic relevance is even less clear. KEY POINTS  • Aldosterone synthesis/secretion is primarily regulated by angiotensin II and potassium in two parallel feedback loops.

SODIUM HOMEOSTASIS Under normal physiological conditions, over 99% of the sodium filtered through the glomerulus is reabsorbed along the nephron: approximately 70% by the proximal tubules, 20% to 30% by the thick ascending limb of Henle, 5% to7% by the distal convoluted tubules (DCTs), and finally by the aldosterone-­sensitive connecting tubules (and cortical collecting ducts.12 As the final arbiter of sodium excretion, these aldosterone-­sensitive segments are thus pivotal. The RAAS contributes to an integrated homeostatic response to fluctuations in dietary sodium intake.13 Sodium deficiency increases adrenal sensitivity to AngII through time; the converse is true of the vasopressor response. Aldosterone-­induced sodium retention restores intravascular volume status by maintaining the balance between volume and vascular capacity.12,13 The response of the individual to aldosterone-­mediated sodium retention is self-­limiting, in that, after 3 to 4 days, the expansion of extracellular volume plateaus and sodium secretion returns to control levels. This process is termed escape.14 It should be noted that the kaliuretic effect persists despite the escape of sodium retention. Intrarenal regulators, particularly prostaglandins, are probably the critical mediators of the escape, although other factors (e.g., ANP) may play a role.15 Other epithelia, particularly the distal colon, contribute to aldosterone-­mediated sodium homeostasis through mechanisms that

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PART 7  Adrenal

parallel those in the distal nephron. In addition, nuclei within the brain contribute to the regulation of salt appetite.16 A local RAAS has been reported to operate in a number of tissues, although the relative physiologic versus pathophysiological importance of these local systems remains unresolved.5

POTASSIUM HOMEOSTASIS Aldosterone is primarily involved in the chronic regulation of plasma potassium levels.17 Acute regulation involves nonrenal mechanisms such as those mediated by insulin and β-­adrenergic agonists. Aldosterone regulates potassium homeostasis through direct effects on transporting epithelia. Potassium flux occurs in the principal cells of the collecting duct in response to aldosterone-­mediated sodium reabsorption, with the resulting electrochemical gradient favoring potassium excretion at the basolateral membrane.17a Aldosterone also regulates potassium transport in the distal nephron independent of the sodium flux. The collecting duct contains intercalated cells, which have well-­recognized roles in potassium and acid–base homeostasis.18

SPECIFICITY-­CONFERRING ENZYMES Prereceptor metabolism (activation or inactivation) is seen for several steroid hormones, including the corticosteroids. The physiological glucocorticoid, cortisol (corticosterone in rodents), is synthesized in the adrenal fasciculata, and also in the periphery, particularly the liver, by the conversion of cortisone (11-­dehydrocorticosterone in rodents) to the active glucocorticoid by the enzyme 11β-­hydroxysteroid dehydrogenase type 1 (11β-­HSD1), a member of the short-­chain dehydrogenase/reductase family of microsomal enzymes. The reverse reaction, mediated by the 11β-­HSD type 2 isoform (11β-­HSD2), mediates the conversion of cortisol to the inactive metabolite, cortisone, thereby enabling the mineralocorticoid actions of aldosterone.19

11β-­Hydroxysteroid Dehydrogenase Type 2 Although the affinity of the mineralocorticoid receptor in vitro20 is equivalent for aldosterone, corticosterone, and cortisol, in vivo cortisol is excluded from such receptors in the kidney, parotid, and colon (but not in the hippocampus).19 This reflects the activity of 11β-­HSD2, which is responsible for the conversion of cortisol to its inactive metabolite, cortisone. In the kidney, this is reflected in the reduced cortisol:cortisone ratio in human renal venous blood. Unlike 11β-­HSD1, 11β-­HSD2 colocalizes with MR in renal distal tubular elements, colon, sweat, and salivary glands; it is also expressed at high abundance in the placenta and in select nuclei in the rat brain.16 It has a low Km (i.e., high affinity) for both corticosterone (∼5 nM) and cortisol (∼50 nM), unlike the micromolar Km of 11β-­HSD1; in vivo, it appears operationally unidirectional, acting uniquely as a dehydrogenase, whereas 11β-­HSD1 acts predominantly as a reductase.19 Deletion of the gene encoding 11β-­HSD2 in mice yields a phenotype19 consistent with the clinical syndrome of apparent mineralocorticoid excess.21,22 Liquorice ingestion has long been known to cause sodium retention, hypokalemia, and hypertension; the mechanism of its action was elucidated by elegant studies23,24 in human volunteers, in whom ingestion of 250 g of liquorice per day for 10 days produced a clinical picture equivalent to a mild form of apparent mineralocorticoid excess. When rats were administered glycyrrhetinic acid (the active principal of liquorice) or carbenoxolone (glycyrrhetinic acid hemisuccinate)25 to block 11β-­HSD, the normal aldosterone selectivity of epithelial MR was abolished. These studies demonstrated the crucial specificity-­ conferring role in mineralocorticoid target tissues for 11β-­HSD2. Aldosterone is not a substrate for 11β-­HSD2, because its 11β-­OH

group is stabilized as an 11,18-­hemiketal by cyclization with the highly reactive aldehyde group unique to aldosterone at C18. KEY POINTS  • Aldosterone selectivity of the MR in epithelial tissues is conferred by 11β-hydroxysteroid dehydrogenase type II.

MINERALOCORTICOID RECEPTORS The classic actions of aldosterone in the distal nephron and distal colon involve epithelial cells that mediate sodium flux. As with other steroid hormones, the principal mode of action, at least in sodium transport, involves an intracellular receptor that, when activated by ligand binding, regulates gene transcription, a so-­called genomic mechanism of action. High-­affinity cytosol and nuclear binding of 3H-­aldosterone was first described in classic mineralocorticoid target tissues such as kidney and parotid gland.4 Spironolactone was shown to block aldosterone binding and action on urinary electrolytes in parallel,26 providing evidence that these sites are physiologic MRs. In contrast to glucocorticoid receptors (GRs), which are expressed ubiquitously, MRs have a tissue-­specific pattern of expression, with the highest levels observed in the distal nephron,20,27 distal colon,28 and hippocampus.20 Lower levels of expression are observed elsewhere in the gastrointestinal tract, in cardiovascular tissues, in regions of the brain, and in a range of other tissues that are both epithelial and nonepithelial.20,27,28 In these nonepithelial tissues, a physiologic effect of MR activation on sodium homeostasis is unlikely (e.g., hippocampus). The MR is unique among the steroid hormone receptors in that it is a receptor for two physiologic agonists, aldosterone and cortisol; indeed, the MR has a higher affinity for cortisol than does the GR.20 Progesterone is a physiological competitive antagonist of the human MR.29 KEY POINTS  • Aldosterone activates the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily.

Evolution The MR is found only in vertebrates; it diverged with the GR as a gene duplication from the ancestral corticoid receptor gene over 450 million years ago.30 This ancestral corticoid receptor first appeared in cartilaginous fish and is found in lamprey and hagfish (jawless fish). Aldosterone first appeared as an active steroid in amphibians. It has been postulated that the MR originated in fish to regulate ion balance under the control of glucocorticoids, and that amphibians evolved mineralocorticoids to appropriate the MR for this function. In most tetrapods (including human, rodent, alligator, and Xenopus) progesterone is an antagonist of MR, as is its pharmacologic derivative spironolactone.29 Although the MR is present in teleost fish that lack aldosterone synthesis, the teleost MR responds to aldosterone, as well as to cortisol and 11-­deoxycorticosterone, consistent with the MR being highly conserved throughout vertebrate evolution.29,31 Characterization of various fish MRs found that, in contrast to terrestrial vertebrate, teleost MRs were activated by both progesterone and spironolactone.29 These findings suggest that progesterone may be a physiological agonist for the MR in teleosts. Curiously, this evolutionary switch of the MR response to progesterone from agonist to antagonist reflects a single amino acid change in the MR31 that occurred at the time of the appearance of aldosterone synthesis in terrestrial vertebrates. The physiological significance of the MR as a progesterone receptor (PR), or perhaps as a receptor for a progesterone metabolite, has not been fully characterized.32

CHAPTER 88  Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance

LBD

NTD

DBD N terminal domain 1

DBD 602 670 734

LBD 984

Fig. 88.2  Domain structure of the mineralocorticoid receptor showing the three principal functional domains, including the N-­terminal domain (NTD), the DNA-­binding domain (DBD), and the ligand-­ binding domain (LBD). A predicted tertiary structure is shown above the linear representation.

KEY POINTS  • The MR is unique, being the receptor for two physiologic ligands: aldosterone and cortisol.

Structure The human MR is a protein of 984 amino acids,20 which, with the GR, PR, and androgen receptor (AR), forms a distinct subfamily within the steroid/thyroid/retinoid/orphan receptor superfamily. A central cysteine-­rich DNA-­binding domain (DBD) defines this receptor superfamily. At the C-­terminus is the ligand-­binding domain (LBD), which has a highly conserved tertiary structure. The N-­ terminal domain has little or no homology between receptors. Within the MR/GR/PR/AR subfamily, MR and GR are closely related, with 94% amino-­acid identity in the central cysteine-­rich DBD and 57% identity in the C-­terminal LBD (Fig. 88.2). The genes encoding MR and GR are, however, located on different chromosomes (MR on 4q31.2; GR on 5q31).33,34 The cysteine residues of the DBD complex around two zinc atoms to form two α-­helices, one of which lies in the major groove and binds with a common consensus sequence in the DNA, the hormone response element (HRE).35 This element is an inverted palindrome that appears to be similar, if not identical, to the well-­ characterized GR response element (GRE). Several groups have sought to characterize the genome-­wide distribution of the HRE for the MR, the MR cistrome, using chromatin immunoprecipitation with massive parallel sequencing, with differing outcomes. A study using a transformed human renal cell line identified 974 MR binding sites, the majority of which, somewhat surprisingly, did not include canonical GRE but did contain recognition motifs for other transcription factors,36 whereas in a murine epithelial cell line 1113 binding sites were identified, with many containing the consensus GRE sequence.37A total of 918 unique MR binding sites were identified in rat hippocampus that included the

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well-­characterized canonical GRE sequence; the MR-­specific binding sites included a motif known to bind the NeuroD family of transcription factors.38 The LBD consists of 11 α-­helices (designated helix 1 to 12 based on the estrogen receptor structure: helix 2 is unstructured in the MR) that form a three-­layered antiparallel structure with the ligand-­binding pocket buried in the lower third of the struture.39-­41 The N-­terminal domain, by contrast, is relatively unstructured, allowing a diversity of interactions.42 The N-­terminal domains are not conserved between steroid receptors, although in several (including the MR) a functional interaction has been described between the N-­and C-­termini.43 The unliganded receptor is predominantly cytoplasmic, being complexed with the heat shock proteins 70 and 90 and their cochaperones.44 This configuration maintains the receptor in a transcriptionally inactive high-­affinity binding state. The interaction of the LBD with this complex is an important determinant of ligand-­binding affinity and specificity. The MR antagonists spironolactone and eplerenone appear to be accommodated into the ligand-­binding pocket without distortion, suggesting that the mechanism of their antagonism differs from that of the estrogen receptor antagonists such as tamoxifen and raloxifene.40 In nonepithelial tissues, the response of MR to cortisol/ corticosterone and aldosterone is often not equivalent, leading to speculation about the relationship of epithelial to nonepithelial MR. Most evidence to date would suggest that, although transcription of the gene encoding MR is activated through multiple, tissue-­specific promoters,45 the coding region is unaltered between tissues, with the possible exception of some minor isoforms.46 The explanation for such differences in MR target gene activation by the various ligands may lie in the nature of the conformation that the MR adopts after ligand binding.47 Such conformational differences may alter some, but not all, transactivation functions, such that tissue-­specific receptor coactivators or corepressors may mediate different responses in different tissues. In contrast to the other steroid receptors, such interactions are only now being characterized for the MR. Several MR coactivator molecules have now been described, including some with ligand specificity.48 The interaction of these coregulators with the LBD, as with other nuclear receptors, involves a surface pocket that interacts with an LxxLL motif (where L is leucine and x is any amino acid) in the coactivator molecule.41 Other factors also appear to modulate MR signaling, including Rac1 (Ras-­related C3 botulinum toxin substrate1), a small RhoGTPase, which may play a role in regulating nuclear translocation of transcription factors and has been reported to activate MR through both ligand-­ dependent and ligand-­independent mechanisms.49 In addition to its effects on ion flux in classic mineralocorticoid target tissues, aldosterone has been shown act via the MR to mediate a range of responses, unrelated to sodium homeostasis, in other tissues. Aldosterone elevates blood pressure in the rat when infused into the cerebral ventricles50; this effect is clearly via MR unprotected by11β-­ HSD2, because it is blocked by simultaneous infusion of low doses of corticosterone. Thus, corticosterone in the anteroventral third ventricle region acts as an aldosterone antagonist on MR, in contrast with the kidney and other epithelia, where its action is to mimic aldosterone. An additional difference between epithelial and nonepithelial tissues is that, in the former, activation of GR has been shown to mimic that of MR, whereas in nonepithelial tissues this is clearly not the case. Mice homozygous for inactivating mutations in the gene encoding MR51 (MR knockout; MRKO) showed classic features of aldosterone deficiency—salt wasting, hyperkalemia, and dehydration—but had marked hyperaldosteronism; these features are also seen in the syndrome of pseudohypoaldosteronism (PHA) type 1. In patients with PHA1, neonatal salt wasting results in hypovolemia with high renin and aldosterone

PART 7  Adrenal

Genomic Versus Nongenomic Aldosterone Actions As with other steroid hormone receptors, the principal mode of action involves the ligand-­activated MR binding to specific responses elements in the genes targeted for transcriptional control. Mice homozygous for an inactivating mutation in the DBD of the MR phenocopy MRKO mice, demonstrating the essential role of DNA binding for MR signaling, at least in sodium-­transporting epithelial tissues.52 For several other steroid hormone receptors there is an alternate mode of action, again involving regulation of gene expression, but where the steroid receptor interacts with other transcription factors, including other nuclear receptors, to modulate their signaling, a mechanism described as transrepression or tethering.53 The relevance of this mode of signaling to the MR remains to be clearly defined. There has also been considerable interest with respect to steroid hormone action as to whether all responses are mediated through the classic nuclear receptor with direct regulation of gene expression, or whether other pathways involving novel cell membrane receptors exist. The evidence for novel receptors is focused on the seven-­ transmembrane G protein–coupled receptor GRP30, also known as the G protein estrogen receptor.54 There is clear evidence both in vitro and in vivo for rapid, nongenomic signaling. This can involve activation of intracellular kinases such as Src kinase associated with the epidermal growth factor receptor, with consequent downstream signaling through the MAP kinase pathway; the signaling appears to require only the LBD of the MR.55 Rapid effects of aldosterone have also been reported to be associated both with protein kinase signaling56 in isolated cell patches from cardiomyocytes57 and in vascular cells58 and with glutamate release from the pyramidal neurons of the hippocampus.59 In each case the receptor involved is the classic MR. The relative contribution of this signaling to the mineralocorticoid response has not yet been evaluated, although it is speculated that these rapid responses may prime the transcriptional response or alter the dynamic range of the response.

CELLULAR MECHANISMS OF ALDOSTERONE ACTION As the term mineralocorticoid implies, the classic effects of aldosterone reflect its role in the regulation of electrolyte flux across transporting epithelia. The molecular basis of the regulation of sodium transport in the distal nephrons is relatively well-­characterized. The molecular mechanisms that mediate the more recently defined roles of aldosterone in the central regulation of blood pressure,50 in salt appetite,16 and in the pathogenesis of cardiac fibrosis60 are less well understood. Emerging evidence points strongly to a proinflammatory, profibrotic effect of MR activation in cardiovascular tissues consistent with the clinical observation that hypertension secondary to hyperaldosteronism is associated with worse cardiovascular outcomes than idiopathic hypertension.60

Sodium Transport Aldosterone increases transepithelial sodium flux in a number of target tissues, of which the amphibian bladder, the mammalian distal nephron, and the mammalian distal colon are the best characterized.1 The temporal pattern of response comprises a lag period (latent phase) of 30 to 60 minutes, followed by an early phase in

Apical

Basal

Spironolactone

Aldo

levels, hyperkalemia, and resistance to exogenous mineralocorticoids (9α-­fludrocortisone). MRKO mice are born at the expected frequency from heterozygote mating; untreated, they begin to deteriorate from day 5, and die between day 8 and 11; salt supplementation allows survival and growth. Mutations of the gene encoding MR have also been reported in the autosomal dominant form of PHA,52 which thus appears to be equivalent to mice heterozygous for the MR gene knockout.

Aldo

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Aldo

MR MR ENaC αβγ

AIP

Amiloride

K+

CHIF

Na.K-ATPase

Na+

Sgk- P Nedd4-2 Degradation

Na+

Sgk PI3-K

?

Ras

Fig. 88.3  Aldosterone (Aldo) acts on epithelial cells of the distal nephron and the distal colon via the mineralocorticoid receptor to induce aldosterone-­induced proteins (AIPs). ENaC, Epithelial sodium channels; sgk, serum and glucocorticoid-­induced kinase; CHIF, channel-­ inducing factor; PI3-­K, phosphoinositide 3-­ kinase; Na+, sodium; K+, potassium.

which preexisting pumps and channels are activated, followed by a so-­called late phase.61 This late phase starts 3 to 6 hours after steroid exposure and is characterized by an increase in the number of pumps and channels; with longer exposure, morphologic as well as functional changes are observed.62 The latent phase is consistent with the concept that the effects of aldosterone are primarily genomic, with the genes regulated by the MR either mediating sodium transport per se or modulating components of the transport pathway. In the classic model of aldosterone-­induced transport, sodium entry at the apical membrane is through an amiloride-­sensitive electrogenic sodium channel, with efflux at the basolateral membrane via the sodium pump, with adenosine triphosphate (ATP) generation needed to drive the process (Fig. 88.3).1 KEY POINTS  • Aldosterone-induced epithelial sodium transport involves the epithelial sodium channel at the apical membrane and Na+/K+-ATPase at the basolateral membrane.

Epithelial Sodium Channels Although a variety of amiloride-­sensitive sodium channels have been identified in epithelia, only one has been clearly shown to be directly involved in aldosterone-­dependent sodium transport. The epithelial sodium channel (ENaC) genes encode three homologous subunits (α, β, and γ).63 Each subunit consists of two transmembrane domains with intracellular N-­and C-­termini. When the α subunit is expressed in Xenopus oocytes, a weak amiloride-­sensitive sodium flux can be demonstrated; expression of the other subunits alone is without activity, and coexpression of all three subunits is required for maximal amiloride-­sensitive sodium transport.63 The subunits exhibit a heterotrimeric complex of the three subunits, α, β, and γ, arranged in a counter-­clockwise manner.64 The genes for the ENaC subunits are members of the degenerin (DEG)/ENaC superfamily of sodium channels; the DEGs mediate mechanosensory transduction in Caenorhabditis elegans.63 The ENaCs are unique among the DEG/ENaC channels in being activated by proteolysis of the extracellular domain to release inhibitory peptides. These serine proteases act synergistically with aldosterone to increase channel opening probability.63

CHAPTER 88  Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance Insights into the structural determinants of function of the ENaC subunits has come from a combination of both naturally occurring and engineered mutations. Deletion of the αENaC gene in transgenic mice results in early neonatal death caused by defective clearance of lung liquid66; partial genetic restoration of αENaC expression in these mice results in a phenotype similar to that seen in PHA. Mice with the βENaC or γENaC gene deleted show a less severe pulmonary phenotype but die between 24 and 36 hours with salt wasting and profound hyperkalaemia, again reminiscent of PHA67,68; low levels of βENaC subunit gene expression in mice in which the βENaC gene has been disrupted also result in a milder phenotype analogous to PHA.69 Autosomal recessive PHA is indeed associated with inactivating mutations of genes encoding all three of the ENaC subunits.70 The molecular characterization of Liddle syndrome71 also provided important insights into the function of the C-­ terminal intracellular domain and emphasized the central role of the channel in amiloride-­ sensitive sodium transport. Liddle syndrome, or pseudoaldosteronism, has the reciprocal pathophysiology to PHA,71 with apparent aldosterone excess (i.e., hypertension, hypokalemia, and suppressed plasma renin activity) but low aldosterone levels, and no response to spironolactone but a response to amiloride. A number of studies have identified mutations in either the β-­ or γENaC genes. The mutations are either nonsense or missense mutations localized to the C-­terminus; the latter are particularly informative, because they identify a key motif, proline-­proline-­proline-­X-­ tyrosine (PY), which is conserved across the subunits and is disrupted in all cases of Liddle syndrome.72 ENaC is a relatively short-­lived protein that is ubiquitinated on residues in the N-­terminus of the α and γ but not the β subunits; the PY motif interacts with Nedd4-­2, a ubiquitin protein-­ligase whose role is to target the channels for proteosomal degradation.73 These studies demonstrated the central role of the ENaC in mediating aldosterone-­induced epithelial sodium transport. ENaC subunit gene expression is regulated by aldosterone.1 Both β-­ and γENaC subunit mRNA levels are increased in the colon by sodium depletion and by dexamethasone or aldosterone treatment; in the kidney, however, levels remained unaltered, although when separate regions of the kidney were distinguished, an increase in αENaC mRNA levels was seen in the inner medulla. Cultured medullary collecting duct cells show increased α-­ but not β-­ or γENaC mRNA levels after 3 hours of aldosterone exposure; in cultured cortical collecting duct cells, a γENaC subunit response to aldosterone required 24 hours of treatment.74 Although it is clear from the aforementioned studies that aldosterone can induce ENaC synthesis (at least in the late phase), a transcriptional effect of the MR in the early phase (i.e., a primary response) has not clearly been demonstrated. Aldosterone treatment in vivo rapidly increases the levels of the serine threonine kinase serum and glucocorticoid-­regulated kinase (Sgk) in the kidney and colon, with a time course consistent with an effect of aldosterone on transcription.75,76 Sgk directly interacts with Nedd4-­2 to block its binding with ENaC and, as a consequence, slows ENaC degradation.77,78 Regulation of αENaC subunit gene expression involves Sgk-­1 through relief of Dotla-­Af9-­mediated transcriptional repression.79 Nedd4-­2 is also regulated by Usp2-­45, a deubiquitinylation enzyme that is itself regulated by aldosterone.80 Sgk requires phosphorylation by the phosphatidylinosital 3-­kinase (PI3 kinase) pathway for full activity. This may be a point at which the signaling of nuclear receptors and membrane-­associated receptors such as the insulin receptor are integrated.81 PI3 kinase may be activated by small monomeric G proteins, including Ras. K-­ras 2A has been identified as an aldosterone-­induced gene in the rodent distal colon.82 The glucocorticoid-­induced leucine zipper protein is also aldosterone-­ induced; it interacts with and inhibits Raf-­1, leading to repression of ERK signaling, a negative regulator of ENaC, as well as directly interacting with Sgk and Nedd 4-­2.83 A further aldosterone-­induced protein, connector

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enhancer of kinase repressor of Ras3, appears to serve as a scaffold protein in the assembly of the ENaC regulatory complex.74

Na+/K+-­ATPase Active extrusion of sodium from the cell reflects sodium pump activity in the basolateral cell membrane. Na+/K+-­ATPase activity is increased in epithelia by aldosterone. In toad bladder and in A6 cells, there is evidence that Na+/K+-­ATPase α and β subunit gene expression is significantly increased in the early phase of the response to aldosterone,61 although this does not appear to be the case in mammalian systems.84 Na+/K+-­ATPase activity is sensitive to intracellular sodium concentration, and in isolated cortical tubules the early Na+/K+-­ATPase response to aldosterone is blocked by amiloride, suggesting that the increased activity is secondary to sodium influx at the apical membrane.85 In the late phase of the aldosterone response, Na+/K+-­ATPase mRNA, protein, and activity levels are all increased.86 Channel-­inducing factor (CHIF) was first identified as a novel corticosteroid-­induced gene in the rat distal colon. CHIF is a member of the FXYD family of small transmembrane proteins, which includes the γ-­subunit of Na+/K+-­ ATPase.87 Under experimental conditions, CHIF is upregulated in the distal colon in response to aldosterone88,89 through a primary transcriptional response. CHIF increases the affinity of Na+/K+-­ATPase for sodium.87 The early aldosterone-­induced increase in Na+/K+-­ATPase activity is mediated by CHIF.

Potassium Transport Aldosterone also regulates potassium transport, independent of its effects on the electroneutral apical sodium transport coupled to the Na+/K+-­ATPase-­mediated basolateral membrane exchange of sodium for potassium. Aldosterone also increases the apical membrane channel density of renal outer medullary potassium channel (ROMK), an ATP-­regulated potassium channel with weak inward rectification in the principal cells of the cortical collecting duct.90 Sgk-­1 has been reported to increase the channel density of ROMK through direct phosphorylation, through Nedd4-­2,91 or perhaps via the with-­no-­lysine (WNK) kinases.92 Insights into the roles of ROMK and the channels regulated by the WNK kinases have come from studies of Bartter, Gitelman, and Gordon syndromes; however, whether the responses in the DCT are mediated via aldosterone directly or via its effects elsewhere in the nephron remains controversial. Other inwardly rectifying potassium channels expressed on the basolateral membrane act as sensors of extracellular potassium to play a central role in potassium and sodium homeostasis.90 This aldosterone-­independent pathway enables extracellular potassium to act through the type 2 mTOR complex to activate Sgk-­1 and stimulate ENaC to enhance ENaC-­mediated sodium transport and, indirectly, potassium excretion.93 The intercalated cells within the collecting duct consist of subtypes α, β, and non-­α/non-­β.94 Intercalated cells express H+-­ATPase on the apical or basolateral plasma membrane, depending on whether the subtype mediates hydrogen ion or bicarbonate secretion. The β-­intercalated cells express the chloride/bicarbonate ion exchanger pendrin on the apical plasma membrane and H+-­ATPase at the basolateral plasma membrane, such that increased activity will promote potassium retention. The response of the β-­intercalated cell to aldosterone is modulated by phosphorylation of a specific serine (serine 843) in the LBD of the MR, which then precludes aldosterone binding.95 Diminished plasma potassium levels promote dephosphorylation of this serine, allowing aldosterone-­induced increases in both pendrin and H+-­ATPase. Dephosphorylation was also described in response to AngII; this has been suggested to provide a mechanism by which the AngII-­stimulated, aldosterone-­mediated response to volume depletion can result in sodium reabsorption with limited potassium depletion.

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PART 7  Adrenal

The H+/K+-­ATPase in the apical membrane of β-­intercalated cells in the collecting duct is composed of catalytic α and regulatory β subunits, with expression of the α2 subunit being regulated by dietary potassium and corticosteroids in both the outer medullary collecting duct and the distal colon.96 Bartter and Gitelman syndromes are inherited conditions of hypokalemia induced by secondary aldosteronism in response to a chloride leak in the thick ascending limb and the DCT, respectively.97 In contrast to primary aldosteronism, they are associated with hypotension rather than hypertension. The syndromes can be viewed as being pharmacologically mimicked by loop diuretics such as frusemide and thiazide diuretics, respectively. Bartter syndrome is associated with a low calcium and a normal magnesium being both uncommon and severe, whereas Giteman syndrome is associated with a normal calcium and a low magnesium, often with a milder and later onset. There are various subcategories of Bartter syndrome, but the predominant mutations are in ROMK or the sodium-­potassium-­chloride cotransporter, whereas mutations in the thiazide-­sensitive sodium-­chloride cotransporter (NCCT) (or less commonly CLCNKB––a member of CLC family of voltage-­gated chloride channels) are seen in Gitleman syndrome, usually as a compound heterozygote. Gordon syndrome (pseudohypoaldosteronism type 2 or familial hyperkalemia and hypertension syndrome) can be viewed as the reciprocal phenotype of Gitleman syndrome, with low-­renin hypertension, hyperkalemia, and metabolic acidosis; the condition responds to thiazide diuretics. It is a monogenic syndrome resulting from mutations in four genes, including those encoding the WNK kinases, that directly or indirectly regulate the NCCT.98 KEY POINTS  • Aldosterone-induced epithelial potassium and hydrogen ion transport involves sodium-dependent and -independent mechanisms.

Hydrogen Ion Transport As for potassium, aldosterone also produces effects on proton excretion beyond a simple cation exchange for sodium across the epithelium to maintain electroneutrality. The targets for this effect are carbonic anhydrase–rich cells, particularly the α-­intercalated cells within the outer medullary collecting ducts. Aldosterone-­ induced transport across the apical membrane is through an H+-­ATPase activity, coupled with increased activity of the basolateral chloride/bicarbonate exchanger (which is upregulated by aldosterone).99 These are largely sodium-­independent effects, as demonstrated by a lack of an effect of amiloride. Aldosterone also produces an effect on the Na+/H+ antiporter in a range of tissues; in some tissues the response is rapid and represents a nongenomic action.100

NONEPITHELIAL TISSUES The presence of the MR in a range of tissues that may not be related to electrolyte homeostasis suggests additional actions for aldosterone ion physiology and pathophysiology. In some tissues, such as the vasculature101 and specific regions of the CNS where 11β-­HSD2 is coexpressed with the MR, the MR response is likely to be attributed to activation by aldosterone. In other tissues such as cardiomyocytes and monocyte/ macrophages where the MR is expressed, but 11β-­HSD2 is not, rather than that of aldosterone, cortisol is the predominant ligand that activates MR, except perhaps in pathological states of aldosterone excess.102 The MR is also expressed in a range of other tissues, including adipocytes, immune cells, and reproductive tissues, where the physiological roles remain to be determined, although in most tissues, in the absence of 11β-­HSD2, they are likely to be responding to cortisol.

Cardiovascular System In recent decades, the pathophysiology of MR in the cardiovascular systems has been defined by the use of pharmacological, genetic, and cell-­selective knockout animal models to reveal cell-­, ligand-­, and disease-­specific roles for the MR. The MR plays an important physiological role in this system to control the response to volume loss. Aldosterone-­ directed MR activation in the myocardium regulates the ionotropic and chronotropic activity of cardiomyocytes, resulting in increased cardiac output. MR in the vascular smooth muscle cells (VSMCs), for example, contributes to vasoconstriction, which, together with the endothelial response, has been argued to reflect aldosterone-­ mediated protection of vascular integrity to maintain volume homeostasis.103 Enhanced contraction of the peripheral vasculature, via direct MR actions or via enhanced signaling of vasoactive peptides, serves to increase peripheral resistance, which, together with an increased cardiac output, will raise blood pressure and increase organ perfusion. Recent studies using transgenic mice null for the MR in VSMCs have also identified a role for the MR in vascular stiffness and blood pressure control in aging.101 Single-­cell studies using patch-­clamp to analyze single channels or ion exchangers in rabbit cardiomyocytes identified ligand-­selective actions of the MR on the sodium-­potassium-­chloride cotransporter. Whereas acute aldosterone acutely induced flux, cortisol did not. However, under oxidative stress, cortisol was an equivalent agonist to aldosterone.104 These studies support the increasing body of evidence that the MR in the heart has ligand-­selective responses. These ligand-­specific responses are important, given that the cardiac MR it is not protected by 11β-­HSD2 and is thus continuously occupied by cortisol under physiological conditions.105 Indeed, several studies have shown that cortisol can oppose aldosterone agonist effects. The question remains as to the physiological role of the MR in the heart in the absence of volume depletion and an adequate sodium/potassium intake. Very recent studies have identified the circadian molecular clock as a target of the MR in the heart and potentially in other tissues (i.e., kidney); this may represent a physiological action of the cortisol-­ activated MR.106 In the vasculature, where the MR is coexpressed with 11β-­HSD2, aldosterone regulates a range of genes associated with inflammation and fibrosis, resulting in vascular remodeling. In vascular endothelial cells, chronic elevation of aldosterone increases transcription and cell surface expression of intracellular adhesion molecule-­1 and vascular adhesion molecule-­1 to promote recruitment of mononuclear cells as part of the profibrotic response.103 These effects occur independent of the aforementioned increases in vascular reactivity. In the heart (a nonepithelial tissue expressing MR), in vivo studies show that levels of aldosterone inappropriately high for the sodium status of the rat produce diffuse perivascular and interstitial fibrosis, an effect that can be antagonized by either corticosterone or spironolactone.107 The clinical correlation of these observations was found in large trials showing a benefit of the addition of a mineralocorticoid antagonist to the conventional regimen in the treatment of severe cardiac failure, with respect to both morbidity and mortality.108-­110 Studies in transgenic mice using tissue-­specific deletion of the MR are starting to clarify the specific roles and mechanisms of signaling through the MR in these tissues.111 KEY POINTS  • Activation of the MR in nonepithelial tissue is associated with a proinflammatory, profibrotic response that drives adverse cardiovascular outcome, as seen with chronic aldosterone excess.

CHAPTER 88  Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance

Central Nervous System Similarly, the MR is expressed extensively in the CNS; indeed, the hippocampus arguably has the highest abundance of MR of any tissue except for the distal colon.28 The function of the MR in the CNS has been the subject of extensive investigation; it appears to mediate diverse behavioral responses, including memory and affect.112 In the majority of brain regions, including the hippocampus, the MR is likely functioning as a receptor for cortisol, given that 11β-­HSD2 is not coexpressed with the MR, and penetration of the blood-­brain barrier by aldosterone is substantially less than that of most steroid hormones.16 There is, however, coexpression of 11β-­HSD2 and the MR in a small number of nuclei in the nucleus of the solitary tract (NTS); in these nuclei the role of the MR is to regulate salt appetite in response to aldosterone, and therefore sodium balance.113 They occupy a subregion of the NTS with a diminished blood-­brain barrier that may afford exposure to circulating aldosterone. These neurons express the AT1R, and MR activation appears to interact synergistically with AngII to promote salt appetite.114

SUMMARY AND FUTURE DIRECTIONS Aldosterone acts through the MR to act as the final critical arbiter of sodium, potassium, and hydrogen ion homeostasis in the distal nephron and the distal colon. Aldosterone also promotes sodium retention, and hence volume homeostasis, through effects on salt appetite and on vascular integrity. The role of the MR in nonepithelial tissues remains to be fully elucidated, including the relative importance of cortisol and aldosterone as the physiological ligand in these tissues. The significance of progesterone as an MR antagonist in terrestrial vertebrates and as an MR agonist in fish suggests yet unappreciated roles for aldosterone and the MR.

REFERENCES 1. Rossier BC, Baker ME, Studer RA. Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited. Physiol Rev. 2015;95:297–340. 2. Simpson SA, Tait JF, Wettstein A, et al. [Isolierung eines neuen kristallisierten hormons aus nebennieren mit besonders hoher wirksamkeit auf den mineral-­stoffwechsel.] Experientia. 1953;9:333–335. 3. Crabbé J. Stimulation of active sodium transport across toad bladder with aldosterone in vitro. J Clin Invest. 1961;76:2103–2110. 4. Feldman D, Funder JW, Edelman IS. Subcellular mechanisms in the action of adrenal steroids. Am J Med. 1972;53:545–560. 5. Paul M, Poyan Mehr A, Kreutz R. Physiology of local renin-­angiotensin systems. Physiol Rev. 2006;86:747–803. 6. Okubo S, Niimura F, Nishimura H, et al. Angiotensin-­independent mechanism for aldosterone synthesis during chronic extracellular fluid volume depletion. J Clin Invest. 1997;99:855–860. 7. De Sousa K, Boulkroun S, Baron S, et al. Genetic, cellular, and molecular heterogeneity in adrenals with aldosterone-­producing adenoma. Hypertension. 2020;75:1034–1044. 8. Hattangady NG, Olala LO, Bollag WB, et al. Acute and chronic regulation of aldosterone production. Mol Cell Endocrinol. 2012;350:151–162. 9. Williams GH, Rose LI, Dluhy RG, et al. Aldosterone response to sodium restriction and ACTH stimulation in panhypopituitarism. J Clin Endocrinol Metab. 1971;32:27–35. 10. Wils J, Duparc C, Cailleux A-­F, et al. The neuropeptide substance P regulates aldosterone secretion in human adrenals. Nat Commun. 2020;11:2673. 11. Lefebvre H, Duparc C, Naccache A, et al. Paracrine regulation of aldosterone secretion in physiological and pathophysiological conditions. Vitam Horm. 2019;109:303–339.

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12. Nishimoto M, Fujita T. Renal mechanisms of salt-­sensitive hypertension: contribution of two steroid receptor-­associated pathways. Am J Physiol Renal Physiol. 2015;308:F377–F387. 13. Kotchen TA, Cowley Jr AW, Frohlich ED. Medical progress: salt in health and disease—A delicate balance. New Engl J Med. 2013;368:1229–1237. 14. Knox F, Burnett JJ, Kohan D, et al. Escape from the sodium-­retaining effects of mineralocorticoids. Kidney Int. 1980;17:263–276. 15. Zimmerman RS, Edwards BS, Schwab TR, et al. Atrial natriuretic peptide during mineralocorticoid escape in the human. J Clin Endocrinol Metab. 1987;64:624–627. 16. Geerling JC, Loewy AD. Aldosterone in the brain. Am J Physiol Renal Physiol. 2009;297:F559–F576. 17. Young DB. Quantitative analysis of aldosterone’s role in potassium regulation. Am J Physiol. 1988;55:F811–F822. 17a Palmer BF. Regulation of potassium homeostasis. Clin J Am Soc Nephrol. 2015;10:1050–1060. 18. Wall SM, Verlander JW, Romero CA. The renal physiology of pendrin-­ positive intercalated cells. Physiol Rev. 2020;100:1119–1147. 19. Odermatt A, Kratschmar DV. Tissue-­specific modulation of mineralocorticoid receptor function by 11β-­hydroxysteroid dehydrogenases: an overview. Mol Cell Endocrinol. 2012;350:168–186. 20. Arriza JL, Weinberger C, Cerelli G, et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 1987;237:268–275. 21. Wilson RC, Krozowski ZS, Obeyesekere VR, et al. A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess. J Clin Endocrinol Metab. 1995;80:2263–2266. 22. Mune T, Rogerson FM, Nikkila H, et al. Human hypertension caused by mutations in the kidney isozyme of 11 beta-­hydroxysteroid dehydrogenase. Nature Genet. 1995;10:394–399. 23. Stewart PM, Valentino R, Wallace AM, et al. Mineralocorticoid activity of licorice: 11β-­hydroxysteroid dehydrogenase activity comes of age. Lancet. 1987;2:821–824. 24. Edwards CRW, Stewart PM, Burt D, et al. Localization of 11β-­ hydroxysteroid dehydrogenase: tissue-­specific protector of the mineralocorticoid receptor. Lancet. 1988;2:986–989. 25. Funder JW, Pearce PT, Smith R, et al. Mineralocorticoid action: target-­tissue specificity is enzyme, not receptor-­mediated. Science. 1988;242:583–585. 26. Marver D, Stewart J, Funder JW, et al. Renal aldosterone receptors: studies with (3H)aldosterone and the anti-­mineralocorticoid (3H)spirolactone (SC-­26304). Proc Natl Acad Sci U S A. 1974;71:1431–1435. 27. Todd-­Turla MD, Schnermann J, Fejes-­Toth G, et al. Distribution of mineralocorticoid glucocorticoid receptor mRNA along the nephron. Am J Physiol. 1993;264:F781–F791. 28. Fuller PJ, Verity K. Mineralocorticoid receptor gene expression in the gastrointestinal tract: distribution and ontogeny. J Steroid Biochem. 1990;36:263–267. 29. Baker ME, Katsu Y. Progesterone: an enigmatic ligand for the mineralocorticoid receptor. Biochem Pharmacol. 2020;177:113976. 30. Bridgham JT, Carroll SM, Thornton JW. Evolution of hormone-­receptor complexity by molecular exploitation. Science. 2006;312:97–101. 31. Fuller PJ, Yao YZ, Jin R, et al. Molecular evolution of the switch for progesterone and spironolactone from mineralocorticoid receptor agonist to antagonist. Proc Natl Acad Sci U S A. 2019;116:18578–18583. 32. Kiilerich P, Geffroy G, Valotaire C, et al. Endogenous regulation of 11-­deoxycorticosterone (DOC) and corticosteroid receptors (CRs) during rainbow trout early development and the effects of corticosteroids on hatching. Gen Comp Endocrinol. 2018;265:22–30. 33. Fan Y-­S, Eddy RL, Byers LL, et al. The human mineralocorticoid receptor gene (MLR) is located on chromosome 4 at q31.2. Cytogenet Cell Genet. 1989;52:83–84. 34. Theriault A, Boyd E, Harrap SB, et al. Regional chromosomal assignment of the human glucocorticoid receptor gene to 5q31. Hum Genet. 1989;83:289–291. 35. Hudson WH, Youn C, Ortlund EA. Crystal structure of the mineralocorticoid receptor DNA binding domain in complex with DNA. PLoS One. 2014;9:e107000.

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36. Le Billan F, Khan JA, Lamribet K, et al. Cistrome of the aldosterone-­ activated mineralocorticoid receptor in human renal cells. FASEB J. 2015;29:3977–3989. 37. Ueda K, Fujiki K, Shirahige K, et al. Genome-­wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor. Biochem Biophys Res Commun. 2014;445:132–137. 38. van Weert LTCM, Buurstede JC, Mahfouz A, et al. NeuroD factors discriminate mineralocorticoid from glucocorticoid receptor DNA binding in the male rat brain. Endocrinology. 2017;158:1511–1522. 39. Fagart J, Huyet J, Pinon GM, et al. Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension. Nature Struct Mol Biol. 2005;12:554–555. 40. Bledsoe RK, Madauss KP, Holt JA, et al. A ligand-­mediated hydrogen bond network required for the activation of the mineralocorticoid receptor. J Biol Chem. 2005;280:31283–31293. 41. Li Y, Suino K, Daugherty J, et al. Structural and biochemical mechanisms for the specificity of hormone binding and coactivator assembly by mineralocorticoid receptor. Mol Cell. 2005;19:367–280. 42. Kumar R, McEwan IJ. Allosteric modulators of steroid hormone receptors: structural dynamics and gene regulation. Endoc Rev. 2012;33:271–299. 43. Pippal JB, Yao Y, Rogerson FM, et al. Structural and functional characterization of the interdomain interaction in the mineralocorticoid receptor. Mol Endocrinol. 2009;23:1360–1370. 44. Bruner KL, Derfoul A, Robertson NM, et al. The unliganded mineralocorticoid receptor is associated with heat shock proteins 70 and 90 and the immunophilin FKBP-­52. Recept Signal Transduct. 1997;7:85–98. 45. Zennaro M-­C, Keightley MC, Kotelevtsev Y, et al. Human mineralocorticoid receptor genomic structure and identification of expressed isoforms. J Biol Chem. 1995;270:21016–21020. 46. Zennaro MC, Souque A, Viengchareun S, et al. A new human MR splice variant is a ligand-­independent transactivator modulating corticosteroid action. Mol Endocrinol. 2001;15:1586–1598. 47. Fuller PJ, Yang J, Young MJ. Mechanisms of mineralocorticoid receptor signaling. Vitam Horm. 2019;109:37–68. 48. Fuller PJ, Yang J, Young MJ. Coregulators as mediators of mineralocorticoid receptor signalling diversity. J Endocrinol. 2017;234:T23–T34. 49. Nishimoto M, Fujita T. Renal mechanisms of salt-­sensitive hypertension: contribution of two steroid receptor-­associated pathways. Am J Physiol Renal Physiol. 2015;308:F377–F387. 50. Gomez-­Sanchez EP, Venkataraman MT, Thwaites D, et al. Intracerebroventricular infusion of corticosterone antagonizes ICV-­aldosterone hypertension. Am J Physiol. 1990;258:E649–E653. 51. Berger S, Bleich M, Schmid W, et al. Mineralocorticoid receptor knockout mice: pathophysiology of Na+ metabolism. Proc Natl Acad Sci U S A. 1998;95:9424–9429. 52. Zennaro MC, Fernandes-­Rosa F. Mineralocorticoid receptor mutations. J Endocrinol. 2017;234:T93–T106. 52. Cole TJ, Terella L, Morgan J, et al. Aldosterone-­mediated renal sodium transport requires intact mineralocorticoid receptor DNA-­binding in the mouse. Endocrinology. 2015;156:2958–2968. 53. De Bosscher K, Desmet SJ, Clarisse D, et al. Nuclear receptor crosstalk -­ defining the mechanisms for therapeutic innovation. Nat Rev Endocrinol. 2020;16:363–377. 54. Feldman RD, Limbird LE. GPER (GPR30): a nongenomic receptor (GPCR) for steroid hormones with implications for cardiovascular disease and cancer. Annu Rev Pharmacol Toxicol. 2017;57:567–584. 55. Grossmann C, Freudinger R, Mildenbergr S, et al. EF-­domains are sufficient for nongenomic mineralocorticoid receptor actions. J Biol Chem. 2008;283:7109–7116. 56. McEneaney V, Harvey BJ, Thomas W. Aldosterone regulates rapid trafficking of ENaC subunits in renal cortical collecting duct cells via protein kinase D activation. Mol Endocrinol. 2008;22:881–882. 57. Mihailidou AS, Mardini M, Funder JW. Rapid, nongenomic effects of aldosterone in the heart mediated by epsilon protein kinase C. Endocrinology. 2003;145:773–780. 58. Alzamora R, Marusic ET, Gonzalez M, et al. Nongenomic effect of aldosterone on Na+,K+-­adenosine triphosphatase in arterial vessels. Endocrinology. 2003;144:1266–1272.

59. Karst H, Berger S, Turiault M, et al. Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone. Proc Natl Acad Sci USA. 2005;102:19204– 19207. 60. Young MJ, Adler GK. Aldosterone, the mineralocorticoid receptor and mechanisms of cardiovascular disease. Vitam Horm. 2019;109:361– 385. 61. Verrey F. Early aldosterone action: toward filling the gap between transcription and transport. Am J Physiol. 1999;277:F319–F327. 62. Wade JB, Stanton BA, Field MJ, et al. Morphological and physiological responses to aldosterone: time course and sodium dependence. Am J Physiol. 1990;259:F88–F94. 63. Canessa CM, Schild L, Buell G, et al. Amiloride-­sensitive epithelial Na+ channel is made of three homologous subunits. Nature. 1994;367:463– 467. 64. Noreng S, Bharadwaj A, Posert R, et al. Structure of the human epithelial sodium channel by cryo-­electron microscopy. Elife. 2018;7:Ge39340. 65. Vuagniaux G, Vallet V, Jaeger NF, et al. Synergistic activation of ENaC by three membrane-­bound channel-­activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum-­and glucocorticoid-­regulated kinase (Sgk1) in Xenopus oocytes. J Gen Physiol. 2002;120:191–201. 66. Hummler E, Barker P, Gatzy J, et al. Early death due to defective neonatal lung liquid clearance in αENaC-­deficient mice. Nat Genet. 1996;12:325–328. 67. Barker PM, Nguyen MS, Gatzy JT, et al. Role of gamma ENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism. J Clin Invest. 1998;102:1634–1640. 68. McDonald FJ, Yang B, Hrstka RF, et al. Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype. Proc Natl Acad Sci U S A. 1999;96:1727–1731. 69. Pradervand S, Barker PM, Wang Q, et al. Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-­ subunit of the amiloride-­sensitive epithelial sodium channel. Proc Natl Acad Sci U S A. 1999;96:1732–1737. 70. Chang SS, Grunder S, Hanukoglu A, et al. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet. 1996;12:248–253. 71. Shimkets RA, Warnock DG, Bositis CM, et al. Liddle’s syndrome: heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel. Cell. 1994;79:407–414. 72. Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell. 2001;104:545–556. 73. Staub O, Gautschi I, Ishikawa T, et al. Regulation of stability and function of the epithelial Na+ channel ENaC by ubiquitination. EMBO J. 1997;16:6325–6336. 74. Soundararajan R, Pearce D, Ziera T. The role of the ENaC-­regulatory complex in aldosterone-­mediated sodium transport. Mol Cell Endocrinol. 2012;350:242–247. 75. Brennan FE, Fuller PJ. Rapid up-­regulation of serum and glucocorticoid-­ regulated kinase (sgk) gene expression by corticosteroids in vivo. Mol Cell Endocrinol. 2000;30:129–136. 76. Bhargava A, Fullerton MJ, Myles K, et al. The serum-­and glucocorticoid-­ induced kinase is a physiological mediator of aldosterone action. Endocrinology. 2001;142:1587–1594. 77. Debonneville C, Flores SY, Kamynina E, et al. Phosphorylation of Nedd4-­2 by Sgk1 regulates epithelial Na+ channel cell surface expression. EMBO J. 2001;20:7052–7059. 78. Snyder PM, Olson DR, Thomas BC. Serum and glucocorticoid-­regulated kinase modulates Nedd4-­2-­mediated inhibition of the epithelial Na+ channel. J Biol Chem. 2002;277:5–8. 79. Zhang W, Xia X, Reisenauer MR, et al. Aldosterone-­induced Sgk1 relieves Dot1a-­Af9-­mediated transcriptional repression of epithelial Na+ channel α. J Clin Invest. 2007;117:773–783. 80. Fakitsas P, Adam G, Daidié D, et al. Early aldosterone-­induced gene product regulates the epithelial sodium channel by deubiquitylation. J Am Soc Nephrol. 2007;18:1084–1092.

CHAPTER 88  Mineralocorticoids: Physiology, Metabolism, Receptors, and Resistance 81. Wang J, Barbry P, Maiyar AC, et al. SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am J Physiol. 2001;280:F303–F313. 82. Brennan FE, Fuller PJ. Mammalian K-­ras2 is a corticosteroid-­induced gene in vivo. Endocrinology. 2006;147:2809–2816. 83. Rashmi P, Colussi G, Ng M, et al. Glucocorticoid-­induced leucine zipper protein regulates sodium and potassium balance in the distal nephron. Kidney Int. 2017;91:1159–1177. 84. Fuller PJ, Verity K. Colonic sodium-­potassium adenosine triphosphate subunit gene expression: ontogeny and regulation by adrenocortical steroids. Endocrinology. 1990;127:32–38. 85. Petty KJ, Kokko JP, Marver D. Secondary effect of aldosterone on Na-­ KATPase activity in the rabbit cortical collecting tubule. J Clin Invest. 1981;68:1514–1521. 86. Sansom SC, O’Neil RG. Mineralocorticoid regulation of apical cell membrane Na+ and K+ transport of the cortical collecting duct. Am J Physiol. 1985;248:F858–F868. 87. Beguin P, Crambert G, Guennoun S, et al. CHIF, a member of the FXYD protein family, is a regulator of Na,K-­ATPase distinct from the gamma-­ subunit. EMBO J. 2001;20:3993–4002. 88. Wald H, Goldstein O, Asher C, et al. Aldosterone induction and epithelial distribution of CHIF. Am J Physiol. 1996;271:F322–F329. 89. Brennan FE, Fuller PJ. Acute regulation by corticosteroids of CHIF mRNA in the distal colon. Endocrinology. 1999;140:1213–1218. 90. Manis AD, Hodges MR, Staruschenko A, et al. Expression, localization, and functional properties of inwardly rectifying K+ channels in the kidney. Am J Physiol Renal Physiol. 2020;318:F332–F337. 91. Yoo D, Kim BY, Campo C, et al. Cell surface expression of the ROMK (Kir 1.1) channel is regulated by the aldosterone-­induced kinase, sgk-­1, and protein kinase A. J Bio Chem. 2003;278:23066– 23075. 92. Glover M, O’Shaughnessy KM. Molecular insights from dysregulation of the thiazide-­sensitive WNK/SPAK/NCC pathway in the kidney: Gordon syndrome and thiazide-­induced hyponatraemia. Clin Exp Pharmacol Physiol. 2013;40:876–884. 93. Sørensen MV, Saha B, Jensen IS, et al. Potassium acts through mTOR to regulate its own secretion. JCI Insight. 2019;5:e126910. 94. Wall SM, Verlander JW, Romero CA. The renal physiology of pendrin-­ positive intercalated cells. Physiol Rev. 2020;100:1119–1147. 95. Shibata S, Rinehart J, Zhang J, et al. Mineralocorticoid receptor phosphorylation regulates ligand binding and renal response to volume depletion and hyperkalemia. Cell Metab. 2013;18:660–671. 96. Greenlee MM, Lynch IJ, Gumz ML, et al. Mineralocorticoids stimulate the activity and expression of renal H+, K+-­ATPases. J Am Soc Nephrol. 2011;22:49–58. 97. Seyberth HW, Weber S, Kömhoff M. Bartter’s and Gitelman’s syndrome. Curr Opin Pediatr. 2017;29:179–186.

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98. Mabillard H, Sayer JA. The molecular genetics of Gordon syndrome. Genes (Basel). 2019;10:986. 99. Hays TR. Mineralocorticoid modulation of apical and basolateralmembrane H+/OH-­/HCO3 transport processes in the rabbit inner stripe of outer medullary collecting duct. J Clin Invest. 1992;90:1–8. 100. Dooley R, Harvey BJ, Thomas W. Non-­genomic actions of aldosterone: from receptors and signals to membrane targets. Mol Cell Endocrinol. 2012;350:223–234. 101. McCurley A, Pires PW, Bender SB, et al. Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors. Nat Med. 2012;18:1429–1433. 102. Iqbal J, Andrew R, Cruden NL, et al. Displacement of cortisol from human heart by acute administration of a mineralocorticoid receptor antagonist. J Clin Endocrinol Metab. 2014;99:915–922. 103. Biwer LA, Wallingford MC, Jaffe IZ. vascular mineralocorticoid receptor: evolutionary mediator of wound healing turned harmful by our modern lifestyle. Am J Hypertens. 2019;32:123–134. 104. Mihailidou AS, Tzakos AG, Ashton AW. Non-­genomic effects of aldosterone. Vitam Horm. 2019;109:133–149. 105. Iqbal J, Andrew R, Cruden NL, et al. Displacement of cortisol from human heart by acute administration of a mineralocorticoid receptor antagonist. J Clin Endocrinol Metab. 2014;99:915–922. 106. Fletcher EK, Morgan J, Kennaway DR, et al. Deoxycorticosterone/salt-­ mediated cardiac inflammation and fibrosis are dependent on functional CLOCK signaling in male mice. Endocrinology. 2017;158:2906–2917. 107. Young MJ, Funder JW. Mineralocorticoid receptors and pathophysiological roles for aldosterone in the cardiovascular system. J Hypertens. 2002;20:1465–1468. 108. Pitt B, Zannad F, Remme WJ, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med. 1999;341:709–717. 109. Pitt B, Remme W, Zannad F, et al. Eplerenone post-­acute myocardial infarction heart failure efficacy and survival study investigators. N Engl J Med. 2003;348:1309–1321. 110. Zannad F, McMurray JJ, Krum H, et al. Eplerenone in patients with systolic heart failure and mild symptoms. N Engl J Med. 2011;364:11–21. 111. Cole TJ, Young MJ. Mineralocorticoid receptor null mice: informing cell-­type-­specific roles. J Endocrinol. 2017;234:T83–T92. 112. de Kloet ER, Joëls M. Brain mineralocorticoid receptor function in control of salt balance and stress-­adaptation. Physiol Behav. 2017;178:13–20. 113. Gasparini S, Resch JM, Narayan SV, et al. Aldosterone-­sensitive HSD2 neurons in mice. Brain Struct Funct. 2019;224:387–417. 114. Resch JM, Fenselau H, Madara JC, et al. Aldosterone-­sensing neurons in the NTS exhibit state-­dependent pacemaker activity and drive sodium appetite via synergy with angiotensin II signaling. Neuron. 2017;96:190– 206.

89 Glucocorticoid Receptors: Mechanisms of Action in Health and Disease Robert H. Oakley, Stephen R. Hammes, Donald B. DeFranco, and John A. Cidlowski

OUTLINE Introduction, 1484 Glucocorticoid Receptor Domain Organization, 1484 Glucocorticoid Receptor Regulation of Transcription, 1484 Glucocorticoid Receptor Polymorphisms, 1486 Glucocorticoid Receptor Isoforms Derived From Single Gene, 1486 Glucocorticoid Receptor Posttranslational Modifications, 1488 Glucocorticoid Receptor Physiology, 1489

Immune System, 1489 Cardiovascular System, 1490 Central Nervous System, 1490 Hepatic Carbohydrate and Lipid Metabolism, 1490 Glucocorticoid Receptor Ligands and Pulsatility, 1491 Glucocorticoid Receptor Resistance, 1492 Summary and Future Directions, 1492

 

INTRODUCTION Glucocorticoids are primary stress hormones necessary for life that affect virtually every aspect of human physiology. They are synthesized by and released from the adrenal glands following activation of the hypothalamic-pituitary-adrenal (HPA) axis in a circadian manner and in response to physical and emotional stress. Synthetic derivatives of glucocorticoids are one of the most commonly prescribed classes of drugs in the world today.1 The physiological actions of glucocorticoids include highly effective antiinflammatory and immunomodulatory actions that are exploited for the treatment of diseases such as asthma, arthritis, allergic rhinitis, ulcerative colitis, multiple sclerosis, and leukemia/lymphoma.2,3 In addition, glucocorticoids have important roles in lung development, skeletal growth, behavior, reproduction, metabolism, and cardiovascular function. Ultimately, glucocorticoids act to maintain homeostasis in the face of stressful stimuli. The broad actions of glucocorticoids account for the serious side effects commonly experienced with chronic glucocorticoid treatment. These include the development of glucocorticoid resistance in diseased tissues, osteoporosis, growth retardation in children, hypertension, muscle atrophy, and metabolic syndrome.1,4 The physiological and pharmacological actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of proteins that regulate gene transcription in a ligand-­dependent manner.

GLUCOCORTICOID RECEPTOR DOMAIN ORGANIZATION Like other members of the nuclear receptor superfamily, GR is a modular protein comprised of an N-­terminal transactivation domain (NTD), a central DNA-­binding domain (DBD), and a C-­terminal ligand-­binding domain (LBD) (Fig. 89.1).5 Separating the DBD and LBD is a flexible region of the protein termed the hinge region. The DBD is the most conserved region across the nuclear receptor superfamily. It contains two zinc finger motifs that recognize and bind target DNA sequences, termed glucocorticoid-­responsive elements (GREs). The NTD is the most variable region among family members and

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contains a strong transcriptional activation function (AF1) that interacts with coregulators and the basal transcription machinery. The LBD forms a hydrophobic pocket for binding glucocorticoids and contains a second transcriptional activation function (AF2) that interacts with coregulators in a ligand-­dependent manner. The LBD also possesses a nuclear localization signal and sites for interaction with chaperone proteins and other transcription factors. An additional nuclear localization signal spans the junction of the DBD and hinge region. The GR protein is a substrate for various types of posttranslational modifications, including phosphorylation, ubiquitination, sumoylation, and acetylation that regulate GR function (Fig. 89.1).

GLUCOCORTICOID RECEPTOR REGULATION OF TRANSCRIPTION Unliganded GR is located in the cytoplasm of cells as part of a multiprotein complex that includes chaperone proteins (HSP90, HSP70, and P23) and immunophilins of the FK506 family (FKBP51 and FKBP52) (Fig. 89.2).6 The chaperone proteins maintain the receptor in a transcriptionally inactive state that favors high-­affinity ligand binding. Upon binding glucocorticoids, GR undergoes a conformational change resulting in the dissociation of the associated proteins, exposure of the nuclear localization signals, and translocation of the receptor into the nucleus via nuclear pores. The receptor then induces or represses gene transcription by binding directly to GREs and/or by interacting with other transcription factors and modulating their activity. The consensus GRE sequence is an imperfect palindrome (GGAA­ CAnnnTGTTCT) that is comprised of two 6-­bp half sites separated by a three-­nucleotide spacer. GR binds this element as a homodimer, with each half site occupied by one receptor subunit (Fig. 89.2A). The interaction of ligand-­bound GR with the GRE results in the enhanced expression of numerous genes. For this reason, the GRE is often referred to as an activating or positive GRE. However, GR occupancy of the canonical GRE can also lead to the repression of target genes, suggesting a critical role for factors outside the GRE sequence, such as epigenetic regulators and chromatin context, in determining the polarity of the transcriptional response.7 A negative GRE (nGRE)

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Ser-134 Ser-141

Ser-203 Ser-211 Ser-226

Lys-277 Lys-293

Ser-404 Lys-419

Lys-494 Lys-495

Lys-703

Ser-113

CHAPTER 89  Glucocorticoid Receptors: Mechanisms of Action in Health and Disease

P P P

P P P

S S

P U

A A

S

DBD

NTD 1

421 AF1

486

H

LBD

528

777 AF2

Dimerization Nuclear localization Hsp90 Figure 89.1  Glucocorticoid receptor (GR) domain structure and sites of posttranslational modification. GR is composed of an N-­terminal transactivation domain (NTD), DNA-­binding domain (DBD), hinge region (H), and ligand-­binding domain (LBD). Regions involved in transactivation (AF1 and AF2), dimerization, nuclear localization, and Hsp90 binding are indicated. Also shown are some of the amino acid residues posttranslationally modified by phosphorylation (P), sumoylation (S), ubiquitination (U), and acetylation (A). Numbers are for the human GR.

Figure 89.2  Glucocorticoid receptor (GR) signaling pathways. The unliganded GR resides in the cytoplasm of cells in a complex with other proteins. Upon binding glucocorticoids, the receptor undergoes a change in conformation (activation), dissociates from accessory proteins, and translocates into nucleus via the nuclear pore complex (NPC). Nuclear GR regulates gene expression in three primary ways: binding directly to DNA (A), tethering itself to other DNA-­bound transcription factors (B), or binding directly to DNA and interacting with neighboring DNA-­bound transcription factors (C). BTM, Basal transcription machinery.

that differs in sequence from the classic GRE has also been described that mediates glucocorticoid-­dependent repression of specific genes (Fig. 89.2A).8 The consensus nGRE sequence, CTCC(n)0-­2GGAGA, is palindromic but has a variable spacer and is occupied by two individual GR monomers rather than a homodimer.9 Binding sites for GR are found not only within promoters of glucocorticoid-­responsive genes but also in intragenic and intergenic regions that can be far removed from the transcription start site.10 For example, glucocorticoids have been shown to induce the expression of the β-­arrestin-­1 gene (ARRB1) via an intron 1 GRE and to repress the expression of the β-­arrestin-­2 gene (ARRB2) via an intron 11 nGRE.11 Glucocorticoid regulation

of the β-­arrestin-­1/β-­arrestin-­2 ratio alters the signaling profiles of G protein-coupled receptors and may account for the superior clinical efficacy of the glucocorticoid/beta-­2-­adrenergic receptor agonist combination therapies used to treat asthma and chronic obstructive pulmonary disease. Another example of clinical importance is that GR occupancy of an nGRE located in intron 6 of the GR gene itself mediates homologous downregulation of GR expression,12 a mechanism of glucocorticoid resistance that limits the therapeutic response to glucocorticoids. Many GREs are found in the genome, yet only a small fraction of these sites is actually bound by the receptor. The specific sites of

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GR occupancy vary in a tissue-­specific manner due to differences in chromatin landscape that regulate the accessibility of the GRE.13 Glucocorticoid concentration can also govern GR binding to specific GREs and subsequent regulation of target gene expression.14 For some GREs, GR occupancy occurs at very low concentrations of hormone. Other GREs, however, require high doses of glucocorticoids for GR binding to take place. Distinguishing hypersensitive from hyposensitive GREs is an important endeavor because low-­dose glucocorticoid therapy targeting hypersensitive sets may avoid the harmful side effects of higher glucocorticoid doses frequently used in the clinic. The GR– GRE interaction is short-­lived, as the receptor rapidly cycles on and off target sites every few seconds.15 Upon binding DNA, GR undergoes a conformational change that leads to the recruitment of coregulators and chromatin remodeling complexes that regulate RNA polymerase II activity and thereby alter the transcription rates of target genes.16-­18 Steroid receptor coactivators (SRC1–3), the histone acetyltransferases CBP/p300, and the nuclear methylase coactivator-­associated arginine methyltransferase (CARM1) are coregulators that mediate transcriptional activation, whereas nuclear receptor corepressor 1 (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT) are corepressors that mediate transcriptional repression. The specific cofactors that are assembled and their activity on glucocorticoid-­ responsive genes depends on the ligand bound to GR, as well as the specific GRE sequence occupied by the receptor.19,20 In response to glucocorticoids, GR can also regulate gene expression by interacting with other transcription factors (Fig. 89.2B,C). The two most studied examples of this form of regulation involve the transcription factors activator protein 1 (AP-­1) and nuclear factor-­κB (NF-­ κB). These two proteins are central mediators of the inflammatory and immune response, and their inhibition by GR is thought to underlie the major antiinflammatory and immunosuppressive actions of glucocorticoids.5 When activated by stress signals such as proinflammatory cytokines, bacterial and viral infectious agents, or proapoptotic stimuli, AP-­1 and NF-­κB bind their cognate response elements and induce the expression of many proinflammatory genes, including those encoding cytokines, cell adhesion molecules, and enzymes involved in tissue destruction. GR represses the activity of AP-­1 and NF-­κB through a direct interaction with the c-­Jun subunit of AP-­1 and the p65 subunit of NF-­κB. For some genes, the inhibition is accomplished by GR tethering itself to these DNA-­bound proteins without the receptor binding to DNA (Fig. 89.2B). For other genes, the repression involves GR acting in a composite manner in which the receptor both binds a GRE and physically associates with AP-­1 or NF-­κB bound to neighboring DNA sites (Fig. 89.2C). Glucocorticoids can also indirectly antagonize the actions of AP-­1 and NF-­κB by inducing the expression of antiinflammatory genes encoding factors such as IκB protein, which sequesters NF-­κB in the cytoplasm,21 MAPK phosphatase 1, which dephosphorylates c-­Jun N-­terminal kinase to prevent activation of AP-­1,22 and tristetraprolin, which destabilizes the mRNA of many AP-­1- and NF-­ κB-induced genes.23 In contrast to its repressive actions on AP-­1 and NF-­κB, GR interacts with specific members of the signal transducer and activator of transcription (STAT) family of transcription factors, either apart from (tethering action) or in conjunction with (composite action) GRE binding, and enhances their activity on responsive genes (Fig. 89.2B,C).24 STAT transcription factors are activated by a range of cytokines through induction of the Janus kinase pathway (JAK) and tyrosine phosphorylation. Upon binding their cognate response elements, STATs regulate genes involved in the immune response, differentiation, survival, and apoptosis. Global gene expression analyses indicate that up to 10% to 20% of the genome is regulated by glucocorticoids.5 Many of these genes, termed primary glucocorticoid-­responsive genes, are regulated by GR

directly binding to DNA (GREs and nGREs) and/or interacting with other DNA-­bound transcription factors that control the expression of the specific gene. However, GR can also have a profound influence on the genome in an indirect manner. These secondary gene changes occur more slowly and are mediated by the protein products of the primary glucocorticoid-­responsive genes. This second wave of gene expression changes can be accomplished by transcription factors that are direct targets of GR regulation. For example, in the heart, glucocorticoid-­ activated GR increases the expression of the zinc finger DNA-­binding transcription factor Kruppel like factor 13 (KLF13) via binding a GRE located in intron 1 of the KLF13 gene. The upregulated KLF13, in turn, regulates a large cohort of genes that protect cardiomyocytes from cell death induced by the antineoplastic drug doxorubicin.25 KEY POINTS  • Upon glucocorticoid binding, cytoplasmic glucocorticoid receptor (GR) translocates to the nucleus. • GR binds directly to glucocorticoid-­responsive elements (GREs) or negative GREs to regulate target gene expression. • GR also interacts with other transcription factors, such as AP-­1 and NF-­κB, to regulate target gene expression.

GLUCOCORTICOID RECEPTOR POLYMORPHISMS Several polymorphisms in the GR gene have been described that change the amino acid sequence of the encoded receptor and alter its transcriptional activity. The single nucleotide polymorphism N363S, which results in an asparagine to serine substitution in the NTD of GR, occurs in approximately 4% of the population and is associated with glucocorticoid hypersensitivity.26,27 N363S carriers have been reported to have an increased risk for metabolic syndrome, type 2 diabetes, decreased bone mineral density, and cardiovascular disease. Genome-­ wide microarrays on isolated macrophages and cultured osteosarcoma cells have revealed unique gene regulatory profiles for the N363S polymorphism compared with wild-­type GR in response to glucocorticoid treatment.28,29 The ER22/23EK polymorphism occurs in approximately 3% of the population and results in an arginine to lysine change at position 23 within the NTD of GR.26,27 The ER22/23EK polymorphism, in contrast to N363S, exhibits reduced transcriptional activity on target genes and has been associated with glucocorticoid insensitivity. Carriers of this polymorphism have a more favorable metabolic profile due to their decreased sensitivity to glucocorticoids and exhibit a lower tendency to develop type 2 diabetes and cardiovascular disease. The mechanism underlying the glucocorticoid resistance in ER22/23EK carriers may involve alterations in the expressed complement of GR isoforms (discussed later).30 These GR polymorphisms may account, at least in part, for the heterogeneous responses observed among individuals on glucocorticoid therapy.

GLUCOCORTICOID RECEPTOR ISOFORMS DERIVED FROM SINGLE GENE GR is expressed throughout the body, yet there is considerable diversity among cell types in terms of their responses to glucocorticoids. The discovery of multiple GR isoforms has provided a molecular mechanism contributing to the specificity of glucocorticoid action. The human GR gene is located on chromosome 5q31-­32 and is comprised of nine exons (Fig. 89.3). The GR NTD is encoded primarily by exon 2, the DBD is encoded by exons 3 and 4, and the hinge region and LBD are encoded by exons 5 to 9. Alternative splicing in exon 9

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GR gene

1H

2

3

4

5

6

7

8

9

Alternative splicing

GRα

5'UTR

GRβ

5'UTR

3'UTR

GR mRNAs 3'UTR

Alternative translation initiation GR isoforms GRα-A

1

GRα-B

27

NTD

DBD H

LBD

777

GRβ-A

1

777

GRβ-B

27

NTD

DBD H

LBD

742 742

GRα-C1

86

777

GRβ-C1

86

742

GRα-C2

90

777

GRβ-C2

90

742

GRα-C3

98

777

GRβ-C3

98

742

777

GRβ-D1

331

777

GRβ-D2

331

742

336

777

GRβ-D3

336

742

GRα-D1 GRα-D2 GRα-D3

316

316

742

Figure 89.3  Multiple glucocorticoid receptor (GR) isoforms generated from single gene. The human GR gene is comprised of nine exons. Alternative splicing at the 3’ end of the primary transcript generates GRα and GRβ mRNAs, which encode GRα and GRβ proteins differing only at their C-­terminus. Alternative translation initiation from eight different AUG start codons derived from exon 2 generates additional protein isoforms with progressively shorter N-­terminal domains. Numbers shown denote the first and last residues for the human GR isoforms. For simplicity, only the most proximal of nine alternate exon 1s (1H) is shown.

generates two receptor isoforms, termed GRα and GRβ, that are identical through amino acid 727 but then diverge at their C-­termini (Fig. 89.3).5,31 The classic, full-­length GRα contains an additional 50 amino acids, whereas the splice variant GRβ has an additional, nonhomologous 15 amino acids. Because of its unique C-­terminus, GRβ does not bind glucocorticoids and resides constitutively in the nucleus of cells. GRβ has been shown to function as a dominant negative inhibitor and repress the transcriptional activity of GRα32,33; therefore, alterations in GRβ expression may contribute to changes in glucocorticoid responsiveness. Indeed, expression of GRβ is selectively increased over GRα in cells exposed to proinflammatory cytokines and microbial superantigens leading to reduced sensitivity to glucocorticoids.5,31 Glucocorticoid-­ resistant forms of inflammatory diseases, such as asthma, rheumatoid arthritis, and ulcerative colitis, have also been associated with elevated expression of GRβ. Furthermore, upregulation of GRβ and diminished GRα signaling is observed in erythroid cells from patients with polycythemia vera. Conversely, methotrexate, an effective drug for treating autoimmune and inflammatory diseases, promotes a selective increase in GRα at the expense of GRβ and improves the glucocorticoid sensitivity of lymphocytes. The mechanisms underlying the dominant negative activity of GRβ are unclear but may involve competition for GRE binding and/or formation of GRα/GRβ heterodimers that are transcriptionally deficient. The splicing factor SRp30c has been shown to play a critical role in the selective increase in GRβ expression.34 In addition, elevated GRβ levels result from the naturally occurring ATTTA to GTTTA

polymorphism (A3669G) in the 3’ untranslated region of GRβ. This nucleotide substitution disrupts an mRNA destabilization motif and leads to increased stability of the GRβ mRNA and enhanced protein expression.35,36 Persons harboring the A3669G allele have an elevated risk for pathologies with inflammatory underpinnings, such as autoimmune disease, coronary artery disease, myocardial infarction, and heart failure, suggesting the rise in GRβ limits the beneficial immunosuppressive actions of GRα.37-­39 Another polymorphism in the GRβ 3’ untranslated region (G3134T) has also been shown to increase GRβ expression by mRNA stabilization.40 Carriers of the G3134T polymorphism have elevated levels of endogenous glucocorticoids, display glucocorticoid resistance, and exhibit alterations in the glucocorticoid-­ regulated transcriptome in isolated macrophages. A broader role for GRβ in cell signaling has recently emerged with the demonstration that this isoform can modulate gene expression apart from its effects on GRα. In a genome-­wide microarray analysis performed in human osteosarcoma cells selectively expressing GRβ, the isoform was found to alter the expression of over 5000 genes.41 Less than 20% of the genes were commonly regulated by ligand-­activated GRα, indicating that GRβ possesses its own unique gene-­regulatory profile. GRβ was also shown to bind the glucocorticoid antagonist mifepristone (RU486), and binding of this ligand abolished most of the GRβ-­mediated changes in gene expression.41 Interestingly, structural analysis of the GRβ/RU486 complex suggests GRβ is found in a conformation that favors corepressor binding.42 To explore the function of GRβ in vivo, the splice variant was expressed in the liver

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of wild-­type mice and in GR liver knockout mice using an adeno-­ associated virus.43 GRβ antagonized the function of GRα and attenuated hepatic gluconeogenesis. Moreover, GRβ regulated many genes by GRα-­independent mechanisms. For example, GRβ upregulated the expression of the STAT1 gene in the livers of both wild-­type and GR liver knockout mice. This induction involved GRβ binding to an intergenic GRE adjacent to the STAT1 gene and was inhibited by RU486 treatment. As a bona fide transcription factor, GRβ may contribute to alterations in glucocorticoid responsiveness in healthy and diseased tissues by genomic effects apart from its dominant negative activity on GRα. Alternative translation initiation of the GR mRNA produces an additional cohort of receptor subtypes (Fig. 89.3).44,45 Eight GR translational isoforms with progressively shorter NTDs are generated from one GRα mRNA transcript via different AUG start codons: GRα-­A, GRα-­B, GRα-­C1, GRα-­C2, GRα-­C3, GRα-­D1, GRα-­D2, and GRα-­D3. The eight initiation sites are located in exon 2 and are highly conserved across species. GRα-­A is the classic, full-­length 777-amino acid human receptor that is generated from the first initiator AUG codon. The GRβ mRNA also contains the identical start codons and would be expected to give rise to a similar complement of subtypes. The GRα isoforms, all of which retain an intact DBD and LBD, bind glucocorticoids with similar affinity and show a similar capacity to interact with GREs.46 Additionally, all eight isoforms occupy the nucleus of cells following glucocorticoid treatment. In the absence of hormone, however, the subcellular distribution of the subtypes differ with the GRα-­D isoforms residing predominantly in the nucleus and the others predominantly in the cytoplasm.44,46 Marked differences have been reported in the transcriptional properties of the GRα translational isoforms.46,47 Global microarray analysis of cells selectively expressing individual GRα isoforms revealed that each receptor subtype displays a unique gene-­regulatory profile. Less than 10% of the glucocorticoid-­responsive transcriptome was commonly regulated by all the isoforms, indicating that the vast majority of genes were regulated in an isoform-­specific manner. The GRα-­C3 isoform is the most transcriptionally active subtype, whereas GRα-­D isoforms are the least active. The heightened activity of GRα-­C3 has been linked to a motif in the NTD that sterically hinders the larger isoforms.48 The isoform-­ unique gene-­regulatory profiles were further shown to produce functional differences in cellular responsiveness to glucocorticoids, as the GRα translational isoforms exhibited distinct capabilities to induce apoptosis.46,47 Cells expressing GRα-­C3 were the most sensitive to the apoptosis-­inducing actions of glucocorticoids, whereas cells expressing GRα-­D3 were the most resistant. Unlike other GRα translational isoforms, GRα-­D3 did not repress NF-­κB activity and failed to inhibit the transcription of certain antiapoptotic genes.49 The GRα translational isoforms show a widespread tissue distribution; however, their relative levels vary across cell types. GRα-­A and GRα-­B are the most highly expressed isoforms in many cells. However, GRα-­C and GRα-­D are abundant in trabecular meshwork cells,50 and the GRα-­D isoform predominates in immature dendritic cells.51 In rodents, GRα-­C isoforms are expressed at high levels in the pancreas and colon, whereas GRα-­D isoforms are abundant in the spleen and lungs.44 In the human brain, the composition of GRα translational isoforms changes during development and aging.52 Additionally, a selective increase in the GRα-­D isoform has been observed in the brains from patients with schizophrenia and bipolar disorder.53,54 Differential expression of GRα translational isoforms may be a major mechanism behind the diverse tissue sensitivity and responses to glucocorticoids. In vivo support for this conclusion comes from knockin mice that exclusively express the GRα-­C3 isoform.55 The GRα-­C3 knockin mice die at birth due to respiratory distress but can be rescued by antenatal

glucocorticoid administration. Rescued GRα-­C3 mice exhibited alterations in sensitivity to lipopolysaccharide (LPS)-­induced inflammation, and gene expression studies revealed a deficiency in the ability of GRα-­C3 to regulate a large cohort of immune and inflammatory response genes in the spleen. KEY POINTS  • The single glucocorticoid receptor (GR) gene gives rise to multiple protein isoforms. • Alternative slicing in exon 9 of the GR gene produces the GRβ splice variant. • Alternative translation initiation of the GR mRNA produces eight GRα translational isoforms with progressively shorter N-­terminal domains. • The various GR isoforms, with their unique expression and gene-­regulatory profiles, contribute to tissue specificity in glucocorticoid responsiveness.

GLUCOCORTICOID RECEPTOR POSTTRANSLATIONAL MODIFICATIONS In addition to the diversity in cellular response to glucocorticoids driven by changes in the coding sequence of GR (i.e., via polymorphisms, splicing, or translational isoforms), posttranslational modifications to the receptor can have a profound effect on glucocorticoid-­regulated transcription.56-­59 Phosphorylation is the most well characterized, but not the only, posttranslational modification of GR and was initially described in the mid-­1970s. It was proven to occur in the late 1980s and early 1990s with the identification of specific phosphorylated amino acid residues within the receptor. The application of sophisticated mass spectrometry methods and assessment of distinct model systems have led to more recent identification of new GR phosphorylation sites previously unrecognized. Currently, 16 highly conserved amino acids within the GR (15 within the amino terminal domain and one within the hinge region) have been shown to be phosphorylated, with 15/16 occurring within serine and/or threonine residues and only one at a tyrosine residue. Many of the GR phosphorylation sites are regulated by ligand binding to the receptor, with two recently identified phosphorylation sites induced by brain-­derived neurotrophic factor (BDNF). Studies using pharmacological approaches to modulate GR phosphorylation first pointed to a role for site-­specific GR phosphorylation in directing distinct transcriptional responses of the receptor. The application of site-­directed mutagenesis, as well as biochemical and genomic approaches, revealed the mechanistic basis for distinct transcriptional responses directed by GR with site-­specific phosphorylation. Thus, each phosphorylation site on GR presents a unique surface that impacts the binding affinity of a wide variety of cofactors.60-­62 These selective phospho-­GR/cofactor complexes are likely to be differentially stabilized at direct or indirect GR binding sites on the genome, ultimately leading to selective modulation of transcriptional output from linked glucocorticoid-­responsive promoters. Furthermore, this modulation of GR transcriptional activity will be responsive to many physiological signals or drugs that impact the activity of intracellular protein kinases and phosphatases that directly act on the receptor. For example, long-­acting beta-­2-­adrenergic agonists, which are used in combination with inhaled corticosteroids to treat moderate or serve persistent asthma, may enhance the antiinflammatory effects of glucocorticoids through modulation of site-­specific GR phosphorylation.63,64 In many cases, site-­directed mutagenesis has confirmed the biological effects of specific GR phosphorylation sites. For example, GR phosphorylation at serine 134 is elevated in triple negative breast cancer

CHAPTER 89  Glucocorticoid Receptors: Mechanisms of Action in Health and Disease (TNBC) relative to other breast cancers.65 Furthermore, in vitro experiments with TNBC cells established the role for serine 134 phosphorylation in the migratory, invasive, and stem cell properties of these cells, demonstrating that GR phosphorylated at serine 134 generates a unique transcriptome. The relevance of these in vitro results was suggested from analysis of gene expression profiles of breast cancer patient samples, which reveal poorer survival outcomes in those stratified patients with relatively higher expression levels of the GR phosphoserine-­ 134 gene signature. In mice, BDNF-­regulated GR phosphorylation plays a role in the retention of newly learned motor skills through a mechanism that involves the stabilization of newly formed postsynaptic dendritic spines in the motor cortex.66 The impact of BDNF on multiple brain circuits regulating resilience to stress could be driven in part by regulation of specific GR phosphorylation events.59 Assessment of site-­specific phosphorylation of GR may be a useful clinical biomarker for diseases influenced by altered GC signaling, as evidenced by studies associating changes in site-­specific receptor phosphorylation with severe asthma67 and type 2 diabetes.68 In addition to dynamic changes in GR phosphorylation in response to physiologic or therapeutic inputs, receptor phosphorylation is also likely influenced by cell-­and tissue-­specific factors. GR phosphorylation is prominent in the central nervous system,69 and many studies in animal models have identified site-­specific modulations of receptor phosphorylation within select brain regions in response to acute and chronic insults or various types of stress.70-­72 Some of the same GR phosphorylation sites implicated in rodent stress responses are altered in peripheral blood mononuclear cells (PBMCs) of humans with neuropsychiatric conditions, in some cases in a sex-­specific manner.73,74 Thus, a comprehensive analysis of GR phosphorylation in PBMCs may provide some insights into changes in GC signaling in the brain that are associated with select neuropsychiatric disorders. Other posttranslational modifications in addition to phosphorylation have been shown to modulate GR function. Degradation of the GR through the ubiquitin-­proteasome pathway requires modification of a specific receptor lysine residue (lysine 419 in human GR) by ubiquitin.75 This posttranslational modification involves the concerted action of three specific enzymes culminating in the covalent addition of the 79-amino acid ubiquitin moiety on GR by an E3 ligase enzyme. A number of E3 ubiquitin ligases have been identified that catalyze the ubiquitylation and subsequent degradation of GR,76-­78 and in some cases, the signaling pathways that regulate GR ubiquitylation and turnover have been identified and biological effects revealed.79 Small ubiquitin-­like modifiers are small proteins that are also covalently attached to GR using an analogous multienzyme pathway related to the ubiquitin pathway. However, rather than target the receptor for degradation, sumoylation of GR influences its promoter-­specific transcriptional regulatory activity.80-­82 Finally, GR has been shown to be subjected to nitration83 and acetylation,84,85 which may regulate the receptor’s action in antiinflammatory and circadian pathways, respectively. KEY POINTS  • Posttranslational modifications of glucocorticoid receptor (GR) include phosphorylation, ubiquitination, sumoylation, nitration, and acetylation. • Site-­specific phosphorylation of GR directs unique transcriptional responses by regulating the receptor’s interaction with various cofactors. • Site-­specific phosphorylation of GR has specific physiological and pathological effects. • Site-­specific ubiquitination of GR alters the degradation properties of the receptor.

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GLUCOCORTICOID RECEPTOR PHYSIOLOGY Glucocorticoids have major effects on the organ systems of the body. Understanding the physiological actions of glucocorticoids has been aided by the development of novel mouse strains with conditional knockout of GR in specific cell types.86 The following sections summarize recent progress elucidating the role of glucocorticoids and GR signaling in the immune system, cardiovascular system, central nervous system, and metabolism.

Immune System Proper modulation of immune system activity and inflammation is critical to normal human function. A blunted immune response leaves the door open for potentially fatal infections, whereas an overstimulated immune response can result in autoimmune activity that damages organs. Glucocorticoids are widely used in the clinic for their ability to suppress the immune system and inhibit inflammation.87,88 The glucocorticoid-­mediated immunosuppression impacts both the innate and adaptive immune response and involves multiple mechanisms, including the stimulation of antiinflammatory genes, repression of proinflammatory genes (primarily by inhibiting NF-­κB and AP-­1 activity), and regulation of genes involved in apoptosis. The innate immune system is the first line of defense in response to pathogen or injury and is mediated by leukocytes, macrophages, and dendritic cells, among others. The adaptive immune response is a second line of defense mediated by T and B lymphocytes. Glucocorticoids achieve their immunosuppressive effects by acting on nearly all cell types of the immune system. Macrophages play a central role in innate immunity via the expression of pattern recognition receptors that sense infectious agents and harmful signals. This leads to activation of the inflammasome complex and the release of proinflammatory cytokines. GR signaling has been shown to suppress the immune response in macrophages as mice lacking GR specifically in macrophages exhibit greater mortality and increased cytokine production in response to LPS than wild-­type mice.89 A critical role for macrophage GR in the downregulation of inflammatory cytokines and chemokines in contact dermatitis has also been demonstrated in macrophage GR knockout mice.90 Interestingly, glucocorticoids have also been shown to augment the proinflammatory response in macrophages by inducing expression of inflammasome components and specific Toll-­like receptors and by promoting macrophage migration via the upregulation of exopeptidase dipeptidyl peptidase-­4.88,91 Thus, macrophage GR signaling may function initially to prime the immune system to respond to injury and then subsequently to limit the inflammatory response and restore homeostasis. Dendritic cells are antigen-­presenting cells that coordinate both innate and adaptive immunity. Glucocorticoids suppress the maturation and migration of dendritic cells and stimulate their death by apoptosis.92 Mice lacking GR in dendritic cells have revealed that glucocorticoids protect against LPS-­induced sepsis by suppressing the expression of the proinflammatory cytokine IL-­12 by dendritic cells.93 Proinflammatory T-­ cells of the adaptive immune response undergo apoptosis in response to glucocorticoids.92 Mice with conditional knockout of GR in T-­cells have revealed that glucocorticoid signaling limits potentially lethal cytokine production by T-­ cells in response to immune activation.94 In addition, studies on T-­cellspecific GR-­deficient mice have shown that the beneficial actions of glucocorticoids in models of rheumatoid arthritis, multiple sclerosis, sepsis, and helminth infection are mediated by GR signaling events that inhibit T-­cell function, alter T-­cell migration, and/or lead to thymocyte cell death.95-­99 Furthermore, it has been demonstrated in these conditional knockout mice that GR signaling promotes T-­cell

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selection.100 The function of B-­cells is also modulated by glucocorticoids. Glucocorticoids regulate B-­cell activation, survival, proliferation, and differentiation.101 Moreover, it was recently demonstrated in mice with B-­cell–specific deletion of GR that glucocorticoid signaling is required for proper migration of mature B-­cells from blood to bone marrow.102 Many immune genes are regulated by GR in a sex-­specific manner.103 In rats, glucocorticoids were shown to regulate a greater number of inflammatory genes and elicit a greater antiinflammatory response in males compared with females.104 Sexual dimorphism in the immunosuppressive actions of glucocorticoids not only provides a mechanistic basis for the predisposition of females to develop autoimmune diseases but also suggests that glucocorticoid dosages used in the clinic should be optimized based on the sex of the patient.

Cardiovascular System Glucocorticoids can have both positive and negative effects on the heart.105 The contractile performance of the heart is improved by glucocorticoid treatment, and cardiomyocyte cell death triggered by various insults can be blocked by glucocorticoids. However, elevated glucocorticoids can also lead to bradycardia, cardiac hypertrophy, and heart failure. Moreover, major risk factors for cardiovascular disease, including metabolic syndrome and hypertension, are associated with increased levels of glucocorticoids. Differences in physiological context and the magnitude and duration of the glucocorticoid exposure likely play a role in whether the actions of glucocorticoids on the cardiovascular system are beneficial or adverse. The cardiovascular outcomes may also depend on whether glucocorticoids are acting directly on the heart or indirectly via their systemic effects on blood pressure and metabolism. New insights into the direct actions that glucocorticoids have on the heart have come from transgenic mouse models with altered expression of GR in cardiomyocytes.105 Mice overexpressing GR in cardiomyocytes have conduction defects, including bradycardia and atrioventricular block.106 Prenatal studies performed on mice with global knockout of GR or selective knockout of GR in cardiomyocytes revealed a critical role for GR in the maturation of the fetal heart.107,108 Glucocorticoid signaling in fetal cardiomyocytes was necessary to maintain appropriate expression levels of genes important for cardiomyocyte architecture and function. Using adult mice bearing a conditional deletion of GR in cardiomyocytes, several laboratories have demonstrated that GR signaling is required for normal heart physiology and function.109-­111 Mice lacking cardiomyocyte GR spontaneously develop heart disease characterized by pathological cardiac hypertrophy, left ventricular systolic dysfunction, heart dilatation, and fibrosis, and these knockout mice die prematurely from heart failure. Mechanistically, the absence of cardiomyocyte GR signaling leads to the dysregulation of key genes involved in calcium handling. Interestingly, cardiomyocyte GR has been shown to act in a sexually dimorphic manner, as male mice lacking the receptor were more sensitive to heart disease than their female counterparts.112 In addition, deleterious signaling by the closely related mineralocorticoid receptor (MR), which can be bound and activated by glucocorticoids, was found to exacerbate the pathology in hearts lacking GR, suggesting that balanced cardiomyocyte GR and MR signaling is critical for normal heart homeostasis.109

Central Nervous System Receptors for glucocorticoids are widely expressed in the central nervous system (CNS) and thereby influence various behaviors, emotional responses, learning and memory, and central control of metabolic and autonomic function. The expression of MR, which is activated by basal levels of natural glucocorticoids, such as cortisol in humans, is

much more restricted in the brain than GR and is limited primarily to structures within the limbic system (i.e., hippocampus, amygdala, and prefrontal cortex).113 Furthermore, MR is responsive to cortisol in the brain, because it lacks 11β-­hydroxysteroid dehydrogenase-­2, the cortisol inactivating enzyme highly expressed in kidney that blocks MR activation by cortisol in kidney. Extensive studies in humans and animal models established a role for MR in various aspects of memory and executive function, as well as cognitive responses to stress.114 Both genomic115 and nongenomic116 actions of MR are likely to mediate its complex regulation of memory, cognition, and emotional state and driven by dynamic changes in connectivity and neurotransmission that accompany a central response to an emerging stress. As GRs are also eventually engaged in the central response to stress, the dynamic shift from MR-­to GR-­driven outputs117 aids in the recognition, processing, and response to variable stresses that is adaptive. Chronic stress, long-­term exposure to synthetic glucocorticoids, or disruption of the ultradian and/or circadian rhythm of endogenous glucocorticoid accumulation can alter the dynamic balance between MR and GR signaling in the CNS, leading to maladaptive responses to stress that can contribute to affective disorders.118 GRs in the CNS regulate behavior, cognition, and affective functions; however, they also control energy metabolism,119 acting through specific neuroendocrine cells within the hypothalamus.120 Regulation of the HPA axis is another function of central GRs, although negative feedback of adrenal glucocorticoid synthesis and secretion also operates at the level of corticotrophs within the anterior pituitary. Glucocorticoid suppression of corticotrophin-­ releasing hormone (CRH) synthesis and release from neuroendocrine cells within the paraventricular nucleus in the hypothalamus involves numerous pathways and circuits regulated directly and indirectly by central GRs.121 In the paraventricular nucleus (PVN), rapid inhibition of CRH release is due to glucocorticoids acting indirectly via a nongenomic mechanism to reduce glutamatergic drive to CRH neurons.121 Specifically, activation of classical GR located within the plasma membrane stimulates the production of endocannabinoids, which act on cannabinoid receptor type 1 within presynaptic terminals that synapse onto CRH neurons to reduce glutamate release.122,123 As in other examples of nongenomic glucocorticoid actions in the brain,124 rapid stimulation of endocannabinoid production by glucocorticoids require nuclear GR localized at the plasma membrane.125 Multiple intracellular pathways are activated by ligand-­bound plasma membrane-associated nuclear GR, although mobilization of intracellular calcium stores is one of the more prominent features of rapid GR signaling in neuronal systems.124,126 In contrast to the inhibitory effects of nongenomic GR signaling on neurotransmission in the PVN, stress-­induced activation of plasma membrane GR and MR via glucocorticoids in limbic areas increases glutamate release and is likely driven by intracellular signaling pathways distinct from those that modulate glucocorticoid effects on endocannabinoid production.127,128 The completion of central negative feedback control of the HPA axis requires GR action within limbic structures, including the prefrontal cortex and hippocampus.121,129 While the importance of “delayed feedback” from limbic areas to regulate HPA axis function had been recognized for many years based upon lesion and pharmacologic studies,129,130 identification of the specific regions and cell types that utilize transcriptional responses of GR to modulate inhibitory inputs into CRH neurons was firmly established using mouse models with cell type–specific deletions of the receptor.86,131,132

Hepatic Carbohydrate and Lipid Metabolism Glucocorticoid effects on carbohydrate and lipid metabolism are widely recognized.133-­135 The homeostatic effects of these hormones

CHAPTER 89  Glucocorticoid Receptors: Mechanisms of Action in Health and Disease on systemic metabolism are due to their promotion of gluconeogenesis in the liver and the mobilization of substrates in peripheral tissues, such as white adipose and skeletal muscle. They also limit glucose update in peripheral tissues and promote glycogen storage in liver. Glucocorticoids also promote gluconeogenesis and lipid metabolism through “permissive” effects on other hormones, including glucagon and catecholamines.133-­135 The mechanisms responsible for glucocorticoid stimulation of gluconeogenesis in liver have been extensively studied and rely primarily, but not exclusively, on the transcriptional induction by GR of many genes in the gluconeogenic pathway.135,136 This transcriptional response is essential to physiologic regulation of glucose homeostasis, as evidenced by hypoglycemia caused by adrenal insufficiency. Much of the current understanding of the molecular mechanisms responsible for hepatocyte-­specific transcriptional regulation of gluconeogenesis comes from extensive molecular studies of the phosphoenolpyruvate carboxykinase (PCK1) gene, which revealed one of the earliest examples of “composite” GREs that include GR and other tissue-­or cell type–enriched transcription factors required for a functional glucocorticoid response.136-­138 Members of the forkhead box transcriptional factor family play a prominent role in both glucocorticoid and insulin regulation of many genes in the gluconeogenesis pathway in hepatocytes.135,139-­141 In mouse liver the C/EBPβ protein is required to “preprogram” chromatin accessible sites to allow for GR binding and an ensuing transcriptional response.142 However, this is only one level of complexity in GR regulation of gluconeogenesis, as the dynamics of GR cooperation with other transcription factors can change in response to the duration of a fasting state.143 Specifically, during a short-­term fast, GR enhances the recruitment of the cyclic AMP response element-binding protein 1 (CREB1) transcription factor to gluconeogenic gene enhancers, leading to synergistic activation of transcription initially mobilized following glucagon-­induced site-­specific phosphorylation and activation of CREB1.144 In contrast, in response to a long-­term fast, GR promotes the transcription of ketogenic and fatty acid oxidation pathway genes through the induction of a major regulator of these pathways, the peroxisome proliferator-­activated receptor alpha (PPARα).144 Glucocorticoids also regulate lipogenesis in hepatocytes and increase triglyceride levels primarily through the inhibition of lipolysis and induction of triglyceride synthesis.136,145 The impact of glucocorticoids on lipid metabolism in the liver is not limited to direct action of the GR on genes in the lipogenesis pathway, as other signaling pathways affecting glucose metabolism and insulin sensitivity influence the effect of glucocorticoids on lipid metabolism.136,143,146 For example, the gene encoding the transcriptional repressor Hes1 is a direct GR target gene, and repression of its transcription by glucocorticoids is essential not only for the regulation of lipid metabolism in hepatocytes,147 but also for many systemic responses to glucocorticoids.148

GLUCOCORTICOID RECEPTOR LIGANDS AND PULSATILITY The physiological response to glucocorticoids occurs in the context of their rhythmic production and secretion from the adrenal gland. Specifically, superimposed upon the daily fluctuations in circulating glucocorticoid levels, which peaks in the early morning and reaches its nadir around midnight in humans, are ultradian rhythms of glucocorticoid secretion that have a periodicity of 60 to 90 minutes.149 Different physiological pathways underlie the complex regulation of daily versus ultradian rhythms of circulating glucocorticoids, with circadian pacemakers in the hypothalamus predominantly regulating the daily rhythm of glucocorticoid secretion from the adrenal,

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while ultradian rhythms are mediated by the balance between feedforward control of adrenal glucocorticoid production by pituitary ACTH and negative feedback by glucocorticoids on ACTH production.150 Various approaches have been used in animal models to reveal an impact of altered glucocorticoid rhythms on metabolism, inflammation, and multiple behavioral outcomes. Alterations in the daily rhythms of glucocorticoids modulate their action on regulators of circadian rhythms (e.g., PER and REV-­ERBA) in peripheral organs contributing to disruptions in carbohydrate and lipid metabolism.151,152 In humans, chronic elevations of endogenous or exogenous glucocorticoids in Cushing syndrome trigger high morbidity and mortality associated with pathophysiological effects on metabolism, cardiovascular function, immune responses, and behavior.149 These adverse effects may be exacerbated by alterations in circadian clock gene expression, which coordinate many external outputs to optimize systemic metabolic activity. This has led to glucocorticoid replacement therapy regimens for patients with adrenal insufficiency that mimic the natural daily and ultradian rhythm of glucocorticoid secretion (i.e., “chronopharmacotherapy”), which have demonstrated clinical benefit.153 Molecular changes in cellular response to glucocorticoid chronotherapy have also been reported. Specifically, analysis of peripheral blood mononuclear cells from patients in the Dual Release Hydrocortisone versus Conventional Glucocorticoid Replacement Treatment in Hypocortisolism (DREAM) trial revealed physiological patterns of CLOCK gene expression by modified release hydrocortisone but not conventional multiple daily doses.154 Modified release patients also had better body weight reduction, normal immune profiles, and improved quality of life. Furthermore, in healthy subjects with pharmacological suppression of glucocorticoid synthesis, the restoration of normal daily and ultradian rhythms of cortisol was most effective at maintaining positive mood and “resting state neural dynamics,” as revealed by functional neuroimaging.155 Finally, the administration of synthetic glucocorticoids (i.e., two daily doses 24 hours apart) to pregnant women “out of phase” with their natural daily rhythm of circulating cortisol can have long-­ lasting effects on the stress responsivity of their infants at 5 years of age.156 Therefore, glucocorticoid chronotherapy that takes into account the natural rhythm of cortisol may have long-­lasting clinical benefit in patients requiring chronic or even acute glucocorticoid treatment. How do the patterns of glucocorticoid rhythms affect the cellular response of the GR? Synthetic glucocorticoids used therapeutically typically exhibit a higher affinity for GR than cortisol and dissociate much more slowly from the receptor. These differences in the kinetics of ligand occupancy of the receptor modulate the dynamics of GR binding to specific target sites in the genome. Cycles of GR occupancy on target genes are further modified by ATP and chaperone proteins operating in the nucleus.157,158 At the molecular level, the dynamics of GR binding to genomic sites in cells occurs at a much more rapid time scale (i.e., seconds) than ultradian pulses of natural glucocorticoids.15 This inherent property of the GR and other transcription factors may aid in the stochastic sampling of preassembled macromolecular complexes within the genome and facilitate the selectivity of transcriptional responses of the receptor. Finally, dynamic chromatin occupancy of the GR in cells and animal models driven by pulsatile exposure to natural glucocorticoids that mimic ultradian rhythms can impact gene-­specific responses to glucocorticoids.159 Therefore, enhanced clinical efficacy of glucocorticoid chronotherapy may be driven by natural cycles in the genomic structure and organization of GR-­ responsive genes that require the appropriate assembly of receptor-­interacting factors for an efficient transcriptional response to glucocorticoid exposure.

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KEY POINTS  • Glucocorticoids are secreted by the adrenal gland in circadian and ultradian rhythms. • Glucocorticoid chronotherapy that mimics the natural rhythm of circulating cortisol has demonstrated clinical benefit.

GLUCOCORTICOID RECEPTOR RESISTANCE Generalized glucocorticoid resistance (GGR) or “Chrousos syndrome” is a rare disorder characterized by ACTH-­dependent hypercortisolism and the absence of clinical features of glucocorticoid excess.160,161 The glucocorticoid sensitivity of all tissues is decreased in GGR. Physiologically normal serum cortisol concentrations are therefore insufficient to suppress CRH and ACTH secretion from the hypothalamus and anterior pituitary, respectively. Consequently, ACTH levels are elevated and stimulate the adrenal glands to produce greater than normal amounts of cortisol, adrenal androgens, and mineralocorticoids. In GGR, glucocorticoid resistance extends to peripheral tissues, although sensitivity to androgens and mineralocorticoids is normal. Hence, the clinical findings are not those of glucocorticoid excess, but rather those of mineralocorticoid excess (hypertension and hypokalemia) or hyperandrogenism. Treatment of Chrousos syndrome consists of suppressing the excess ACTH secretion through administration of mineralocorticoid-­sparing glucocorticoids such as dexamethasone. Loss of function mutations within the GR gene have been identified in a majority of individuals with GGR, with the LBD as the most common site of mutation.161,162 While many LBD mutations in GGR reduce hormone-­binding affinity, the transcriptional regulatory activity of GR can also be affected by LBD mutations that do not impact ligand binding.163 Loss-­of-­function mutations have also been found in other GR domains in GGR individuals leading to reduced DNA binding, nuclear translocation, or expression.161 In some individuals with GGR, GR gene mutations have not been identified, suggesting that other components of the glucocorticoid signaling pathway in GGR may be uniquely sensitive to specific gene mutations that do not affect other endocrine systems.164,165 Acquired glucocorticoid resistance is much more common than GGR and is often characterized by tissue-­selective resistance of GR to endogenous or synthetic glucocorticoids.166 This form of resistance is often associated with chronic inflammatory conditions,167,168 corticotropinomas,169,170 or hematological malignancies.166,171 This resistance reduces the therapeutic benefit of glucocorticoids to treat leukemia, lymphoma, and myeloma, as well as many chronic inflammatory conditions. Although uncommon, somatic GR mutations have been identified that are responsible for malignant cell resistance to glucocorticoid therapy, although they are not associated with GGR, as expected from their somatic origin.166 More common mechanisms responsible for glucocorticoid resistance in malignant cells are reductions in GR expression and the generation of alternative GR mRNA splice isoforms, such as GRβ discussed earlier. Recently, genome-­wide analysis identified novel genes and pathways contributing to glucocorticoid resistance in acute lymphoblastic leukemia.172 Such comprehensive genomic analysis of glucocorticoid resistance is likely to generate novel therapeutic strategies and drug combinations that could mitigate malignant cell resistance to glucocorticoid therapy.172 Most severe asthma patients do not respond well to glucocorticoid treatment and are designated as having glucocorticoid-­unresponsive, refractory, or resistant asthma.173-­175 The molecular basis of glucocorticoid-­resistant asthma has been extensively studied, revealing several molecular and cellular mechanisms that may contribute to

the failure of this typically highly effective class of agents to control asthma symptoms and exacerbations.168,173,175 Mutations or polymorphisms within the GR gene are unlikely to be a major contributor to glucocorticoid-­resistant asthma, although some rare cases in specific ethnic populations may exist.176 However, genome-­ wide association studies have identified a functional variant in a glucocorticoid-­ regulated gene (i.e., the glucocorticoid-­induced transcript 1) that is associated with reduced lung function response to inhaled glucocorticoids.177 Additional rare genetic variants that contribute to the development of glucocorticoid-­resistant asthma may be uncovered with more extensive pharmacogenetic and pharmacogenomic analyses in distinct populations.178 As mentioned earlier for other forms of glucocorticoid resistance, an increase in the GRβ/GRα ratio plays a significant role in glucocorticoid-­resistant asthma.179-­182 The induction of GRβ gene expression in airway epithelial cells that could generate glucocorticoid resistance is promoted by specific T-­helper-type 17 cytokines.183,184 Activation of the p38 mitogen-­activated protein kinase (MAPK) by proinflammatory cytokines is one intracellular pathway that contributes to upregulation of GRβ expression and the development of glucocorticoid-­resistant asthma.185,186 The impact of proinflammatory cytokines and activated MAPK pathways on glucocorticoid-­resistant asthma are not limited to the regulation of GRβ expression and extend to inhibitory effects on various components of the GRα intracellular signaling, including DNA binding,187 nuclear translocation,188 and interaction with transcription factor or coregulator partners.187,189,190 Some of these effects may be mediated by direct posttranslational modification of GRα (e.g., phosphorylation191) or alterations in receptor or partner protein oxidation as a result of oxidative or nitrative stress.192 KEY POINTS  • Generalized glucocorticoid resistance affects all tissues and is most often caused by mutations in the glucocorticoid receptor (GR) ligand-­binding domain, resulting in reduced hormone-­binding affinity. • Acquired glucocorticoid resistance is tissue-­specific and is commonly caused by reductions in GR expression and/or increased expression of the dominant-­negative splice variant GRβ.

SUMMARY AND FUTURE DIRECTIONS Glucocorticoids act through GR to regulate numerous physiological processes, and synthetic derivatives of these hormones are widely prescribed for their antiinflammatory and immunosuppressive actions. Tissue responses to glucocorticoids are markedly different, and this heterogeneity in glucocorticoid signaling presents a challenge to clinicians in managing patients on glucocorticoid therapy. The diversity in glucocorticoid signaling is achieved by multiple processes, including polymorphisms in the GR gene, the generation of various GR isoforms by alternative splicing and alternative translation initiation, and posttranslational modifications of the receptor protein. The capacity of a cell to generate dozens of GR isoforms with unique gene-­regulatory profiles provides enormous potential for variations in GR signaling. Moreover, the glucocorticoid response is impacted by the glucocorticoid secretion rhythm and by both general and acquired glucocorticoid resistance. A critical goal of future studies will be to use genetic approaches to assess the contribution that an individual GR isoform makes to the actions of glucocorticoids in the whole animal. Additionally, it will be important to determine whether changes in the tissue complement of GR isoforms underlie pathologies characterized by glucocorticoid resistance and/or the severe side effects

CHAPTER 89  Glucocorticoid Receptors: Mechanisms of Action in Health and Disease that accompany glucocorticoid treatment. Finally, it will be crucial to determine the transcriptome for individual GR isoforms and to assess how it changes in response to receptor posttranslational modifications. A greater understanding of the factors and molecular processes that control tissue-­specific responses to glucocorticoids should aid in the development of safer and more effective glucocorticoid therapies.

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109. Oakley RH, Cruz-­Topete D, He B, et al. Cardiomyocyte glucocorticoid and mineralocorticoid receptors directly and antagonistically regulate heart disease in mice. Sci Signal. 2019;12:eaau9685. 110. Oakley RH, Ren R, Cruz-­Topete D, et al. Essential role of stress hormone signaling in cardiomyocytes for the prevention of heart disease. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:17035–17040. 111. Richardson RV, Batchen EJ, Thomson AJ, et al. Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis. J Endocrinol. 2017;232:437–450. 112. Cruz-­Topete D, Oakley RH, Carroll NG, et al. Deletion of the cardiomyocyte glucocorticoid receptor leads to sexually dimorphic changes in cardiac gene expression and progression to heart failure. J Am Heart Assoc. 2019;8:e011012. 113. Reul JM, de Kloet ER. Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology. 1985;117:2505–2511. 114. de Kloet ER, Otte C, Kumsta R, et al. Stress and depression: a crucial role of the mineralocorticoid receptor. J Neuroendocrinol. 2016;28:1–12. 115. Mifsud KR, Reul J. Mineralocorticoid and glucocorticoid receptor-­ mediated control of genomic responses to stress in the brain. Stress. 2018;21:389–402. 116. Karst H, Berger S, Turiault M, et al. Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:19204–19207. 117. de Kloet ER. From receptor balance to rational glucocorticoid therapy. Endocrinology. 2014;155:2754–2769. 118. McEwen BS, Akil H. Revisiting the stress concept: implications for affective disorders. J Neurosci. 2020;40:12–21. 119. Kellendonk C, Eiden S, Kretz O, et al. Inactivation of the GR in the nervous system affects energy accumulation. Endocrinology. 2002;143:2333– 2340. 120. Shibata M, Banno R, Sugiyama M, et al. AgRP neuron-­specific deletion of glucocorticoid receptor leads to increased energy expenditure and decreased body weight in female mice on a high-­fat diet. Endocrinology. 2016;157:1457–1466. 121. Herman JP, McKlveen JM, Ghosal S, et al. Regulation of the hypothalamic-­pituitary-­adrenocortical stress response. Compr Physiol. 2016;6:603–621. 122. Di S, Malcher-­Lopes R, Halmos KC, et al. Nongenomic glucocorticoid inhibition via endocannabinoid release in the hypothalamus: a fast feedback mechanism. J Neurosci. 2003;23:4850–4857. 123. Evanson NK, Tasker JG, Hill MN, et al. Fast feedback inhibition of the HPA axis by glucocorticoids is mediated by endocannabinoid signaling. Endocrinology. 2010;151:4811–4819. 124. Samarasinghe RA, Di Maio R, Volonte D, et al. Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:16657–16662. 125. Nahar J, Haam J, Chen C, et al. Rapid nongenomic glucocorticoid actions in male mouse hypothalamic neuroendocrine cells are dependent on the nuclear glucocorticoid receptor. Endocrinology. 2015;156:2831– 2842. 126. Harris C, Weiss GL, Di S, Tasker JG. Cell signaling dependence of rapid glucocorticoid-­induced endocannabinoid synthesis in hypothalamic neuroendocrine cells. Neurobiol Stress. 2019;10:100158. 127. Gray JD, Kogan JF, Marrocco J, et al. Genomic and epigenomic mechanisms of glucocorticoids in the brain. Nat Rev Endocrinol. 2017;13:661– 673. 128. Popoli M, Yan Z, McEwen BS, et al. The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission. Nat Rev Neurosci. 2011;13:22–37. 129. Diorio D, Viau V, Meaney MJ. The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-­pituitary-­adrenal responses to stress. J Neurosci. 1993;13:3839–3847. 130. Keller-­Wood ME, Dallman MF. Corticosteroid inhibition of ACTH secretion. Endocr Rev. 1984;5:1–24.

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131. Boyle MP, Kolber BJ, Vogt SK, et al. Forebrain glucocorticoid receptors modulate anxiety-­associated locomotor activation and adrenal responsiveness. J Neurosci. 2006;26:1971–1978. 132. Solomon MB, Furay AR, Jones K, et al. Deletion of forebrain glucocorticoid receptors impairs neuroendocrine stress responses and induces depression-­like behavior in males but not females. Neuroscience. 2012;203:135–143. 133. Exton JH. Regulation of gluconeogenesis by glucocorticoids. Monogr Endocrinol. 1979;12:535–546. 134. Kraus-­Friedmann N. Hormonal regulation of hepatic gluconeogenesis. Physiol Rev. 1984;64:170–259. 135. Kuo T, McQueen A, Chen TC, et al. Regulation of Glucose Homeostasis by Glucocorticoids. Adv Exp Med Biol. 2015;872:99–126. 136. Patel R, Williams-­Dautovich J, Cummins CL. Minireview: new molecular mediators of glucocorticoid receptor activity in metabolic tissues. Mol Endocrinol. 2014;28:999–1011. 137. Imai E, Stromstedt PE, Quinn PG, et al. Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate carboxykinase gene. Molecular and Cellular Biology. 1990;10:4712–4719. 138. Imai E, Miner JN, Mitchell JA, et al. Glucocorticoid receptor-­cAMP response element-­binding protein interaction and the response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids. J Biol Chem. 1993;268:5353–5356. 139. Nakae J, Kitamura T, Silver DL, et al. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-­6-­phosphatase expression. J Clin Invest. 2001;108:1359–1367. 140. Wolfrum C, Asilmaz E, Luca E, et al. Foxa2 regulates lipid metabolism and ketogenesis in the liver during fasting and in diabetes. Nature. 2004;432:1027–1032. 141. Puigserver P, Rhee J, Donovan J, et al. Insulin-­regulated hepatic gluconeogenesis through FOXO1-­PGC-­1alpha interaction. Nature. 2003;423:550–555. 142. Grontved L, John S, Baek S, et al. C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements. EMBO J. 2013;32:1568–1583. 143. Xiao Y, Kim M, Lazar MA. Nuclear receptors and transcriptional regulation in non-­alcoholic fatty liver disease. Mol Metab. 2020;101119. 144. Goldstein I, Baek S, Presman DM, et al. Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response. Genome Res. 2017;27:427–439. 145. Dolinsky VW, Douglas DN, Lehner R, et al. Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-­esterification by the glucocorticoid dexamethasone. Biochem J. 2004;378:967–974. 146. Patel R, Patel M, Tsai R, et al. LXRbeta is required for glucocorticoid-­ induced hyperglycemia and hepatosteatosis in mice. J Clin Invest. 2011;121:431–441. 147. Lemke U, Krones-­Herzig A, Berriel Diaz M, et al. The glucocorticoid receptor controls hepatic dyslipidemia through Hes1. Cell Metab. 2008;8:212–223. 148. Revollo JR, Oakley RH, Lu NZ, et al. HES1 is a master regulator of glucocorticoid receptor-­dependent gene expression. Sci Signal. 2013;6:ra103. 149. Lightman SL, Wiles CC, Atkinson HC, et al. The significance of glucocorticoid pulsatility. Eur J Pharmacol. 2008;583:255–262. 150. Lightman SL, Birnie MT, Conway-­Campbell BL. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev. 2020;41:470–490. 151. Quagliarini F, Mir AA, Balazs K, et al. Cistromic reprogramming of the diurnal glucocorticoid hormone response by high-­fat diet. Molecular Cell. 2019;76:531–545. e535. 152. Bahrami-­Nejad Z, Zhao ML, Tholen S, et al. A transcriptional circuit filters oscillating circadian hormonal inputs to regulate fat cell differentiation. Cell. Metab. 2018;27:854–868. e858. 153. Minnetti M, Hasenmajer V, Pofi R, et al. Fixing the broken clock in adrenal disorders: focus on glucocorticoids and chronotherapy. J Endocrinol. 2020;246:R13–R31. 154. Venneri MA, Hasenmajer V, Fiore D, et al. Circadian rhythm of glucocorticoid administration entrains clock genes in immune cells: a DREAM trial ancillary study. The Journal of Clinical Endocrinology and Metabolism. 2018;103:2998–3009.

155. Kalafatakis K, Russell GM, Ferguson SG, et al. Glucocorticoid ultradian rhythmicity differentially regulates mood and resting state networks in the human brain: a randomised controlled clinical trial. Psychoneuroendocrinology. 2021;124:105096. 156. Astiz M, Heyde I, Fortmann MI, et al. The circadian phase of antenatal glucocorticoid treatment affects the risk of behavioral disorders. Nat Commun. 2020;11:3593. 157. Elbi C, Walker DA, Romero G, et al. Molecular chaperones function as steroid receptor nuclear mobility factors. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:2876–2881. 158. Conway-­Campbell BL, George CL, Pooley JR, et al. The HSP90 molecular chaperone cycle regulates cyclical transcriptional dynamics of the glucocorticoid receptor and its coregulatory molecules CBP/p300 during ultradian ligand treatment. Mol Endocrinol. 2011;25:944–954. 159. Stavreva DA, Wiench M, John S, et al. Ultradian hormone stimulation induces glucocorticoid receptor-­mediated pulses of gene transcription. Nat Cell Biol. 2009;11:1093–1102. 160. Chrousos GP, Vingerhoeds A, Brandon D, et al. Primary cortisol resistance in man. A glucocorticoid receptor-­mediated disease. J Clin Invest. 1982;69:1261–1269. 161. Martins CS, de Castro M. Generalized and tissue specific glucocorticoid resistance. Mol Cell Endocrinol. 2021;530:111277. 162. Charmandari E, Kino T, Souvatzoglou E, et al. Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular genotype, genetic transmission, and clinical phenotype. The Journal of Clinical Endocrinology and Metabolism. 2004;89:1939–1949. 163. Charmandari E, Raji A, Kino T, et al. A novel point mutation in the ligand-­binding domain (LBD) of the human glucocorticoid receptor (hGR) causing generalized glucocorticoid resistance: the importance of the C terminus of hGR LBD in conferring transactivational activity. The Journal of Clinical Endocrinology and Metabolism. 2005;90:3696–3705. 164. Huizenga NA, de Lange P, Koper JW, et al. Five patients with biochemical and/or clinical generalized glucocorticoid resistance without alterations in the glucocorticoid receptor gene. The Journal of Clinical Endocrinology and Metabolism. 2000;85:2076–2081. 165. Nicolaides NC, Roberts ML, Kino T, et al. A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: dissociation of the transactivating and transreppressive activities. The Journal of Clinical Endocrinology and Metabolism. 2014;99:E902–E907. 166. Gross KL, Lu NZ, Cidlowski JA. Molecular mechanisms regulating glucocorticoid sensitivity and resistance. Mol Cell Endocrinol. 2009;300:7–16. 167. Vandewalle J, Luypaert A, De Bosscher K, et al. Therapeutic mechanisms of glucocorticoids. Trends Endocrinol Metab. 2018;29:42–54. 168. Barnes PJ, Adcock IM. Glucocorticoid resistance in inflammatory diseases. Lancet. 2009;373:1905–1917. 169. Antonini SR, Latronico AC, Elias LL, et al. Glucocorticoid receptor gene polymorphisms in ACTH-­secreting pituitary tumours. Clin Endocrinol (Oxf). 2002;57:657–662. 170. Ciato D, Albani A. Molecular mechanisms of glucocorticoid resistance in corticotropinomas: new developments and drug targets. Front Endocrinol (Lausanne). 2020;11:21. 171. Renner K, Ausserlechner MJ, Kofler R. A conceptual view on glucocorticoid-­induced apoptosis, cell cycle arrest and glucocorticoid resistance in lymphoblastic leukemia. Curr Mol Med. 2003;3:707–717. 172. Autry RJ, Paugh SW, Carter R, et al. Integrative genomic analyses reveal mechanisms of glucocorticoid resistance in acute lymphoblastic leukemia. Nat Cancer. 2020;1:329–344. 173. Barnes PJ, Greening AP, Crompton GK. Glucocorticoid resistance in asthma. Am J Respir Crit Care Med. 1995;152:S125–S140. 174. Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med. 2012;18:716–725. 1 75. Henderson I, Caiazzo E, McSharry C, et al. Why do some asthma patients respond poorly to glucocorticoid therapy? Pharmacol Res. 2020;160:105189. 176. Zhao F, Zhou G, Ouyang H, et al. Association of the glucocorticoid receptor D641V variant with steroid-­resistant asthma: a case-­control study. Pharmacogenet Genomics. 2015;25:289–295.

CHAPTER 89  Glucocorticoid Receptors: Mechanisms of Action in Health and Disease 177. Tantisira KG, Lasky-­Su J, Harada M, et al. Genomewide association between GLCCI1 and response to glucocorticoid therapy in asthma. N Engl J Med. 2011;365:1173–1183. 178. Edris A, de Roos EW, McGeachie MJ, et al. Pharmacogenetics of inhaled corticosteroids and exacerbation risk in adults with asthma. Clin Exp Allergy. 2021 http://dx.doi.10.1111/cea.13829. 179. Hamid QA, Wenzel SE, Hauk PJ, et al. Increased glucocorticoid receptor beta in airway cells of glucocorticoid-­insensitive asthma. Am J Respir Crit Care Med. 1999;159:1600–1604. 180. Leung DY, Hamid Q, Vottero A, et al. Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta. J Exp Med. 1997;186:1567–1574. 181. Sousa AR, Lane SJ, Cidlowski JA, et al. Glucocorticoid resistance in asthma is associated with elevated in vivo expression of the glucocorticoid receptor beta-­isoform. The Journal of Allergy and Clinical Immunology. 2000;105:943–950. 182. Goleva E, Li LB, Eves PT, et al. Increased glucocorticoid receptor beta alters steroid response in glucocorticoid-­insensitive asthma. Am J Respir Crit Care Med. 2006;173:607–616. 183. Al Heialy S, Gaudet M, Ramakrishnan RK, et al. Contribution of IL-­17 in steroid hyporesponsiveness in obese asthmatics through dysregulation of glucocorticoid receptors alpha and beta. Front Immunol. 2020;11:1724. 184. Vazquez-­Tello A, Semlali A, Chakir J, et al. Induction of glucocorticoid receptor-­beta expression in epithelial cells of asthmatic airways by T-­ helper type 17 cytokines. Clin Exp Allergy. 2010;40:1312–1322.

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185. Bhavsar P, Khorasani N, Hew M, et al. Effect of p38 MAPK inhibition on corticosteroid suppression of cytokine release in severe asthma. Eur Respir J. 2010;35:750–756. 186. Mercado N, To Y, Kobayashi Y, et al. p38 mitogen-­activated protein kinase-­gamma inhibition by long-­acting beta2 adrenergic agonists reversed steroid insensitivity in severe asthma. Mol Pharmacol. 2011;80:1128–1135. 187. Adcock IM, Lane SJ, Brown CR, et al. Differences in binding of glucocorticoid receptor to DNA in steroid-­resistant asthma. J Immunol. 1995;154:3500–3505. 188. Chang PJ, Michaeloudes C, Zhu J, et al. Impaired nuclear translocation of the glucocorticoid receptor in corticosteroid-­insensitive airway smooth muscle in severe asthma. Am J Respir Crit Care Med. 2015;191:54–62. 189. Adcock IM, Ito K, Barnes PJ. Histone deacetylation: an important mechanism in inflammatory lung diseases. COPD. 2005;2:445–455. 190. Qiu W, Guo F, Glass K, et al. Differential connectivity of gene regulatory networks distinguishes corticosteroid response in asthma. The Journal of Allergy and Clinical Immunology. 2018;141:1250–1258. 191. Irusen E, Matthews JG, Takahashi A, et al. p38 Mitogen-­activated protein kinase-­induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-­insensitive asthma. The Journal of Allergy and Clinical Immunology. 2002;109:649–657. 192. Barnes PJ. Mechanisms and resistance in glucocorticoid control of inflammation. J Steroid Biochem Mol Biol. 2010;120:76–85.

90 Adrenal Androgens, Adrenarche, and Adrenopause Selma Feldman Witchel, Anne Claire Burghard, and Sharon E. Oberfield

OUTLINE Adrenarche, 1498 The Adrenal Glands, 1498 The Fetal Adrenal Gland, 1498 Postnatal Adrenal Morphology and Function, 1499 Clinical Facets of Adrenarche, 1500 Adrenal Steroidogenesis, 1501 Dehydroepiandrosterone and in Vitro Fertilization, 1502 Adrenopause, 1503

Epidemiology/Associations with Dehydroepiandrosterone Sulfate Concentrations, Aging, and Cardiovascular Risks, 1503 Neurobiology of Dehydroepiandrosterone and Dehydroepiandrosterone Sulfate, 1504 Clinical Implications of Dehydroepiandrosterone Sulfate/Dehydroepiandrosterone, 1505 Dehydroepiandrosterone Sulfate/Dehydroepiandrosterone and “The Fountain of Youth”, 1505 Conclusion, 1505

 

ADRENARCHE The morphologic and functional characteristics of the adrenal glands change dramatically through the different stages of prenatal and postnatal development. Adrenarche is defined as the increased production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) by the zona reticularis of the adrenal glands between 6 and 8 years of age. However, with newer data indicating that urinary excretion of DHEA and its metabolites begins to increase at as early as 3 years of age, the age at onset of adrenarche is being reexamined.1 The phenomenon of adrenarche is observed in humans and some nonhuman primates. Current data suggest that the great apes and other nonhuman primates may also experience adrenarche. Yet, sustained postnatal increased DHEAS concentrations are limited to humans, chimpanzees, and bonobos.2,3 It has been speculated that one function of adrenarche is to modify the neural, behavioral, and psychosocial development as part of the human transition from child to adult.4 Clarifying the physiologic roles of the C19 adrenal steroid hormones, especially DHEAS, during adulthood is an ongoing process. Investigating the mechanisms regulating changes in DHEA and DHEAS production will improve understanding of zona reticularis function in health and disease. KEY POINTS  • Adrenarche is defined as the rise in adrenal androgens that occurs in midchildhood and is responsible for the appearance of pubic hair. • The term “adrenal androgens” refers to 19-­carbon steroids (C19 steroids) including dehydroepiandrosterone, dehydroepiandrosterone sul­fate, and androstenedione. These steroids do not bind to the androgen receptor. Rather, these steroids are converted to more potent sex steroids.

THE ADRENAL GLANDS The adrenal cortex synthesizes and secretes diverse C21 and C19 steroid hormones beginning in utero and ending with the demise of the

1498

individual. The Italian anatomist and physician Bartolomeo Eustachio was the first to describe and provide anatomic depictions of the adrenal glands, in 1543.5 Approximately 300 years later, Thomas Addison described the phenotype associated with adrenal insufficiency.6 Subsequently, Charles Edouard Brown-­Séquard confirmed the importance of the adrenal glands; he demonstrated that bilateral adrenalectomy led to rapid death attributed to loss of vital hormones.7 In 1950, the Nobel Prize in Physiology and Medicine was awarded to Edward Calvin Kendall, Tadeusz Reichstein, and Philip Showalter Hench for their discoveries of the adrenal cortical hormones and their biological effects.

THE FETAL ADRENAL GLAND The adrenal glands consist of two separate components, the cortex and the medulla, which have distinct embryonic origins. In the human fetus, the cortex develops around 28 to 30 days postconception from cells in the intermediate mesoderm located between the coelomic epithelium and the dorsal aorta. These cells develop into the adrenogonadal primordium. Around 32 to 40 days postconception, the adrenogonadal primordium separates. Cells that migrate retroperitoneally to the cranial pole of the mesonephros develop into the adrenal primordium. Subsequently, neural crest derived cells invade the adrenal primordium; these cells develop into the catecholamine secreting chromaffin cells of the adrenal medulla.8 An outer mesenchymal capsule surrounds the entire adrenal gland. In the fetus, two distinct concentric zones, the outer definitive and inner fetal zones, can be identified. Beginning at approximately 8 weeks of gestation, the fetal adrenal is an active endocrine organ. The clear demarcation in adrenal weights for normal infants compared to those with anencephaly and congenital adrenal hyperplasia highlights the important role of adrenocorticotropic hormone (ACTH) as a tropic factor influencing the growth of the fetal adrenal gland (Fig. 90.1).9 Due to minimal CYP17A1 activity, the human placenta cannot synthesize estrogen de novo. Hence, one major activity of the human

CHAPTER 90  Adrenal Androgens, Adrenarche, and Adrenopause

estradiol synthesis.12 Of note, 16α-­hydroxyl-­DHEAS, metabolized by the fetal liver, provides the substrate for placental estriol synthesis. Enzymes expressed in the fetal zone include steroidogenic acute regulatory protein (StAR), P450c11A1, P450c17A1, P450c21A2, P450c11B1/ B2, and sulfotransferase family 2A member 1 (SULT2A1). Two enzymes, 3β-­HSD2 and aldo-­keto reductase family 1 member C3 (ARK1C3), are constitutively expressed throughout the first and second trimesters of gestation.13 The outer definitive/adult zone is largely inactive during the first two trimesters of gestation and begins to produce cortisol during the third trimester. After 24 to 28 weeks of gestation, the fetal adrenal directly secretes cortisol. Evaluation of second-­trimester human adrenals showed the presence of cortisol. Intraadrenal steroid content, measured in second-­ trimester human fetal adrenal glands, showed that pregnenolone was the most abundant steroid; 17-­OHP, progesterone, DHEAS, and cortisol were detectable, whereas aldosterone was undetectable.14 Cortisol promotes terminal differentiation of specific tissues such as lung, thyroid, and liver in anticipation of postnatal life.15 Regarding the lung, cortisol promotes structural maturation of the lung and stimulates fetal lung surfactant synthesis.16 At birth, the adrenals weigh 8 to 9 g, which is comparable to the weight of adult adrenals. Over the few months following birth, the cells in the fetal zone undergo apoptosis, leading to involution of the fetal adrenal zone.17 The timing of this involution appears to be more closely tied to gestational age rather than the birth of the infant.18

4.5 = Normal = Anencephalic = CAH fetuses

Combined adrenal weight (g)

4

3

2

1

0 12

14

16

18

20

22

24

26

28

30

GA (wk) Figure 90.1  Combined fetal adrenal weights according to gestational age. CAH, Congenital adrenal hyperplasia; GA, gestational age. (Data from Young MC, Laurence KM, Hughes IA. Relationship between fetal adrenal morphology and anterior pituitary function, Horm Res. 1989;32:130–135.)

POSTNATAL ADRENAL MORPHOLOGY AND FUNCTION

fetal adrenal is to provide substrates to the placenta, with its robust aromatase activity, for estrogen biosynthesis. During a brief period at around 8 to 12 weeks of gestation, the enzyme 3β hydroxysteroid dehydrogenase type 2 (HSD3B2) is expressed, leading to cortisol synthesis to presumably prevent virilization of female fetuses.10 In concert with the morphologic changes in the fetal adrenal gland, DHEA, pregnenolone, 170H-­pregnenolone, and their respective sulfated conjugates are secreted. The steroid output amounts to approximately 200 mg/day, of which 60% is DHEAS.11 As noted above, DHEAS is a major substrate for estrogen biosynthesis via the fetoplacental unit (Fig. 90.2). The fetal adrenal gland provides approximately 60% of the DHEAS for placental Maternal

DHEAS

Postnatally, a precipitous fall in adrenal weight by approximately 50% occurs due to the involution of the fetal zone. The definitive zone persists and develops into the adult adrenal cortex. The adult adrenal cortex is organized into three functionally distinct concentric zones, which were initially described in 1866.19 The names of the zones reflect the organization of the cells within each region. Cells of the outer zone, the zona glomerulosa, are organized into round clusters (Latin glomus, ball). Cells of the middle zone, the zona fasciculata, are arranged in radial pillars or cords (Latin fascicle, bundle). Cells of the innermost zone, zona reticularis, form a mesh-­like structure (Latin rete, net).20 Lineage-­tracing

Placental Sulphatase

Fetal Sulphatase

DHEA

DHEAS 16α-hydroxylase (liver)

3βHSD

Androstenedione

16αOH-DHEAS

16αOH-DHEAS

Sulphatase 16αOH-DHEA

P450 aromatase

Estrone

1499

Estradiol

Estriol

Figure 90.2  The fetal-­placental-­maternal steroid unit. DHEA, Dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate.

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PART 7  Adrenal

studies have confirmed that adrenocortical cells originate in the outer portion of the cortex and progressively move towards the medulla. These functional status of these cells changes during this process.21 The outer zone, the zona glomerulosa, secretes aldosterone. Zona glomerulosa function is regulated by the renin-­angiotensin system and serum potassium concentrations. The enzyme aldosterone synthase encoded by the aldosterone synthase (CYP11B2) gene is only expressed in the zona glomerulosa. The zona fasciculata secretes cortisol in response to ACTH acting through the ACTH receptor encoded by the melanocortin-­2 receptor (MC2R) gene. ACTH has chronic and acute effects. Chronic or tonic ACTH stimulation is essential to promote transcription and translation of adrenal steroidogenic enzymes. ACTH acts acutely to stimulate cortisol secretion. Cortisol induces negative feedback inhibition at the hypothalamus and pituitary. Activity of the hypothalamic–­pituitary–­ adrenal (HPA) axis is organized into circadian and ultradian rhythms, with the highest ACTH and cortisol secretion in the early morning hours prior to awakening.22 Due to its lipophilic nature, cortisol must be synthesized de novo because it cannot be stored in vesicles. The inner zone, the zona reticularis, secretes DHEA, DHEAS, and androstenedione. Despite being labeled as “adrenal androgens,” these C19 steroid hormones do not activate the androgen receptor. Rather, these hormones can be considered to be “prohormones” that can be converted to androgens and estrogens in peripheral tissues. DHEAS, the steroid hormone circulating in the greatest abundance, shows no diurnal variation. The 7-­to 10-­hour half-­life of DHEAS likely contributes to this lack of diurnal variation.23 Extraadrenal conversion between DHEA and DHEAS through reversible sulfoconjugation by SULTA2 (adrenal and liver) and steroid sulfatase (peripheral tissues) activity. These reversible enzyme activities enable tissue-­specific regulation of steroid hormone exposure, also known as intracrinology.24,25 The human adrenals are the primary source of the 11-­oxygenated metabolites of testosterone and androstenedione, 11β-­hydroxy­ androstenedione (11OHA4) and 11β-­hydroxytestosterone (11OHT). In peripheral tissues, these hormones are converted to 11-­ketoan­drostenedione (11KA4), 11-­ketotestosterone (11KT), and 11-­ ketodihydrotestosterone (11KDHT). Collectively, these 19-­carbon steroids containing an oxygen atom on carbon 11 are referred to as the 11-­oxyandrogens (11oAs). The androgenic potencies of 11KT and 11KDHT are comparable to those of testosterone and dihydrotestosterone, respectively.26-­28 KEY POINTS  • Postnatally, the fetal zone involutes leaving the definitive zone to develop into the three distinct zones of the adult adrenal cortex: the zona glomerulosa, the zona fasciculata, and the zona reticularis, which secrete aldosterone, cortisol, and C19 steroid hormones, respectively.

Clinical Facets of Adrenarche Adrenarche represents the peripubertal maturation of the adrenal cortex. It has been arbitrarily defined as the rise in adrenal androgens, DHEA, DHEAS, and androstenedione that occurs between 6 and 8 years of age. Despite this commonly used definition, the maturation of the adrenal cortex may truly be a more gradual process; urinary steroid markers of adrenarche show a continuous rise starting at 3 to 4 years of age.1 This concept is supported by a report regarding a small group of Finnish children in whom DHEAS concentrations were measurable at 1 year of age and correlated with DHEAS concentrations at 6 years of age.29 Despite much investigation, the factors initiating the onset of adrenarche and

subsequently regulating adrenal C19 steroid secretion remain unknown. Studies involving monozygotic and dizygotic twins indicate heritability for onset of adrenarche to be 58% to 61%.30,31 Although small case-­control studies assessed for genetic markers associated with age at adrenarche, no distinct genetic markers were consistently identified and validated.32,33 Pubarche is the clinical manifestation of adrenarche. Pubarche is characterized by the development of pubic or axillary hair, adult apocrine odor, acne, and increased oiliness of the skin and hair. In girls, the pubic hair growth typically starts on the labia. Among boys, both adrenal and testicular C19 steroids promote pubic and axillary hair development. The most recent large cross-­sectional study, the Third National Health and Nutrition Study conducted between 1988 and 1994, reported mean ages for pubic hair development among American youth. For girls, the mean ages for pubic hair development were 9.5 years for non-­Hispanic Blacks, 10.3 years for Mexican Americans, and 10.5 years for non-­Hispanic Whites. For boys, mean ages for pubic hair development were 11.1 years for non-­Hispanic Blacks, 12.3 years for Mexican Americans, and 12 years for non-­Hispanic Whites.34 Premature pubarche is arbitrarily defined as occurring when pubic hair growth appears before 8 years of age in girls and before 9 years of age in boys. The most common cause of premature pubarche is premature adrenarche. Because premature adrenarche is a diagnosis of exclusion, other disorders associated with androgen excess need to be excluded. The differential diagnosis includes congenital adrenal hyperplasia, androgen-­secreting tumors, cortisone reductase deficiency, apparent cortisone reductase deficiency, 3’-­phospho-­adenosine 5’-­phosphosulfate synthase deficiency, glucocorticoid resistance, Cushing syndrome, and exposure to exogenous androgens (Table 90.1). Premature adrenarche is more common in girls than in boys.35 Children with premature adrenarche tend to be taller. Skeletal maturation is normal or minimally advanced. Skeletal maturation is assessed by an x-­ray of the left hand and wrist to evaluate the ossification centers.36 When the skeletal maturation is significantly advanced, greater than two standard deviations (SDs), concerns arise regarding the potential for adult short stature. Despite conflicting outcome studies, most children achieve an adult height appropriate for their predicted midparental height.37-­40 The mini growth spurt in normal children that also occurs at around 6 to 8 years of age is probably not a function of adrenarche.41 Importantly, the finding of a significantly advanced bone age (>2 SDs) in a child with premature adrenarche should prompt evaluation for other disorders associated with premature pubarche, such as nonclassic congenital adrenal hyperplasia and androgen-­secreting tumors.42 Because all children have an increase in adrenal androgen production between 6 and 8 years of age, why hair development occurs in a small minority of children remains puzzling. In addition to DHEA, DHEAS, and androstenedione, the 11-­oxygenated C19 steroid adrenal androgens play a role in the clinical signs of adrenarche.

TABLE 90.1  Differential Diagnosis of

Premature Pubarche

Premature adrenarche Congenital adrenal hyperplasia Androgen-­secreting tumor (adrenal, ovary, or testis) Human chorionic gonadotropin-secreting tumor Apparent cortisone reductase deficiency Cortisone reductase deficiency Apparent dehydroepiandrosterone sulfate sulfotransferase deficiency Cushing syndrome Familial glucocorticoid resistance Exposure to exogenous androgens

CHAPTER 90  Adrenal Androgens, Adrenarche, and Adrenopause

KEY POINTS  • The factors governing onset of adrenarche remain unclear. • The diagnosis of premature adrenarche can be made by the presence of pubic hair before the age of 8 years in girls and 9 years in boys. • Premature adrenarche should be differentiated from more serious conditions such as congenital adrenal hyperplasia and virilizing tumors.

Associations reported between low birth weight and premature adrenarche have been inconsistent.43 Sexual dimorphism is not apparent in normal children, in whom a relationship between lower birth weight and higher adrenal androgen levels is present in both sexes.44 Androgens and estrogens affect skeletal maturation and bone health through their actions on osteoblast, osteocytes, and osteoclasts.45 In a small study of girls with premature adrenarche, increased linear growth was already evident in the first 2 years of life and associated with higher IGF-­1 levels.46 Obesity can further accelerate the rate of skeletal maturation in premature adrenarche. Levels of 11oAs have been measured in several disorders, including castration-­resistant prostate cancer,47 21-­hydroxylase deficiency,48-­50 and polycystic ovarian syndrome (PCOS).26,51 A recent study showed that 11oAs correlate with surrogate markers of metabolic risk in a large cohort of women with PCOS.51 The 11oAs also appear to significantly contribute to the clinical signs observed in adrenarche,26,27,47 including premature adrenarche.52 Recent findings suggest that elevated 11oAs–– rather than DHEA, DHEAS, and A4––may be of particular diagnostic and clinical significance in cases of clinical signs of adrenarche (premature pubarche) without the biochemical diagnosis of premature adrenarche based on DHEAS levels (for premature adrenarche definition, see “Clinical Facets of Adrenarche” later).53 Wise-­Oringer et al. have proposed that measurement of serum 11oAs may be a more accurate way to screen and classify children with premature adrenarche than measurement of serum DHEAS.53 These findings could potentially also be broadly applied to other disease states of androgen excess. Obesity, hyperinsulinemia, and/or insulin resistance have been observed in children with premature adrenarche, and the molecular basis of these findings is likely multifactorial. Obesity exacerbates insulin resistance and hyperinsulinemia, which can promote increased adrenal and gonadal steroid secretion. Adiposity may influence the association between elevated adrenal androgens and signs of metabolic syndrome. Children evaluated for premature adrenarche should be clinically followed for signs of metabolic syndrome, although these relationships remain to be fully elucidated.54-­59 These studies report associations without establishing causality. The question regarding the risk for girls with premature adrenarche to develop PCOS in later life was initially based on findings in a cohort of Catalan girls. Of these girls who had been previously diagnosed with premature pubarche, 45% developed functional ovarian hyperandrogenism.60 Additional features of dyslipidemia, increased visceral fat, hyperinsulinemia, inflammatory marker changes, and early menarche were also identified in this cohort.61 Daughters of mothers with PCOS are at higher risk for developing this syndrome, and a significant number exhibit exaggerated adrenarche.62 Studies investigating the relationship between premature adrenarche and PCOS have led to a proposed developmental sequence beginning with reduced fetal growth, followed by a postnatal catch-­up in height and weight, premature adrenarche, and finally clinical androgen excess and PCOS, with hyperinsulinemia and increased hepatovisceral fat stores playing a key role.63-­66 Still, among retrospective studies, the development of PCOS in girls with premature adrenarche varies.67-­69 Not all girls with premature adrenarche develop PCOS, and conversely, not all girls with PCOS have a history of premature adrenarche.70 Inquiry into the relationship

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of premature adrenarche to PCOS can be a challenge, because the diagnostic criteria for PCOS include normal features for peripubertal development during adolescence, such as irregular menses, polycystic ovary morphology, and mild hyperandrogenism.71 Anti-­Müllerian hormone (AMH) concentrations are typically elevated in women with PCOS, and AMH has been assessed as a possible tool to predict PCOS in girls with premature adrenarche. Whereas one study reported that AMH concentrations were significantly higher in girls with premature adrenarche and correlated with maternal history of PCOS,72 another study suggests that AMH may not have adequate predictive value for the diagnosis of PCOS.73 Currently, AMH should not be used to predict and diagnose PCOS. Currently, no blinded randomized controlled trials exist to provide evidence-­based data regarding the use of insulin sensitizers in a child presenting with typical clinical and biochemical features of premature adrenarche. It is appropriate to counsel parents that some girls with premature adrenarche may later experience menstrual disturbances in adolescence. Early intervention after menarche with insulin sensitizers may prevent progression to PCOS and reduce the risk for long-­ term cardiovascular problems.74 Counseling regarding healthy lifestyle choices is also important. KEY POINTS  • Associations between premature adrenarche and bone age advancement, obesity, hyperinsulinemia, insulin resistance, and polycystic ovary syndrome have been described.

Adrenal Steroidogenesis The initial step in steroidogenesis is the conversion of cholesterol to pregnenolone. The P450c17 enzyme functions as the qualitative regulator of steroidogenesis.75 This enzyme uniquely catalyzes both 17α-­ hydroxylase and 17,20-­lyase activities. It is the latter enzyme activity that preferentially ensures C19 steroid production and, in particular, DHEA synthesis and adrenarche. A number of factors enhance the differential activities of P450c17. These include posttranslational regulation of P450c17 by phosphorylation on serine/threonine residues and the role of electron transfer proteins.76-­78 The principal electron donor to microsomal P450 enzymes, including P450c17, is nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase. This enzyme cofactor augments both 17α-­hydroxylase and 17,20-­lyase activities but is not the principal determinant of increased DHEA synthesis. Enhancement of 17,20-­lyase activity is linked specifically to the action of cytochrome b5 in the presence of adequate P450 oxidoreductase.75 This effect is far more substantial with Δ5 substrates such as 170H-­pregnenolone as compared with a Δ4 substrate such as 170H-­progesterone, hence the predominance of DHEA synthesis. Coupled to the promotion of DHEA synthesis is a relative deficiency of 3β-­hydroxysteroid dehydrogenase (HSDB2) in the fetal adrenal and postnatal zona reticularis. This enzyme would normally compete with P450c17 to convert Δ5 to Δ4 steroids.79 Mutations in the gene encoding P450 oxidoreductase lead to a biochemical profile reminiscent of combined partial 17α-­hydroxylase and 21-­hydroxylase deficiencies with virilization in affected girls and undervirilization in affected boys.80,81 Antley–Bixler syndrome, a condition characterized by severe skeletal abnormalities, genital anomalies, and adrenal dysfunction, is also a manifestation of this enzyme deficiency.82 More than 99% of DHEA is sulfated to DHEAS through the action of dehydroepiandrosterone sulfatase (SULT2A1).83 Sulfated steroids are unavailable as substrates for HSDB2 activity, thus maintaining the high levels of DHEAS. Immunohistochemical

.

l

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↑ cytb5

DHEA-S (µmol/L)

10

↑ SULT2A1 5

= Adrenarche B = Birth P = Puberty

↑ SULT2A1

6

↑ P450 OR

3

↑ cytb5 ↑ cytb5

0

↓ 3βHSD2

↓ cytb5

↓ 3βHSD (z.reticularis)

Fetus B Childhood P Adulthood Figure 90.3  Schematic of dehydroepiandrosterone sulfate (DHEAS) production from fetal to adult life. The arrows indicate temporal changes in factors controlling DHEAS.

Cholesterol StAR P450scc Pregnenolone

P450c17 P450-OR

170H-pregnenolone

P450c17 P450-OR/cytb5

DHEA

SULT2A1

DHEA-S

Low 3βHSD2

(aldosterone)

(cortisol)

Figure 90.4  Pathway of dehydroepiandrosterone sulfate (DHEAS) synthesis in fetal adrenal and adult zona reticularis. Also depicted is the effect of low expression of 3β-­hydroxysteroid dehydrogenase 2 (3β-­ HSD2) in these tissues.

studies in the fetal adrenal gland and in age-­related postnatal adrenal glands have demonstrated a developmental pattern of enzyme and cofactor expression that may explain the intraadrenal control of adrenarche.84-­86 In summary, the pattern of increased DHEAS secretion is associated with increased expression of P450c17, P450 oxidoreductase, cytochrome b5, SULT2A1, and decreased expression of HSDB2 in the fetal adrenal gland and again in the postnatal zona reticularis after 5 years of age. In contrast, a reversal of this pattern occurs in the postnatal adrenal gland between infancy and 5 years of age. Expression of cytochrome b5 and SULT2A1 is maintained beyond the adult age, when DHEAS levels start to decline. The zona reticularis becomes smaller with increasing age, suggesting that the decline in DHEAS levels is associated with a decrease in cell number rather than a change in enzyme content. What factors stimulate the changes in expression of key proteins controlling DHEAS synthesis remain to be determined. HSDB2 has the important function of determining the ratio of Δ5 and Δ4 steroid production. A family of orphan nuclear receptors (nerve growth factor-induced clone B) is expressed in parallel with HSDB2 and directly acts on the promoter to upregulate the HSD3B2 gene (Figs. 90.3 and 90.4).87

DEHYDROEPIANDROSTERONE AND IN VITRO FERTILIZATION DHEA has been used as a supplemental treatment for women undergoing in vitro fertilization, particularly in patients who respond

poorly to gonadotropin stimulation. Regarding the definition of poor ovarian response (POR), The European Society of Human Reproduction and Embryology has recommended that at least two of the following three criteria should be present in identifying a patient as a potential poor responder to ovarian stimulation: (i) advanced maternal age or any other risk factor for POR; (ii) a previous POR; and (iii) an abnormal ovarian reserve test.88 However, defining POR is still unclear, as randomized trials on POR have often used differing definitions.89 The use of DHEA supplementation for POR was first suggested in 2000 in a case series.90 A worldwide survey found that 25.8% of in vitro fertilization (IVF) clinicians in 45 different countries use DHEA to supplement IVF treatment protocols in women with POR.91 Studies have shown that DHEA treatment may increase the number of oocytes, increase embryo numbers and quality, increase pregnancy rate, increase live birth rate, and decrease miscarriage rate.92-­96 In a systematic review and metaanalysis of six randomized controlled trials (RCTs) of DHEA supplementation in women undergoing IVF (five studies in women with POR, one in women without POR), the pooled estimates suggested that DHEA supplementation was associated with a significant increase in clinical pregnancy, endometrial thickness, and retrieved oocytes. No relationship was identified to an improvement in live birth rate, embryos transferred, or an increase of estradiol on human chorionic gonadotropin day.97 Additionally, several studies have found no significant differences in the parameters mentioned; one RCT, for example, failed to show that DHEA supplementation significantly improved IVF outcomes.98 Another study found that, in

CHAPTER 90  Adrenal Androgens, Adrenarche, and Adrenopause women undergoing DHEA supplementation, the markers of very early (AMH) and early differentiated (inhibin B) follicles were not different compared with controls, whereas antral follicle count increased significantly, suggesting that DHEA specifically targets the 2-­to 10-­mm antral cohort of follicles that have escaped atresia for that particular cycle, improving follicular survival.99 The presumed major mechanism of DHEA in improving reproductive outcomes is the increase in androgen levels, because DHEA serves as a precursor to estrogen and testosterone. Androgen receptors are expressed in the granulosa cells, theca cells, and oocytes as follicles mature.100 In this situation, DHEA may act through intracrine mechanisms, allowing for tissue-­specific modifications compared with androgens such as testosterone. This may explain the more precise actions and fewer adverse effects of DHEA supplementation compared with testosterone supplementation.24 Studies have suggested that DHEA could have mitochondrial effects, increasing mitochondrial mass, reducing mitochondrial apoptosis, and restoring mitochondrial homeostasis in cumulus cells and granulosa cells.101,102 The exact mechanism of DHEA supplementation in augmenting IVF in poor responders is therefore likely multifaceted and remains to be fully understood.103 More studies, particularly randomized controlled trials, are needed to establish a clearer picture of the mechanisms of DHEA on improving reproductive outcomes in women undergoing IVF and the efficacy and safety of this intervention. KEY POINTS  • Dehydroepiandrosterone has been used as supplementation for women undergoing in vitro fertilization, possibly working through intracrine mechanisms as a precursor to estrogen and testosterone. More randomized controlled trials are needed to determine the efficacy and safety of this intervention.

Adrenopause The term “adrenopause” refers the observation that DHEA and DHEAS concentrations decline following peak DHEA and DHEAS during the mid-­20s. DHEA and DHEAS concentrations at 80 years of age are only 20% of those at age 25 years.104 Despite decreasing DHEA and DHEAS concentrations, glucocorticoid and mineralocorticoid secretion are largely unchanged. In a longitudinal study of women (Study of Women’s Health Across the Nation), a modest perimenopausal increase was noted in DHEAS concentrations; this was accompanied by stable testosterone concentrations and minimal decreases in androstenedione concentrations.105 During the perimenopausal period, DHEAS concentrations were lower in Black women than in White women.106 In a study involving 100 premenopausal and 100 postmenopausal women, sulfated steroids, i.e., DHEAS, 17-­hydroxypregnenolone, and androstenediol-­3-­sulfate, showed a negative correlation with age.107 Yet, despite the decline in DHEA and DHEAS concentrations, the 11oAs did not decline among aging women. Among these women, no significant associations between steroid concentrations, body mass index (BMI), hypertension, dyslipidemia, hyperglycemia, or bone density were identified. With expansion of this dataset to include more women and men, 11OHA4 and 11OHT concentrations were noted to slightly increase in women while remaining stable in men across adult ages. In this population, 11KT concentrations showed positive correlation with 11KA4, 11OHT, and 11OHA4 in both sexes.108 After adjusting for age, androstenedione concentrations declined and were inversely related to BMI in both sexes. Of the 11oAs, 11OHT increased with BMI in women, whereas 11KT increased with higher BMI in

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men.108 As would be anticipated, testosterone concentrations were higher in men compared with women; 11OHA4, 11KA4, and 11KT concentrations were minimally higher among men.108 The factors responsible for the decreased DHEA/DHEAS production and the dissociation of DHEA/DHEAS production from the 11-­oxygenated C19 steroids are indeterminate. This decline in DHEA and DHEAS concentrations is accompanied by immunohistochemical changes in the adrenal cortex of older adults. Specifically, using human adrenal tissues obtained from reproductive-­ aged adults and older adults, expression of both CYB5A2 and SULT2A1 in the adrenal glands from older individuals was reduced. Similar to the findings in adrenal glands obtained from older women, adrenals from older men demonstrated a less distinct zona fasciculata/zona reticularis zonation and more commonly displayed areas where cells expressing HSD3B2 and CYB5A intermingled.108 The molecular basis for these changes in zona reticularis function is unknown. The small sample size, cross-­sectional nature, and incomplete clinical information limit conclusions from these studies. KEY POINTS  • Adrenopause refers to the fall in adrenal production of dehydroepiandrosterone and dehydroepiandrosterone sulfate that occurs in later life.

Epidemiology/Associations with Dehydroepiandrosterone Sulfate Concentrations, Aging, and Cardiovascular Risks Cross-­sectional epidemiologic studies have queried the relationship between declining DHEAS concentrations and various aspects of aging. A 1986 study showed DHEAS concentrations to be inversely related to death from any cause and to death from cardiovascular disease in men over 50 years of age.109 These findings were not replicated in adult women in the Rancho Bernando Study.110 The Osteoporotic Fractures in Men study in Sweden was a 5-­year longitudinal study that involved 2416 men between 69 to 81 years of age; this study reported that low DHEA and DHEAS concentrations predicted an increased risk for coronary heart disease but not cerebrovascular disease.111 Results from the Women’s Ischemia Syndrome Evaluation reported a greater than 2-­fold increased risk of cardiovascular disease mortality among women in the lowest tertile of DHEAS compared to those in higher categories (hazard ratio = 2.55; 95% confidence interval: 1.19–5.45).112 A metaanalysis involving 18 studies with a total of 92,489 patients concluded that the prognosis was poorer for patients with cardiovascular disease and lower DHEAS levels compared with those with higher DHEAS concentrations.113 Hence, current data suggest that lower DHEAS concentrations are associated with an increased risk for cardiovascular disease. The Atherosclerosis Risk in Communities (ARIC) study assessed some features of the cardiovascular disease. In the ARIC study, lower DHEAS concentrations were associated with increased risk for subclinical myocardial injury, heart failure hospitalization, and death in men and women.114 White participants tended to have lower DHEAS concentrations compared with Black participants. Within this cohort, men who experienced a greater decline in DHEAS concentrations showed an increased risk for hospitalization for heart failure. Their data suggest a threshold effect in which low DHEAS concentrations are associated with increased risk, but higher concentrations do not modify the risk for heart failure and death. Among postmenopausal women, DHEAS concentrations were weakly but significantly related to endothelial function independent of other coronary risk factors, suggesting a protective effect of DHEA

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on the endothelium with improved endothelial cell function.115 To elucidate the basis for the association of low DHEAS concentrations with myocardial injury, Jia and colleagues utilized a proteomics assay and Ingenuity Pathway Analysis. Significant associations between DHEAS concentrations and proteins with known functions pathways involved in the extracellular matrix and cardiac remodeling were identified. The association of DHEAS concentrations with POMC and Agouti-­related protein, which are melanocortin receptor agonists and antagonists, respectively, suggests that DHEAS concentrations may indicate HPA axis dysfunction in older adults.114 Low DHEAS concentrations appear to predict risk for heart failure in older adults.114 However, whether DHEAS has a direct, indirect, or passive bystander role in myocardial remodeling remains to be determined. DHEAS concentrations appear to be associated with risk for ischemic strokes. In the Nurses’ Health Study, lower DHEAS concentrations were associated with increased risk for ischemic strokes.116 Potential mechanisms for DHEAS actions could include inhibiting migration and proliferation of cells within the vascular wall, thereby increasing vascular smooth muscle apoptosis and decreasing vascular remodeling following injury.117 In addition, animal models suggest that DHEAS may influence insulin resistance by affecting hepatocyte glucose production and improving peripheral tissue glucose clearance.118,119 Nevertheless, the question as to whether DHEAS plays an active role in the metabolic pathways or is merely a marker remains to be answered. KEY POINTS  • Adrenopause is accompanied by immunohistochemical changes in the adrenal cortex but is not accompanied by changes in glucocorticoid, mineralocorticoid, or 11-­oxyandrogen production. • Declining dehydroepiandrosterone sulfate concentrations may be associated with various aspects of aging, including increased risk for cardiovascular disease and ischemic strokes.

NEUROBIOLOGY OF DEHYDROEPIANDROSTERONE AND DEHYDROEPIANDROSTERONE SULFATE DHEA and DHEAS concentrations are higher in the brain than in peripheral circulation.120 One likely explanation for this finding is that both DHEA and DHEAS are synthesized de novo in the brain in astrocytes and neurons. For this reason, DHEA and DHEAS are considered to be “neurosteroids.” Another source of brain DHEA is peripheral DHEA, which can readily cross blood–brain barrier due to its lipophilic nature. In contrast, brain DHEAS is more likely synthesized in the brain because its hydrophilic nature interferes with crossing the blood–brain barrier.121 Several mechanisms are likely responsible for DHEA/DHEAS actions. The most obvious mechanism is conversion of DHEA/ DHEAS to more potent sex steroids, followed by activation of androgen or estrogen receptors in specific target tissues.24 In addition, DHEA/DHEAS can bind to the tropomyosin-­related kinase (TrkA), γ-­aminobutyric acid type A (GABAA), sigma subtype 1, and NMDA receptors.122 DHEA/DHEAS appear to have inhibitory actions at the GABAA receptor, whereas they act as agonists at the sigma 1 receptor.123 DHEA has been described as a modulator of neurotransmission because it can affect serotonin, γ-­amino butyric acid (GABA), glutamate, and dopamine levels. In the central nervous system, it can be metabolized to ADIOL, which can bind to ERβ, negatively regulating proinflammatory gene expression.124

Neurobiological actions of DHEA/DHEAS include neuroprotection, neurite growth, neurogenesis, and neuronal survival. At low nanomolar concentrations, in primary cultures of mouse embryonic neocortical neurons, DHEA promoted growth of Tau-­immunopositive axons; in contrast, DHEAS had no effect on axonal growth and stimulated dendritic growth.123 In human fetal cortical neural stem cells, DHEA promoted neurogenesis and neuronal survival; this effect was blocked by an NMDA receptor antagonist and sigma 1 receptor antagonists.125 In addition, DHEA has antiglucocorticoid and neuroprotective effects. Animal studies, both in vivo and in vitro, have demonstrated that DHEA protects against the neurotoxic effects of corticosterone.126 Neurotrophins and their tyrosine kinase receptors influence neuronal differentiation, growth, and survival. DHEA binds to TrkA and TrkC receptors and induces receptor phosphorylation. The functional significance of this finding in human biology is unclear.127 In a mouse model involving lipopolysaccharide stimulation, DHEA appeared to act as an endogenous regulator of neuroinflammatory microglial responses. The proposed mechanism involves interactions with the tropomyosin-­ related kinase receptor (TrkA), which activated the Akt1/Akts2-­CREB pathway, ultimately resulting in enhanced expression of the histone 3 lysine 27 (H3K27) demethylase Jumonji d3 (Jmjd3).128 Preclinical findings have led to clinical investigations assessing DHEA/DHEAS concentrations and potential for therapeutic interventions. For example, researchers in traumatic stress have been intrigued by the potential relationship between post-­traumatic stress disorder (PTSD) and DHEA/DHEAS. Elevated DHEA and low cortisol/DHEA or low cortisol/DHEAS concentrations have been described in some studies. A metaanalysis reviewing a modest number of publications found no consistent relationship between PTSD and DHEA/DHEAS concentrations. The heterogeneity of the results and use of only a single hormone determination without regard to time of day/diurnal rhythm confound interpretation of these studies. Nevertheless, there appeared to be an association between increased basal DHEA/ DHEAS concentrations with a trauma exposure irrespective of PTSD development.129 Measuring hormone samples is hair samples provides a mechanism to circumvent the methodological issues associated with using a single hormone determination to characterize a dynamic hormone. In a study involving self-­reports on psychopathology and coping in 92 Palestinian girls aged 11 to 16 years affected by the Israeli–­Palestinian conflict, trauma-­exposed girls had significantly higher DHEA and lower cortisol/DHEA ratios in their hair than non-trauma-­exposed girls.130 DHEA has been found to have effect on immune and endothelial function. Available data are accumulating indicating that the cortisol-­ to-­DHEAS ratio may be a useful indicator of chronic stress in humans. Indeed, this ratio may provide an indicator of chronic stress in nonhuman animal species and may be useful for animal welfare studies.131 In animal models for multiple sclerosis, DHEA and DHEAS may be beneficial. DHEA suppresses Th17 cell responses, reduces microglia-­ mediated neuroinflammation, and expands IL-­ 10-producing regulatory T-­cell populations in an ERβ-­dependent manner. DHEAS decreases demyelination and axonal loss.124 KEY POINTS  • Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are synthesized de novo in the brain, and their actions are regulated by several mechanisms. • DHEA/DHEAS can contribute to neuroprotection, neurite growth, neurogenesis, and neuron survival and have been shown to be increased in traumatic stress.

CHAPTER 90  Adrenal Androgens, Adrenarche, and Adrenopause

Clinical Implications of Dehydroepiandrosterone Sulfate/Dehydroepiandrosterone Data were obtained in the English Longitudinal Study of Aging using a community-­based population to assess for an association between DHEAS concentrations and depression; data were obtained at baseline and at 4 years. Although no associations were apparent between DHEAS concentrations and depression at baseline, low DHEAS concentrations obtained at baseline predicted future depressive symptoms independent of age, gender, economic circumstances, and health behaviors and characteristics.132 DHEAS concentrations fall after acute injury/trauma. Whereas cortisol concentrations typically increase during septic shock, DHEAS concentrations fall.133 Among patients with schizophrenia, increased serum DHEA and lower cortisol/DHEA ratios were reported and associated with an inverse relationship between hippocampal volume and cortisol/DHEA ratio.134

Dehydroepiandrosterone Sulfate/Dehydroepiandrosterone and “The Fountain of Youth” The observation that DHEA/DHEAS concentrations declined concomitant with onset of aging led to the hypothesis that provision of supplemental DHEA/DHEAS could restore physical health and protect the brain from the degenerative changes due to age. This hypothesis was supported by association studies linking decreased DHEA and DHEAS concentrations to decreased bone mineral density, increased risk of fracture, decrease cognitive function, decreased quality of life, and increased risk of cardiovascular disease.135 Based on this hypothesis, DHEA/DHEAS replacement therapy was tried in older adults. In the study by Nair et al., administering DHEA for 2 years had no effect on body composition, physical performance, or insulin sensitivity.136 No effects were observed on insulin secretion, insulin action, and suppression of systemic lipolysis following mixed meal or glucose tolerance tests.137,138 Most clinical studies of DHEAS administration in Alzheimer disease enrolled more females than males; no improvement in cognition was seen with DHEAS treatment.139 No consistent data supporting a benefit of DHEA supplementation on body composition, cognition, mood, or sexual function in older men.140 RCTs of DHEA supplementation have yielded inconsistent results regarding clinical benefits. Improved arterial elasticity was observed among a heterogeneous group of elderly men and women treated with DHEA (50 mg/day) for 1 year.141 One potential clinical indication for DHEA treatment is vaginal dryness; daily intravaginal 0.05% DHEA improved vaginal dryness and decreased dyspareunia.142 Hence, no evidence-­based data exist substantiating that DHEA and DHEAS are the proverbial “fountain of youth.”143 However, the US Food and Drug Administration classifies these steroids as dietary supplements rather than drugs (DHEA Uses, Benefits, and Side Effects; verywellhealth.com). This means that distribution of these medications is unregulated and does not require a prescription. Hence, DHEA is available for personal use; these preparations have been found characterized to contain 0–­150% of the amount stated on the package.144 KEY POINTS  • There is limited evidence that supplemental dehydroepiandrosterone can improve quality of life.

CONCLUSION Despite being the most abundant circulating hormones in the circulation, the precise physiologic actions of DHEA and DHEAS remain mysteries. DHEA and DHEAS are secreted by the adrenal cortex and

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synthesized de novo in the brain. These hormones play a major role as substrates for placental estrogen synthesis in the fetus. DHEAS production escalates at adrenarche, peaking in early adulthood. Yet, the factors influencing adrenocortical DHEA/DHEAS secretion remain indeterminate. Low DHEAS concentrations appear to be associated with an increased risk for cardiovascular disease in older adults. Investigation into DHEA/DHEAS actions are uncovering effects on endothelial cells, immune function, and neuronal development. Much, however, remains to be elucidated regarding the physiologic roles and regulation of DHEA and DHEAS.

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129. van Zuiden M, Haverkort SQ, Tan Z, Daams J, et al. DHEA and DHEA-­S levels in posttraumatic stress disorder: a meta-­analytic review. Psychoneuroendocrinology. 2017;84:76–82. 130. Schindler L, Shaheen M, Saar-­Ashkenazy R, et al. Victims of war: dehydroepiandrosterone concentrations in hair and their associations with trauma sequelae in Palestinian adolescents living in the West Bank. Brain Sci. 2019;9:20. 131. Whitham JC, Bryant JL, Miller LJ. Beyond glucocorticoids: integrating dehydroepiandrosterone (DHEA) into animal welfare research. Animals (Basel). 2020;10:1381. 132. Souza-­Teodoro LH, de Oliveira C, Walters K, et al. Higher serum dehydroepiandrosterone sulfate protects against the onset of depression in the elderly: findings from the English Longitudinal Study of Aging (ELSA). Psychoneuroendocrinology. 2016;64:40–46. 133. Arlt W, Hammer F, Sanning P, et al. Dissociation of serum dehydroepiandrosterone and dehydroepiandrosterone sulfate in septic shock. J Clin Endocrinol Metab. 2006;91:2548–2554. 134. Ji E, Weickert CS, Purves-­Tyson T, et al. Cortisol-­ dehydroepiandrosterone ratios are inversely associated with hippocampal and prefrontal brain volume in schizophrenia. Psychoneuroendocrinology. 2021;123:104916. 135. Arlt W. Dehydroepiandrosterone and ageing. Best Pract Res Clin Endocrinol Metab. 2004;18:363–380. 136. Nair KS, Rizza RA, O’Brien P, et al. DHEA in elderly women and DHEA or testosterone in elderly men. N Engl J Med. 2006;355:1647–1659. 137. Basu R, Dalla Man C, Campioni M, et al. Two years of treatment with dehydroepiandrosterone does not improve insulin secretion, insulin action, or postprandial glucose turnover in elderly men or women. Diabetes. 2007;56:753–766. Erratum in: Diabetes. 2007;56:1486. 138. Espinosa De Ycaza AE, Rizza RA, Nair KS, et al. Effect of dehydroepiandrosterone and testosterone supplementation on systemic lipolysis. J Clin Endocrinol Metab. 2016;101:1719–1728. 139. Wolkowitz OM, Kramer JH, Reus VI, et al. DHEA-­Alzheimer’s Disease Collaborative Research. DHEA treatment of Alzheimer’s disease: a randomized, double-­blind, placebo-­controlled study. Neurology. 2003;60:1071–1076. 140. Walther A, Seuffert J. Testosterone and dehydroepiandrosterone treatment in ageing men: are we all set? World J Mens Health. 2020;38:178– 190. 141. Weiss EP, Villareal DT, Ehsani AA, et al. Dehydroepiandrosterone replacement therapy in older adults improves indices of arterial stiffness. Aging Cell. 2012;11:876–884. 142. Labrie F, Archer DF, Koltun W, et al. Efficacy of intravaginal dehydroepiandrosterone (DHEA) on moderate to severe dyspareunia and vaginal dryness, symptoms of vulvovaginal atrophy, and of the genitourinary syndrome of menopause. Menopause. 2018;25:1339–1353. 143. Stewart PM. Aging and fountain-­of-­youth hormones. N Engl J Med. 2006;355:1724–1726. 144. Parasrampuria J, Schwartz K, Petesch R. Quality control of dehydroepiandrosterone dietary supplement products. J Am Med Assoc. 1998;280:1565.

91 Adrenal Pathology Ozgur Mete and Lori A. Erickson

OUTLINE Anatomic Pathology in Developmental Abnormalities of the ­Adrenal Gland, 1509 Adrenal Aplasia or Hypoplasia, 1509 Accessory or Heterotopic Adrenal Tissue, 1509 Tumor-­Like Lesions of the Adrenal Gland, 1510 Adrenal Myelolipoma, 1510 Ectopic Thyroid Tissue, 1510 Ovarian Thecal Metaplasia, 1510 Adrenal Amyloidosis, 1511 Adrenal Calcification, 1511 Adrenal Cysts, 1511 Adrenal Infection and Abscess, 1512 Congenital Adrenal Hyperplasia, 1513 Adrenal Cytomegaly, 1513 Inflammatory Adrenalitis, 1513

Functional Approach in Adrenal Pathology, 1513 Pathological Correlates of Primary Aldosteronism, 1514 Pathological Correlates of Adrenal Cushing Syndrome, 1516 Pathological Correlates of Virilism/Feminization, 1517 Pathological Correlates of Catecholamine Excess, 1518 Pathology in the Distinction of Adrenocortical Carcinoma, 1519 Diagnostic and Predictive Immunohistochemical Biomarkers in Adrenal Pathology, 1523 Confirmation of Adrenocortical and Medullary Origin, 1523 Confirmation of Functional Sites in Primary ­Aldosteronism, 1523 Diagnostic and Predictive Biomarkers of Adrenocortical ­Carcinoma, 1523 Molecular Immunohistochemistry in Pheochromocytoma, 1524 Synoptic Reporting in Adrenocortical Carcinoma and ­Pheochromocytoma, 1525

 

ANATOMIC PATHOLOGY IN DEVELOPMENTAL ABNORMALITIES OF THE ADRENAL GLAND The adrenal cortex consists of steroidogenic cells that originate from the intermediate mesoderm.1 A web of factors, including transcriptional factors (e.g., DAX1 encoded by NR0B1, SF1/Ad4BP encoded by NR5A1, and GATA4/6 encoded by GATA4 and GATA6), play a crucial role during the organogenesis and functional differentiation of adrenocortical cells.1,2,3 This complex developmental process stems from the formation of adrenogonadal primordia within the urogenital ridge and is followed by the separation of surrounding mesenchyme and formation of an encapsulated adrenal anlagen (fetal adrenal zone), which later develops into the adult adrenal cortex via the expansion and maturation of the definitive adrenocortical zones.1 The adrenal medulla consists of chromaffin cells that are surrounded by mesodermal steroidogenic cortical cells. Recent evidence suggests that only a small fraction of the adrenal medulla originates directly from the migratory neural crest cells, whereas preganglionic nerve fiber–related pluripotent Schwann cell precursors at the adrenal anlage provide the major path for the development of chromaffin cells.4,5 Disruption in various steps of the organogenesis results in developmental abnormalities. This section provides a brief summary of developmental conditions related to anatomic pathology.

Adrenal Aplasia or Hypoplasia Adrenal aplasia (agenesis) is a very rare condition that refers to the absence of the adrenal gland, whereas adrenal hypoplasia refers to adrenocortical atrophy. Anencephaly is a well-­known cause of neonatal

KEY POINTS  • Defects in primary and secondary developmental regulators of the adrenal cortex can lead to congenital adrenal aplasia or hypoplasia.

adrenal hypoplasia or aplasia.6 In humans, NR5A1 mutations are rare, but they have been reported in bilateral adrenal aplasia or hypoplasia.2,7 Patients with Pollister–Hall syndrome harbor GLI3 mutations and can exhibit adrenal hypoplasia.2 Morphologically, hypoplastic adrenal glands appear to have a prominent medulla due to significantly reduced thickness of the cortex without normal cortical zonation. Several etiologies can result in adrenal hypoplasia (e.g., exogenous glucocorticoid administration, insufficiency related to the hypothalamic-­ pituitary-­ adrenal axis, including but not limited to adrenocorticotropic hormone [ACTH] resistance and familial glucocorticoid deficiency syndromes). Congenital adrenal hypoplasia is also linked to NR0B1-­related pathogenesis. The latter can either manifest with hemizygous pathogenic NR0B1 variants that cause X-­linked adrenal hypoplasia congenita with hypogonadotropic hypogonadism or Xp21 deletion, which results in deletion of NR0B1 and GK (complex glycerol kinase deficiency), and sometimes DMD (Duchenne muscular dystrophy).8 Cytomegalic cells with lipid depletion pattern have been reported in hypoplastic adrenal glands.

Accessory or Heterotopic Adrenal Tissue Accessory or heterotopic adrenocortical tissue is not an uncommon finding. These consist of isolated adrenocortical tissue rests and may have associated adrenal medullary cells (chromaffin cells), as seen in an orthotopic adrenal gland. The occurrence of chromaffin cells in heterotopic adrenal tissue also underscores the crosstalk between

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adrenal cortex and medulla during embryogenesis. Delamination in the adrenogonadal primordia leads to the separation and differential migration of embryonic cells towards the gonadal anlagen and the adrenal anlagen. Therefore, failure in encapsulation of steroidogenic factor 1 (SF1)-­expressing developmental cells that are responsible for the formation of the fetal adrenal zone may explain the origin of adrenocortical tissue rests that are identified in the abdomen (e.g., along the celiac axis, kidney and kidney capsule, periadrenal, spleen and splenic hilum, mesentery), as well as in various anatomic locations that are aligned with the developmental tract of the gonads (e.g., spermatic cord, scrotum, broad ligament, uterus, ovarian and spermatic vessels, gonads).9,10 However, the occurrence of adrenocortical rests in unusual locations such as brain, pituitary, spinal nerves, lung, liver, and pancreas also expands the spectrum of adrenocortical tissue rests that cannot be explained from an embryological point of view.9,10,11 The term “adrenocortical choristoma” is applied to unusual heterotopic adrenocortical rests (e.g., adrenocortical choristoma in a silent corticotroph tumor).11 Failure to recognize these atypical locations may lead to diagnostic dilemmas. Mechanisms that alter with the successful separation of the developmental periadrenal mesenchyme may result in adrenal fusions (union) or adhesions. Adrenal fusions (unions) occur between the adrenal gland and parenchymal elements of the liver or kidney (e.g., adrenohepatic fusion, adrenorenal fusion) (Fig. 91.1). Morphologically, in the setting of an adrenal fusion, there is no intervening connective tissue capsule separating cellular elements of the organ that is united with the adrenal gland. Therefore, adrenocortical elements are intermingled with cellular components of the other organ. Midline adrenal fusions also occur between both adrenal glands. The latter leads to the gross appearance of a single horseshoe-­shaped midline adrenal gland. Other developmental defects, including those of central nervous system, can be identified in patients with midline adrenal unions.9 In adrenal adhesions, there is an intact intervening connective tissue capsule separating parenchymal elements of the adrenal gland and the other organ (e.g., adrenohepatic adhesion). As a consequence, the adrenal gland displays a normal medulla and preserved cortical zonation. Failure to recognize the spectrum of developmental abnormalities such as adrenohepatic fusion or adrenohepatic adhesion, and adrenocortical rests in the liver can result in misinterpretation of a benign developmental process as invasion or metastasis of an adrenocortical carcinoma. The latter may be diagnostically challenging, especially in small biopsy specimens.

TUMOR-­LIKE LESIONS OF THE ADRENAL GLAND KEY POINTS  • Tumor-­like lesions of the adrenal gland encompass a large spectrum of lesions, including adrenal myelolipoma, ectopic thyroid tissue, ovarian thecal metaplasia, amyloid deposition, adrenal hematoma and hemorrhage, adrenal calcification, adrenal cysts, infection with or without abscess, congenital adrenal hyperplasia, and adrenal cytomegaly with or without Beckwith–Wiedemann syndrome.

Adrenal Myelolipoma Myelolipoma consists of a mature adipose tissue admixed with variable number of hematopoietic cells, including megakaryocytes (Fig. 91.2). Myelolipomas with extensive hemorrhage can simulate an adrenal hematoma or malignant process. Microscopic foci of myelolipomatous change can occur in adrenocortical proliferations. Bilateral adrenal gland involvement was recorded in association with congenital adrenal hyperplasia (CAH) and primary bilateral macronodular adrenocortical disease (also known as primary bilateral macronodular adrenocortical hyperplasia).12,13 Giant adrenal myelolipomas exceeding 30 cm have been defined in patients with CAH.12 Increased ACTH-­and androgen-­ mediated stimulus and overexpression of melanocortin 2 receptor and androgen receptor have been postulated in the developmental pathogenesis of some adrenal myelolipomas, especially in the setting of CAH.14

Ectopic Thyroid Tissue The presence of thyroid tissue within the adrenal gland expands the unusual spectrum of ectopic thyroid tissue outside the usual migratory path of the thyroid anlage that typically lies between the foramen cecum and mediastinum.15,16 An abnormal descent of the thyroid anlage can be the source of ectopic thyroid tissue that can be identified in adrenals (Fig. 91.3). While diagnosticians are still required to exclude the possibility of metastatic differentiated thyroid carcinoma in any unusual location, failure to recognize the occurrence of ectopic thyroid tissue in abdominal organs may result in misdiagnosis.15

Ovarian Thecal Metaplasia Ovarian thecal metaplasia is a relatively rare tumor-­like lesion of the adrenal gland. Originally described in postmenopausal women with markedly elevated gonadotropins, these abnormal cellular nests were defined as wedge-­shaped cortical lesions that are composed of ovarian stroma-­like elements. However, these lesions have been defined in both women and men. Subsequent evidence also suggested that these

*

*

* * Fig. 91.1  Adrenorenal fusion. The adrenal cortex (*) is in continuity with the renal parenchyma (**). There is no limiting connective tissue between adrenal gland and kidney. The arrow illustrates renal elements in the zona reticularis.

Fig. 91.2  Adrenal myelolipoma. Myelolipoma consists of a mature adipose tissue admixed with variable number of hematopoietic cells including megakaryocytes. Asterisk indicates the nonlesional adrenal cortex; arrows indicate multinucleated megakaryocytes.

CHAPTER 91  Adrenal Pathology

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lesions rarely contain true ovarian stromal elements, and are composed of fibroblastic and/or myofibroblastic mesenchymal cells. Some cases featuring radial scar-­like appearance have also been defined.17

calcifications.9,18 Necrosis and fibrosis can also occur. GM1 gangliosidosis presents with bilateral adrenal calcifications restricted to the medulla, given the accumulation of ganglioside in neuronal tissues.10,19

Adrenal Amyloidosis

Adrenal Cysts

Adrenal amyloidosis is a rare cause of adrenal insufficiency.9 Affected individuals may exhibit adrenal enlargement, depending on the degree of the involvement. Histologically, amyloid deposition is characterized by a homogenous acellular pink hyaline material. It typically occurs in the wall of vascular structures, but also in the extracellular stroma between adrenocortical cells. The later results in diffuse replacement of cortical cells, depending on the extent of the disease. Special histochemical stains are frequently used to distinguish amyloid from fibrosis or stromal hyalinization, which can be identified in cortical nodular disease as well as in autoimmune adrenalitis.

Primary adrenal cysts have four histologic variants, including (i) vascular (endothelial) adrenal cysts, (ii) epithelial adrenal cysts, (iii) parasitic adrenal cysts, and (iv) adrenal pseudocysts.9,10,20 Most primary adrenal cysts are unilateral disease. Pseudocysts and vascular (endothelial) adrenal cysts are the most common subtypes that can simulate adrenocortical or medullary neoplasm. Absence of epithelial or endothelial lining distinguishes adrenal pseudocysts from other types of adrenal cysts9,20 (Fig. 91.4). These often have hemorrhagic, fibrinoid, or necrotic cyst content that can be mistaken for an adrenocortical carcinoma on imaging studies.9,20 Vascular adrenal cysts are typically lined by endothelial cells that are commonly of lymphatic endothelial cell origin (Fig. 91.5). Vascular cysts may mimic pheochromocytomas. Epithelial cysts are lined by keratin-­expressing cells of mesothelial origin (Fig. 91.6). Parasitic cysts occur in the course of parasitic adrenal infections (e.g., hydatid disease). Adrenal cysts should be distinguished from other adrenal lesions that can manifest with cystic degeneration, including adrenocortical neoplasms, pheochromocytomas, and neuroblastic tumors, as well as some metastatic carcinomas. Beckwith–Wiedeman syndrome can also show hemorrhagic multiloculated adrenal cysts in the background of adrenal enlargement.

Adrenal Calcification Adrenocortical neoplasms and medullary tumors may contain dystrophic calcifications and even metaplastic bone formation. The nonneoplastic correlates of adrenal calcifications can be seen in the setting of infectious disease (e.g., histoplasma), adrenal cysts (see later), Wolman disease (lysosomal acid lipase deficiency) and GM1 gangliosidosis (β-­ galactosidase deficiency).9,10,18,19 Wolman disease manifests with hepatosplenomegaly and infantile bilateral adrenal enlargement with dystrophic

*

Fig. 91.3  Ectopic thyroid tissue in the adrenal. Adrenal gland can host ectopic thyroid tissue. The lack of cytological and architectural atypia warrants a diagnosis of benign ectopic thyroid tissue. The asterisk indicates the nonlesional adrenocortical cells in the zona fasciculata.

A

Fig. 91.4  Adrenal pseudocyst. Adrenal pseudocysts have no lining and are composed of hemorrhagic fibrinoid material.

B

Fig. 91.5  Vascular (endothelial) cyst of the adrenal. This composite photomicrograph illustrates a vascular cyst surrounded by nonlesional adrenocortical cells (A). The lumen is acellular (A). CD31 (not illustrated herein) and D2-­40 stain the vascular endothelial lining of this cyst (B).

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Adrenal Infection and Abscess Primary adrenal infection is a rare clinical presentation, because most develop as a result of widespread or locoregional infections.9,10,21-­24 Immunocompromised individuals have an increased risk of adrenal infection. On computed tomography (CT) scans, infection-­related adrenal enlargement can be mistaken for an adrenal tumor because of a mass-­like appearance or diffuse enlargement with variable heterogeneity.21,22 Adrenal insufficiency (Addison disease) occurs, depending on the extent of tissue destruction. Various microorganisms can cause adrenal infections. Parasitic infestations caused by Echinococcus, amebic species, Trypanosoma, Leishmania, and Microsporidia have been reported in adrenals.9,23 Parasites can manifest with adrenal cystic disease (e.g., hydatid disease). Among fungal pathogens, Cryptococcus and Histoplasma are the most common pathogens in nontropical geography, whereas Paracoccidioides predominates tropic geographic zones.9,10,23,25 Histologically, necrotizing granulomatous inflammation is common in fungal infections.

A

Enlargement of the adrenal glands with extensive adrenal destruction in association with extensive caseous necrosis and calcification is a feature of disseminated Histoplasma infection9,23 (Fig. 91.7). In addition to cultures, histochemical stains (periodic acid–Schiff and Grocott’s methenamine silver stains) are used to confirm fungal elements. Immunocompromised hosts are more likely to develop viral adrenal infections. Among these, cytomegalovirus (CMV)-­related adrenalitis is well-­recognized; however, other viruses can also involve adrenal glands.9,23 Unlike fungal and mycobacterial infections, adrenals show no marked enlargement in viral infections. Mixed inflammation in the corticomedullary junction and/or hemorrhagic necrosis (more frequent in Herpesviridae) can be seen.9,10,24 Demonstration of viral cytopathic changes (e.g., Cowdry type A inclusions in CMV) confirms the diagnosis of a viral adrenal infection. Immunohistochemical antibodies against viral antigens are also very useful in the detection of viral antigens. Bacterial adrenal infections typically occur either due to mycobacteria infections (e.g., immunocompromised or tuberculosis reactivation) or in the context of hematogenous spread in bacterial

B

Fig. 91.6  Epithelial cyst of the adrenal. This composite photomicrograph illustrates an epithelial cyst surrounded by nonlesional adrenocortical cells (A). Pankeratin stains the lining of this cyst, as well as some cortical cells (B).

*

*

A

B

Fig. 91.7  Histoplasma infection in the adrenal. This composite photomicrograph illustrates a necrotizing granulomatous inflammation (A). Asterisks indicate the necrotic center of granulomatous inflammation. Some macrophages in the lesion contain multiple fungi, consistent with Histoplasma (B). Circled area represents fungal elements on hematoxylin and eosin–stained sections.

CHAPTER 91  Adrenal Pathology

Fig. 91.8  Congenital adrenal hyperplasia. Bilateral compact (lipid-­ poor) cell adrenocortical hyperplasia is a characteristic morphologic feature of this disorder.

sepsis (e.g., Streptococcus, Haemophilus, Staphylococcus, Meningococcus, Escherichia, Pseudomonas, and Klebsiella).9 Bacterial adrenal infections can manifest with adrenal enlargement, hemorrhage, necrosis, abscess, and malakoplakia.9 Abscess occurs as a result of hematogenous spread of bacterial pathogens. Unilateral adrenal abscess can be mistaken for a tumor. Adrenal malakoplakia is a rare tumor-­like inflammatory condition caused by gram-­negative bacteria. Histologically, large collections of foamy macrophages with intracytoplasmic basophilic inclusions (Michaelis–Gutmann bodies) are characteristics features of malakoplakia. Waterhouse–Friderichsen syndrome refers to bilateral adrenal hemorrhage and necrosis that is caused by bacterial sepsis. Historically, mycobacterium tuberculosis has been recognized to account an important etiology of Addison disease.9,10 Bilateral marked adrenal enlargement with variable degree of necrotizing granulomatous inflammation is a frequent finding in mycobacterial adrenal infections. Special histochemical stains (e.g., Ziehl–Neelsen) are required to visualize acid-­fast bacilli in the cytoplasm of macrophages. However, histochemical stains fail to detect a very low number of bacteria; therefore, tissue cultures, PCR studies, and interferon-­gamma release assays such as QuantiFERON have been frequently used as the golden standards in the distinction of mycobacterial infection (including the latent infection), as well as for the accurate characterization of mycobacteria.

Congenital Adrenal Hyperplasia Defects in the enzymes involved steroid hormone biosynthesis cause CAH. Most common forms have been linked to CYP21-­related 21-­hydroxylase deficiency.9,10,24 Patients who are not adequately treated with exogenous steroids manifest with marked bilateral adrenal enlargement due to ACTH-­driven bilateral diffuse compact cell adrenocortical hyperplasia9,10,24 (Fig. 91.8). Heterotopic adrenocortical tissue can become hyperplastic in patients with CAH. These hyperplastic tissues often simulate neoplastic disease in various locations.

Adrenal Cytomegaly Adrenal cytomegaly is characterized by variable amount of enlarged adrenocortical cells with pleomorphic hyperchromatic nuclei with or without pseudoinclusions. Adrenal cytomegaly is distinguished from viral cytopathic changes of adrenal CMV infections by the presence of a single eosinophilic nuclear inclusion that is typically surrounded by a clear halo (Cowdry A) in infected cortical cells.9 Premature infants and neonates can display areas of adrenal cytomegaly.9,24 This finding does not qualify as a precursor or malignant process, and adrenal cytomegaly often resolves within months or years. However, focal adrenal cytomegaly rarely persists in other age groups. Bilateral adrenal gland enlargement due to cortical proliferations (usually adrenocortical hyperplasia) with prominent adrenal

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Fig. 91.9  Adrenal cytomegaly in Beckwith–Wiedemann syndrome. Bilateral adrenal gland enlargement due to cortical proliferations (usually adrenocortical hyperplasia) with prominent adrenal cytomegaly (cortical cells with enlarged nuclei) is one of the characteristic features of Beckwith– Wiedemann syndrome.

cytomegaly, hemorrhagic cortical cysts, and prominence of chromaffin cells of the adrenal medulla are characteristic features of Beckwith– Wiedemann syndrome (Fig. 91.9). This congenital overgrowth syndrome is caused by altered imprinting of genes in chromosome 11p15.5 and has also been regarded as a predisposing condition for adrenocortical carcinoma.26

Inflammatory Adrenalitis Focal adrenalitis is not an uncommon histologic finding, and it is often of no clinical significance. Noninfectious inflammatory changes are often seen in the context of autoimmune adrenalitis, which is typically seen in association with autoimmune polyglandular endocrinopathy types I and II9,10 (Fig. 91.10). Depending of the disease stage, the cortex of both adrenals shows marked mixed chronic inflammation composed of lymphocytes, plasma cells, and macrophages.9,10 The inflammatory adrenocortical destruction results in fibrohyalinization and small adrenal glands. The distinction of adrenalitis from a lymphoproliferative disorder is required when inflammation predominates the histology. Ancillary tools (e.g., immunohistochemistry and molecular pathology techniques) can be used to distinguish a lymphoproliferative disease from adrenalitis.

FUNCTIONAL APPROACH IN ADRENAL PATHOLOGY The morphological spectrum of adrenal lesions includes hyperplastic and neoplastic disorders that are associated with hormonally active or nonfunctional “silent” clinicopathologic manifestations. Hormonally active adrenocortical lesions can cause mineralocorticoid excess (primary aldosteronism), glucocorticoid excess (adrenal Cushing syndrome and subclinical adrenal Cushing syndrome), sex hormone excess (virilism and feminization), and a combination of the these hormones excess syndromes.9,27 Adrenal medullary lesions are often functional and result in catecholamine excess–related clinical signs and symptoms. KEY POINTS  • Adrenocortical lesions are classified into three diagnostic morphological categories: (1) adrenocortical hyperplasia, (2) benign adrenocortical neoplasm, which includes adrenocortical adenomas and adrenocortical nodular disease, and (3) adrenocortical carcinoma. Micronodular and macronodular forms of adrenocortical nodular disease and diffuse adrenocortical hyperplasia are almost always functional disorders involving both adrenal glands. Nonfunctional lesions of the adrenal gland are more frequent in a subset of adrenocortical neoplasms that are enriched in Wnt pathway alterations.28,29

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A

B

Fig. 91.10  Inflammatory adrenalitis. This composite photomicrograph illustrates marked mixed chronic inflammation composed of lymphocytes, plasma cells, and macrophages (A). The inflammatory adrenocortical destruction results in fibrohyalinization (A, B).

KEY POINTS  • The endocrine lesions of the adrenal medulla can be divided into two morphological categories: (1) adrenal medullary hyperplasia and (2) pheochromocytoma (adrenal paraganglioma). Pheochromocytomas are frequently associated with mature (adrenergic or mixed adrenergic and noradrenergic); however, immature (dopaminergic, noradrenergic, or mixed dopaminergic and noradrenergic) secretory biochemical phenotypes can also be identified.30

This section will provide a brief overview of the morphological entities that are reflected in functional adrenal disease. The details on molecular correlates of these lesions29 are discussed in several other chapters of this book.

Pathological Correlates of Primary Aldosteronism Discovery of ion channel mutations that result in the transcription of CYP11B2 (encoding aldosterone synthase [CYP11B2]), occurrence of aldosterone-­producing microscopic adenoma(s) that cannot be identified on CT imaging studies from patients with uniglandular disease, and application of CYP11B2 immunohistochemistry in adrenals from patients with primary aldosteronism have helped diagnosticians to better appreciate the clinicopathologic manifestations of primary aldosteronism.29,31-­34 The current evidence suggests that aldosterone-­producing cortical lesions encompass a wide spectrum of lesions that range from microscopic to gross disease that can manifest with unifocal or multifocal disease with synchronous or asynchronous contralateral gland involvement.29 Traditionally, the most common clinical manifestation of primary aldosteronism is thought to represent bilateral hyperplasia of the zona glomerulosa27,33; however, the latter also includes bilateral microscopic aldosterone-­ producing adrenocortical adenomas that are not detected on imaging studies.29 Bilateral diffuse zona glomerulosa hyperplasia is uncommon in the pathologist’s practice, because affected patients often do not require bilateral adrenalectomy, but instead are treated with lifelong antimineralocorticoid therapy. Among neoplastic correlates of this disorder, aldosterone-­producing carcinomas are extremely rare. As a consequence, aldosterone-­producing benign cortical neoplasms account for most of the diagnoses of primary aldosteronism in the pathologist’s practice (Fig. 91.11). Aldosterone-­ producing adrenocortical adenomas show cytomorphological and epidemiological heterogeneity with respect to their underlying somatic mutations (Table 91.1).29,32 For instance,

aldosterone-­producing adenomas that harbor KCNJ5 mutations are enriched in lipid-­rich zona fasciculata–type cortical cells. These tumors are more frequent in females, and are often solitary large tumors.29,32 In contrast, adenomas that are associated with a nonmutated KCNJ5 gene are enriched in compact cortical cells in the setting of multifocal disease with smaller tumor sizes when compared with KCNJ5-­related disease. These tumors are also reported to be more frequent in males.29,32 The preoperative prediction of bilateral disease is still an ongoing challenge in the clinical management of patients with primary aldosteronism. Despite continued efforts to effectively preoperatively lateralize unilateral aldosterone excess via adrenal venous sampling, biochemical recurrence can still occur in such cases. Recently, genotype–phenotype correlations (Table 91.1) have provided critical insights into the genetic and cellular features underlying unilateral versus bilateral disease. Furthermore, CYP11B2 immunohistochemical features underscore morphological findings that may help predict the potential risk for bilateral disease.29,31,34 As a consequence, the modern endocrine pathology practice of primary aldosteronism requires a thorough cytomorphological examination of the entire adrenal gland. For example, in patients with preoperative spironolactone treatment, functional histologic foci can display variable amount of spironolactone bodies that may provide clues to multifocality and bilaterality of disease.27,33 However, the accurate distinction of functional sites requires the use of CYP11B2 immunohistochemistry in adrenalectomy specimens.31,32,34 Mixed aldosterone and glucocorticoid secretion from an adrenocortical adenoma can occur. This manifestation is more frequent in a subset of adrenocortical neoplasms with KCNJ5 mutations.35,36 Unlike aldosterone-­producing benign cortical disease, aldosterone-­producing adrenocortical carcinomas lack ion channel mutations but display genetic alterations as seen in other adrenocortical carcinomas.37 KEY POINTS  • The HISTALDO classification34 has been introduced by a group of international disease experts to provide a unified diagnostic nomenclature relevant to reporting of surgical pathological findings in adrenalectomy specimens from patients with unilateral primary aldosteronism.34 The conventional histopathological examination and CYP11B2 staining patterns identified in adrenal nodules and the adjacent cortex form the basis of this classification system.

CHAPTER 91  Adrenal Pathology

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A

C

B

Fig. 91.11  Aldosterone-­producing adrenocortical adenoma (Conn syndrome). The morphological heterogeneity in primary aldosteronism is reflected in this composite photomicrograph. Aldosterone-­producing adenomas that harbor KCNJ5 mutations are enriched in zona fasciculata–like clear cells (A), whereas KCNJ5– wild-­type adenomas are enriched in zona reticularis– and zona glomerulosa–like cells (B). Patients treated with spironolactone often show tumor cells with eosinophilic lamellated inclusions that are also known as spironolactone bodies (C). The arrow indicates one of the many inclusions.

TABLE 91.1  Genotype–Phenotype Correlations in Primary Aldosteronism29,32 Molecular Subgroups

Gene(s)

Characteristics

Germline Variants*

KCNJ5-­related PA

KCNJ5

FHA type III

KCNJ5–wild-­type PA

ATP1A1 ATP2B3 ATP2B4** CACNA1H CACNA1D CLCN2

CTTNB1-­related PA

CTTNB1

-­ Most frequent cause of PA -­ More frequent in females -­ Pronounced PA -­ Often earlier age of disease onset -­ Often unilateral disease -­ Often solitary or dominant adenoma -­ Enriched in ZF-­like cortical cells -­ Higher CYP17A1 and CYP11B1 transcripts compared with KCNJ5–wild-­type PA -­ Can feature cortisol cosecretion -­ More frequent in males -­ Often less pronounced PA -­ Often later age of disease onset -­ Germline pathogenic CACNA1H and CACNA1D variants manifest with early age of disease onset -­ Smaller tumor size; increased frequency of multifocal disease -­ Increased risk of recurrence -­ Enriched in ZG-­ and ZR-­like compact cells -­ Increased CYP11B2 to CYP11B1 ratio -­ With the exception of CLCN2-­mutant tumors, lower levels of CYP17A1 and CYP11B1 expression -­ Higher levels of CYP11B2 mRNA transcripts compared with KCNJ5-­related disease -­ Rare cause of PA (∼5% of PA) -­ More frequent in females -­ Often later age of disease onset -­ Higher risk of recurrence -­ Heterogenous cytomorphology -­ Heterogenous CYP11B2 expression -­ Nuclear and cytoplasmic beta-­catenin

FHA type II: CLCN2 FHA type IV: CACNA1H PASNA syndrome: CACNA1D ATP2B4 variants

*Germline pathogenic variants that can be seen in this molecular subgroup. **Rare germline variants. PA, Primary aldosteronism; ZF, zona fasciculata; ZG, zona glomerulosa; ZR, zona reticularis; FHA, familial hyperaldosteronism; PASNA syndrome, early onset of primary aldosteronism with seizure and neurological abnormalities.

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Fig. 91.12  Pituitary ACTH-­related diffuse adrenocortical hyperplasia. Pituitary ACTH-­related diffuse adrenocortical hyperplasia is associated with bilateral adrenal gland enlargement. While zona reticularis is slightly expanded, all three cortical zones are preserved.

Fig. 91.13  Ectopic adrenocorticotropic hormone (ACTH)-­ related diffuse adrenocortical hyperplasia. Unlike the pituitary ACTH-­related pathogenesis, the adrenocortical zonation is not preserved. The adrenal cortex in both adrenals show a lipid depletion pattern.

HISTALDO Classification Aldosterone-producing carcinoma (APACC) CYP11B2-expressing adrenocortical carcinoma Aldosterone-producing adenoma (APA) CYP11B2-expressing solitary benign adrenocortical neoplasm (≥1.0 cm) Aldosterone-producing nodule (APN) CYP11B2-expressing subcentimeter benign adrenocortical nodular disease; CYP11B2 staining is often stronger at the periphery Aldosterone-producing micronodule (APM) CYP11B2-expressing subcentimeter microscopic cortical proliferation consisting of zona glomerulosa–like cells that tend to display a gradient staining as seen in APN Multiple aldosterone-producing nodules (MAPN) or multiple aldosterone-producing micronodules (MAPM) Aldosterone-producing diffuse hyperplasia (APDH) CYP11B2-expressing nonnodular broad or continuous zona glomerulosa–layer hyperplasia that accounts for >50% of the zona glomerulosa layer

The pathological correlates of adrenal Cushing syndrome (v) Diffuse adrenocortical hyperplasia a) ACTH dependent (Pituitary, ectopic) b) CRH dependent (Hypothalamic, ectopic) (vi) Bilateral nodular adrenocortical disease c) Micronodular form d) Macronodular form (vii) Adrenocortical adenoma (viii) Adrenocortical carcinoma

Pathological Correlates of Adrenal Cushing Syndrome Diffuse adrenocortical hyperplasia is the only “true hyperplastic” disorder of the adrenal cortex in patients with Cushing syndrome (Fig. 91.12). The latter also accounts for the most frequent cause of endogenous Cushing syndrome.32,38 From a pathogenic perspective, ACTH-­dependent disease is due to functional pituitary corticotroph tumor in the vast majority of patients.9,32,38 However, ectopic ACTH or corticotropin-­releasing hormone (CRH) production in neuroendocrine neoplasms,27,38 and CRH-­ producing hypothalamic tumors39 can also result in diffuse adrenocortical hyperplasia. At the morphologic level, the adrenal cortex in both adrenal glands is expanded with preserved adrenocortical zonation in the setting of pituitary ACTH-­dependent pathogenesis. Endocrine pathologists can distinguish pituitary ACTH-­dependent diffuse hyperplasia from ectopic

*

*

* *

*

* *

*

*

Fig. 91.14  Primary bilateral micronodular adrenocortical disease. Both adrenal glands are slightly enlarged and contain compact cell– rich micronodules that are variably pigmented. The illustration is from an individual with primary pigmented nodular adrenocortical disease. Asterisks indicate micronodules.

ACTH-­related diffuse hyperplasia, because in the latter the lack of cortical zonation and the presence of prominent zona reticularis–like compact cell hyperplasia result in lipid depletion pattern that gives an appearance that is often referred to “pink adrenal cortices”9,27 (Fig. 91.13). Bilateral nodular adrenocortical disease consists of two clinicopathologically significant forms, including (i) primary bilateral micronodular adrenocortical disease (Fig. 91.14) and (ii) primary bilateral macronodular adrenocortical disease (Fig. 91.15) (Table 91.2). At the morphological level, the distinction between the two forms is made based on the size of the cortical nodules. The micronodular forms consists of bilateral small cortical nodular proliferations (measuring less than 1.0 cm; usually ranging in size from 0.1 to 0.4 cm). The micronodules are enriched in compact (lipid-­poor) cortical cells that can be pigmented in some cases (e.g., primary pigmented nodular adrenocortical disease) (Fig. 91.14). These are often located in the deep zona fasciculata and/or zona reticularis.27,29,32 In contrast to micronodular forms of this disease, the macronodular forms are almost always KEY POINTS  • Traditional diagnostic terminologies such as primary micronodular or macronodular hyperplasia of the adrenal cortex are no longer credible nomenclatures for primary bilateral nodular forms of adrenocortical disease, because those nodules are composed of genetically transformed adrenocortical cells; therefore, modern endocrine pathology practices classify them within the spectrum of adrenocortical neoplasia, and they are referred to as bilateral nodular adrenocortical disease.27,29

CHAPTER 91  Adrenal Pathology associated with significantly enlarged adrenals that are composed of several nodules that generously exceed 1.0 cm in size. The nodular proliferations are enriched in zona fasciculata–like clear cells, but variations in cytomorphological findings can occur27,32 (Fig. 91.15). The distinction of primary bilateral nodular adrenocortical disease in Cushing syndrome is of clinical significance given the increased frequency of germline susceptibility in both micro-­and macronodular forms of this manifestation.27,29,32,38 Cortisol-­producing adrenocortical adenomas are often unilateral and solitary neoplasms. At the morphological level, they can display cytomorphological heterogeneity; however, most are composed predominantly of zona fasciculata–like lipid rich cortical cells.9,27,32,38 Oncocytic change and pigmentation can feature in some examples.9,32 Cortisol-­producing adenomas are distinguished from cortisol-­ producing adrenocortical carcinomas using multiparameter scoring systems and ancillary tools9,27 that are summarized later in this chapter. At a molecular level, autonomous overt cortisol excess from adrenal neoplasms (e.g., cortisol-­producing adrenocortical adenomas and bilateral micronodular adrenocortical disease) are often associated with protein kinase A pathway activation,27,29,32,38 which has also been correlated with high expression of the DLK1-­MEG3 miRNA in a

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KEY POINTS  • From a functional histopathology approach, the nontumorous adrenocortical atrophy is a reliable sign of autonomous glucocorticoid secretion from an adrenocortical neoplasm in patients with no history of exogenous glucocorticoid administration (Fig. 91.16). Therefore, assessment of the nontumorous adrenal cortex is a critical responsibility of pathologists to prevent an Addisonian crisis, especially in patients with unrecognized mild autonomous glucocorticoid-­secreting adrenocortical neoplasms.9,27,40

recent genomic studies.28 Most patients with primary macronodular adrenocortical disease have been linked to ARMC5-­driven pathogenesis27-­29,41 that is also correlated with global CpG hypermethylation status and an ovarian gene expression signature in a recent genomic studies28; however, rare examples of primary macronodular disease have been reported in association with MEN1, FH, GNAS, PDE8B, PDE11A, and PRKACA variants.29,32,38 Adrenocortical adenomas causing mild autonomous cortisol excess (subclinical Cushing syndrome) frequently harbor CTNNB1 mutations and chromosome 17q losses.28

Pathological Correlates of Virilism/Feminization KEY POINTS  • In both adult and pediatric age groups, the identification of virilism/feminization due to an adrenal mass requires exclusion of a functional adrenocortical carcinoma,9,27 most of which tend to cosecrete glucocorticoids.

Fig. 91.15  Primary bilateral macronodular adrenocortical disease. Both adrenal glands are significantly enlarged due to multiple benign cortical nodules; each nodule exceeds 1 cm.

Rare examples of testosterone-­producing adrenocortical adenomas have been reported in adults.42 Unlike adults, this phenomenon has been relatively more frequently reported in pediatric adrenocortical adenomas,43-­46 including some examples, especially oncocytic adrenocortical adenomas, that feature synchronous virilization and adrenal Cushing syndrome.44,45 Given the well-­known challenges in the distinction of malignant behavior in adrenocortical neoplasms47 and the fact that this functional group is more frequent in adrenocortical

TABLE 91.2  Clinicopathological Features of Primary Bilateral Nodular Adrenocortical

Disease27-­29,32 Pathology

Gene Alterations or Variants

Primary bilateral micronodular adrenal cortical disease -­ PPNAD -­ MAD

PRKAR1A CNC2 locus/2p16 PDE11A PDE8B PRKACA PRKACB ARCM5*

Primary bilateral macronodular adrenal cortical disease

ARMC5 MEN1** FH** APC** PDE8** PDE11A** PRKAR1A** PRKACA**

Characteristics -­ Bilateral multiple compact cell small nodules ranging in size from 0.1 to 0.4 cm -­ Nodules are located in the zona fasciculata layer or in the intersection of zona fasciculata and reticularis layers -­ PPNAD is distinguished histologically from MAD based on the presence of pigmented micronodules -­ PPNAD may be associated with Carney complex -­ Bead-­like appearance of micronodules on imaging studies -­ Internodular cortex shows atrophy in PPNAD -­ PRKAR1A-­related pathogenesis is associated with an earlier age disease onset compared to CNC2 locus-­ related pathogenesis -­ Paradoxical response on Liddle’s test -­ Marked bilateral enlargement of adrenals -­ Multiple clear cell–rich clonal nodules exceeding 1.0 cm -­ Childhood-­onset disease shows more pronounced Cushing syndrome compared with adult-­onset disease -­ ARCM5-­related disease is more common in adults -­ ARMC5-­related disease shows significantly more enlargement of adrenals with more nodularity and hypercortisolism compared with ARMC5–wild-­type disease -­ Ovarian gene expression signature (FOXL2, CYP19A1, PTHLH) and global CpG hypermethylation

*Rare missense variant. **Rare events; can be more frequent in childhood manifestations. PPNAD, Primary pigmented micronodular adrenocortical disease; MAD, micronodular adrenocortical disease.

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carcinomas,48 one should still be careful when rendering a diagnosis of adenoma in this setting. The morphological criteria that help distinguish adrenocortical carcinoma and ancillary tools available to diagnosticians are discussed later in this chapter.

Pathological Correlates of Catecholamine Excess KEY POINTS  • Pheochromocytoma (adrenal paraganglioma) is the most common pathological correlate of catecholamine excess from the adrenal gland.

At the morphological level, these tumors are no different than their extraadrenal counterparts, which are termed as paragangliomas. They display heterogenous cytology (e.g., conventional, clear cell, oncocytic) and growth patterns (e.g., Zellballen-­like small nests, solid diffuse, trabecular, large confluent nests, cystic) (Figs. 91.17 and 91.18). The term composite pheochromocytoma is applied to pheochromocytomas that are admixed with composite elements, including neuroblastoma, ganglioneuroblastoma, ganglioneuroma, and peripheral nerve sheath tumor (Fig. 91.19). Pathologists are required to determine the presence/absence of composite elements. Adrenal medullary hyperplasia is restricted to diffuse expansion of the adrenal medulla that exceeds one third of the adrenal gland thickness or

when adrenal medulla is identified in the tail of the adrenal gland.49-­51 In the past, microscopic adrenal medullary nodules measuring less than 1.0 cm were referred to as nodular medullary hyperplasia; however, the latter is no longer accepted. Any distinct microscopic nodules (5.0 cm), immature secretory phenotype (e.g., dopaminergic, noradrenergic, or mixed dopaminergic and noradrenergic), tumor necrosis, increased proliferation (e.g., Ki67 labeling index >3%), and molecular characteristics (e.g., somatic MAML3 fusions or CSDE1 alterations related to altered Wnt-­ pathway clusters, SDHB-­related pseudohypoxic pathway disease, select alterations in the kinase signaling pathway, and somatic SETD2, ATRX, and TERT alterations) are considered important risk factors in dynamic risk stratification.60,64-­68

Adrenocortical carcinoma is defined as a malignant epithelial tumor of adrenocortical cells.69 These are rare tumors comprising less than 3% of endocrine tumors and affect all ages, but are more common in those 40 to 50 years of age. Adrenocortical carcinomas affect females more commonly than males. Approximately half of these tumors are functional, and functional tumors are more commonly seen in females. Adrenocortical carcinomas are usually large tumors, often 10 cm or greater in size, but the size can vary greatly from 1 cm up to 30 cm or more. Size alone is a significant feature in adrenocortical neoplasms. Adrenocortical neoplasms weighing 50 g or more are of concern, and those weighing 100 g or more are of very significant concern by size in weight alone. However, adrenocortical carcinomas weighing less than 50 g can occur, and the size and weight alone are not sufficient to differentiate benign from malignant adrenocortical neoplasms. Adrenocortical carcinomas are usually solid and may show necrosis, but cystic adrenocortical carcinomas have been reported.70 Adrenocortical carcinomas can also be myxoid (Fig. 91.23), and the behavior of these tumors may be more difficult to predict than in conventional adrenocortical neoplasms (Fig. 91.24).71 The behavior of oncocytic adrenocortical tumors may also be difficult to predict by conventional classification; thus, a separate classification (the Lin–Weiss–Biscgelia system) is used for these tumors.72,73 A separate classification system is also utilized in diagnosis of malignancy in adrenocortical neoplasms in children.74 Distinction of adrenocortical adenoma from carcinoma is generally straightforward (Figs. 91.23, 91.24, and 91.25). However, some cases can be extraordinarily difficult, as reflected in the numerous classification systems that have been proposed for the diagnosis of adrenocortical carcinoma.75-­78 In 1984, Dr. Weiss and colleagues evaluated 43 adult adrenocortical neoplasms with 5 years of follow-­up for nine histologic parameters.76 Eighteen of 19 tumors with a score of 4 or more were associated with recurrence or metastasis. All 24 tumors with a score of 2 or less were associated with benign outcome. They found no single feature that was diagnostic of malignancy. The most significant features were mitotic rate of greater than five per 50 high-power fields (10 mm2), atypical mitoses, and vascular invasion (venous angioinvasion). In 1989, the Weiss score was lowered from 4 to 3, as a patient with a score of 3 had recurrent disease and died of disease.77 Weiss and colleagues found that only mitotic rate had a significant association with outcome. Tumors with mitotic rate of greater than 20 per 50 high-­power fields were regarded as high-­grade and associated with a median survival of 14 months. Tumors with a mitotic rate of less than or equal to 20 per

Weiss system* 1. Fuhrman nuclear grade III or IV 2. Mitotic activity >5 per 50 high-power fields 3. Atypical mitotic figures 4. Clear cells ≤25% of the tumor volume 5. Diffuse architecture >30% of the tumor volume 6. Tumor necrosis 7. Venous angioinvasion 8. Lymphatic (sinusoidal) invasion 9. Capsular invasion *Malignant: Score ≥3 out of 9 Applied only to nononcocytic and adult-onset tumors

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A

B

C

Fig. 91.21  VHL-­related pheochromocytoma. VHL-­related pheochromocytomas are often encapsulated tumors enriched in clear cells (A). Positivity for neuroendocrine markers (not illustrated herein) and tyrosine hydroxylase (B) distinguishes this tumor from a clear cell–rich adrenocortical neoplasm. Membranous carbonic anhydrase IX (C) expression is a feature of VHL-­related pathogenesis.

Fig. 91.22  Morphology of SDHx-­related pheochromocytoma. SDHx-­ related tumors are composed of tumor cells with variable cytoplasmic eosinophilia and cytoplasmic vacuoles.

Fig. 91.24  Conventional adrenocortical carcinoma. Most adrenocortical carcinomas are composed of lipid poor cells that are arranged in sheets. A tripolar atypical mitotic figure (circled area) is illustrated in this photomicrograph.

*

Fig. 91.23  Myxoid change in adrenocortical carcinoma. Myxoid change can obscure signs of malignancy; however, a detailed morphological assessment often helps in the distinction of carcinoma. Adrenocortical carcinomas are driven by proliferation. The circled area represents an atypical mitotic figure. This tumor had increased mitotic activity (>5 per 50 high-­power fields), including several atypical mitoses.

Fig. 91.25  Vascular invasion in adrenocortical carcinoma. The identification of a bona fide vascular invasion (venous angioinvasion) is not only a sign of malignancy but is also a prognostic factor for metastatic spread. The presence of intravascular tumor cells admixed with fibrin thrombus is illustrated (asterisk).

CHAPTER 91  Adrenal Pathology 50 high-­power fields were regarded as low-­grade, with median survival of 58 months. Numerous other systems have been suggested previously and subsequently, but the system proposed by Weiss in 1984 and its subsequent modification in 1989 with three or more of the nine features as malignant remains the gold standard today.77,79 In 2002, another modified Weiss system, or “Weiss revisited” system, suggested by matching functional status of 24 adrenocortical carcinomas with distant metastasis, gross local invasion, or recurrence with 25 benign tumors.80 This is a modified system based on the most reliable criteria of the prior Weiss studies from 1984 and 1989, including mitotic rate of greater than five per 50 high-­power fields, atypical mitotic figures, clear cells in less than or equal to 25% of the tumor, necrosis, and capsular invasion. A score of 3 or more out 7 in this study was considered diagnostic of malignancy. Still, the 1989 Weiss system remains the diagnostic standard for adult nononcocytic adrenocortical neoplasms.

Modified Weiss System* •  mitotic rate (score: 2) •  compact cytoplasm (score: 2) •  atypical mitoses (score: 1) •  necrosis (score: 1) •  capsular invasion (score: 1) *Malignant: score ≥3 out 7

The “reticulin algorithm” evaluated disruption of the reticular network (highlighted by reticulin staining) (Fig. 91.26) and one or more of the following criteria (mitoses greater than five per 50 high-­power fields, necrosis, or vascular invasion) in 92 carcinomas in 47 adenomas and found sensitivity and specificity of 100%.81 Volante and colleagues found tumor stage III or IV and mitoses greater than nine per 50 high-­ power fields had a strong adverse impact on disease-­free and overall survival, and were able to divide the tumors into three risk groups. A subsequent multicenter study in 2013 by Duregon and colleagues validated the reticulin algorithm in a study of 184 adrenocortical carcinomas and 61 adenomas.82 The reticulin algorithm may be helpful in the evaluation of myxoid and oncocytic adrenocortical tumors. In a study by Mete and colleagues, loss of the reticulin framework, rather

A

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than qualitative alterations of the reticulin framework, was found to differ between adrenocortical carcinoma and adenomas.83

Reticulin Algorithm Altered reticulin framework in association with one of the following parameters: •  Vascular invasion •  Necrosis •  Mitoses exceeding 5 per 50 high-power fields

The Helsinki score was proposed in 2015 for predicting metastasis in adrenocortical carcinomas. Adrenocortical neoplasms from 177 adults with 5 years of follow-­up were evaluated with the Weiss score, Weiss revisited score, and proliferation index by computer-­assisted image analysis.84 A weighted calculation of three parameters (mitotic activity, necrosis, and Ki67-­labeling index) had 100% sensitivity and 99.4% specificity for diagnosing metastatic adrenocortical carcinoma, with a cutoff of 8.5. In a validation study the following year of 225 adrenocortical carcinomas, the Helsinki and Weiss scores were found to be predictive of poor prognosis.85 Reports suggest that the Helsinki scoring system may be helpful in the evaluation of myxoid and oncycotic adrenocortical tumors.85,86

Helsinki Scoring System* •  Mitoses >5 per 50 high-power fields: score 3 •  Necrosis: score 5 •  Ki67 labeling index in hot spots *Malignant: score ≥8.5

Myxoid adrenocortical neoplasms are uncommon, with most cases published as small series or case reports. These tumors are often functional. In a study from Mayo Clinic of 14 cases of adrenocortical neoplasms with a myxoid component comprising 10% to 95% of the tumor, all six characterized as adenomas were associated with benign behavior, while four of the eight carcinomas were associated with a fatal outcome, with two alive with disease and one alive without

B

Fig. 91.26  Reticulin histochemistry in adrenocortical tumors. Adrenocortical adenomas are associated with retained acinar reticulin framework (A), whereas most adrenocortical carcinomas show loss of reticulin framework (B). The latter forms the basis of the reticulin algorithm. However, the identification of altered reticulin framework is often coupled with one of the three criteria proposed on this algorithm.

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disease.87 The immunohistochemical findings are similar to other adrenocortical neoplasms. A subsequent study from Italy by Papotti et al. evaluated four conventional adrenocortical carcinomas with focal myxoid change (5%–20% of the tumor), and found one of the four patients died of disease, and three were alive without disease at the end of the study.71 They also evaluated 10 myxoid adrenocortical carcinomas with small cells, mild atypia, cords, and microcysts, and six of the 10 died of disease, one was alive with disease, and three were alive with no disease at follow-­up. The most striking part of this study is that one of the two tumors with a Weiss score of 1 had a fatal outcome. Thus, the behavior of myxoid adrenocortical tumors is difficult to predict with standard morphological criteria. However, myxoid change does not make an adrenocortical neoplasm as malignant, but the behavior is difficult to predict with standard diagnostic systems. Oncocytic adrenocortical tumors may be overdiagnosed as malignant by applying the standard Weiss systems. In 1998 Lin et al. evaluated seven oncocytic adrenocortical tumors (5–13.5 cm), all showing nuclear atypia and increased Ki67 proliferative index, but none had invasion or metastasis, and all behaved in a benign manner.73 In 2004, Bisceglia and colleagues evaluated 10 oncocytic adrenocortical tumors and found that two criteria (clear cells less than or equal to 25% of the tumor and high nuclear grade) were present in all of the oncocytic tumors studied, and diffuse architecture was present in nine of 10.72 They proposed an algorithm including the remaining six Weiss parameters and an additional criterion of “large size and huge weight” (greater than 10 cm and/or greater than 200 g). The current system used to evaluate oncocytic adrenocortical carcinoma is referred to as the “Lin–Weiss–Bisceglia system,” which is comprised of major criteria and minor criteria. The presence of one or more major criteria (high mitotic rate, atypical mitoses, or vascular invasion) is diagnostic of malignancy. One to four minor criteria (large size and huge weight, necrosis, capsular invasion, or sinusoidal invasion) indicates a tumor of uncertain malignant potential. Tumors with no major or minor criteria are regarded as benign.72 In 2011, a subsequent study of 13 oncocytic adrenocortical neoplasms and all published oncocytic adrenocortical neoplasms confirm the Lin–Weiss–Bisceglia system.88 They also found that 30% of these tumors were functional, and these tumors are associated with median survival of 58 months (which is comparatively longer than for conventional adrenocortical carcinomas). A recent multicenter retrospective study from France matching oncocytic adrenocortical tumors with conventional adrenocortical carcinomas for age, sex, European Network for the Study of Adrenal Tumors (ENSAT) stage, and resection status also showed oncocytic adrenocortical carcinomas to be associated with better overall survival than conventional adrenocortical carcinomas.86 Similar to all adrenocortical

Lin–Weiss–Bisceglia Criteria for Oncocytic Adrenocortical Tumors* Major Criteria: - Mitoses >5 per 50 high-power fields - Atypical mitoses - Vascular invasion Minor Criteria: - Large tumor size (>10 cm) and weight >200 g - Capsular invasion - Necrosis - Sinusoidal invasion Malignant: at least one major criterion Uncertain malignant potential: at least one minor criterion Benign: absence of major and minor criteria

neoplasms, extensive sampling of oncocytic adrenocortical neoplasms is required so that the correct criteria are utilized, as the behavior does vary with the amount of oncocytic change. What is regarded as a pure oncocytic tumor requires greater than 90% of the tumor be oncocytic, requiring extensive sampling of an adrenocortical neoplasm. Mixed oncocytic adrenocortical tumors are characterized as clear cell component of 10% to 50% of cells, and in an adrenocortical tumor with focal oncocytic change the oncocytic component comprises less than 50% of the tumor.27 Sarcomatoid adrenocortical carcinomas are extremely rare and highly aggressive tumors that occur over a wide age range and are generally nonfunctioning.89 Sarcomatoid adrenocortical carcinomas may have a component of more conventional adrenocortical carcinoma or can be completely sarcomatoid in appearance. These tumors must be differentiated from retroperitoneal sarcomas. Most sarcomatoid adrenocortical carcinomas exist in the literature as case reports. A study of six cases, three of which had been previously reported, evaluated the pathologic and immunohistochemical features of these tumors and had targeted next-­generation sequencing analysis performed.89 Among the six sarcomatoid adrenocortical carcinomas, the authors identified 16 morphologically distinct tumor components, eight of which were epithelial, and eight of which were sarcomatoid.89 Thus, the morphologic heterogeneity of these tumors is quite remarkable and may account for some of the difficulty in differentiating these tumors from retroperitoneal sarcoma or other tumors. In the series above, patients ranged in age from 23 to 79 years, with a mean age of approximately 54 years. The tumors were large in size, averaging 14 cm (range: 6.5–24 cm). Some of the tumors had spindle cell components, with some having foci of osteosarcomatous differentiation, rhabdomyosarcomatous differentiation, or even PNET-­like areas. Purely mesenchymal components such as osteosarcomatous or rhabdomyosarcomatous were less common. Keratin (AE1/ AE3) showed focal positivity in the epithelial component of one tumor and in the sarcomatoid components of two tumors, but was otherwise negative. SF1 was present in epithelial component of all cases but negative in the sarcomatoid areas. Interestingly, β-­catenin showed aberrant nuclear staining in the sarcomatoid component of five of the six cases. They also found TP53 and CTNNB1 mutations in both epithelial and sarcomatoid components, which they note supports a common clonal origin in at least a subset of these tumors.89

Wieneke Criteria for Pediatric Adrenocortical Tumors* 1. Tumor weight >400 g 2. Tumor size >10.5 cm 3. Mitoses >15 per 20 high-power fields 4. Atypical mitotic figures 5. Vena cava invasion 6. Capsular invasion 7. Vascular invasion 8. Extension into adrenal soft tissue or adjacent organs 9. Necrosis Malignant/poor outcome: score ≥4 Uncertain malignant potential: score 3 Benign behavior: score ≤2

Pediatric adrenocortical neoplasms are not evaluated with the same classification systems as adult adrenocortical neoplasms. Pediatric tumors may have features that in an adult adrenocortical tumor would be very worrisome for malignancy. Thus, the Wieneke system is often utilized for these challenging tumors.74

CHAPTER 91  Adrenal Pathology

DIAGNOSTIC AND PREDICTIVE IMMUNOHISTOCHEMICAL BIOMARKERS IN ADRENAL PATHOLOGY Confirmation of Adrenocortical and Medullary Origin KEY POINTS  • Biomarkers are frequently used in the distinction of nonfunctional adrenocortical neoplasms from other tumors. As adrenocortical carcinoma is an exceptionally rare tumor, a frankly malignant neoplasm in the adrenal gland would more likely be a metastasis from another site than a primary adrenocortical carcinoma. Immunohistochemical studies to characterize the tumor as a primary adrenocortical tumor are critical.

Adrenocortical neoplasms will often show variable staining for keratins; however, significant staining for keratin is not always identified, rather it is often focal and weak. SF1 is the most reliable biomarker that should be used to confirm the adrenocortical origin31,90 (Fig. 91.27). However, several other somewhat less specific immunohistochemical stains (melan-­A, synaptophysin, alpha-­inhibin, and calretinin) are often used in traditional practices.91,92 Nevertheless, one should recognize that synaptophysin31 and alpha-­inhibin93 can be expressed in pheochromocytomas.

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Pheochromocytomas are often positive for neuroendocrine differentiation markers (e.g., chromogranin-­A, synaptophysin, and INSM1) and negative for keratins. When the distinction from other neuroendocrine neoplasms is indicated, the application of tyrosine hydroxylase (rate-­limiting enzyme in catecholamine biosynthesis) or dopamine β-­hydroxylase, along with GATA3 (variable), helps diagnosticians confirm the diagnosis of pheochromocytoma.94-­96 Most pheochromocytomas also show variable amounts of intratumoral sustentacular cells that are typically positive for SOX10 and S100. Evidence also suggests that sustentacular cells are not neoplastic in these tumors.97 This may be helpful in distinguishing multifocal primary paragangliomas from metastasis in some anatomic sites such as liver and lung, where both primary and metastatic disease can occur.

Confirmation of Functional Sites in Primary ­Aldosteronism Commercially available antibodies against steroidogenic enzymes have gained interest in the workup of functional sites in adrenalectomy specimens. The use of CYP11B2 immunohistochemistry adds great value to the workup of adrenalectomy specimens with primary aldosteronism.31,34 Although assessment for this biomarker is generally not routinely performed in pathology laboratories, the use of this biomarker may be especially useful when dealing with multiple adrenal nodules or when no nodules are noted in the gross examination.

Diagnostic and Predictive Biomarkers of Adrenocortical Carcinoma

Fig. 91.27  Confirmation of the adrenocortical origin. Steroidogenic factor 1 is the most reliable biomarker that should be used to confirm the adrenocortical origin.

A

Numerous biomarkers have been evaluated diagnostically and prognostically in adrenal pathology.31,83 Proliferation markers such as Ki67 (MIB1), proteins (IGF2, CDKN1C/p57, H19) associated with genes on chromosome 11p15 (which is also involved in Beckwith–Wiedemann syndrome); p53, for which TP53 (17q13) mutations occur in Li–Fraumeni syndrome, and somatic TP53 mutations and allelic loss of 17p13 may be seen in sporadic adrenocortical carcinomas; and alterations involving the Wnt/β-­ catenin-­signaling pathway, among others.9,29,31,83 Advances in our understanding of the molecular pathogenesis of adrenocortical carcinoma underscored the importance of p53 and β-­catenin as prognostic biomarkers, because most carcinomas with adverse molecular clusters tend to display nuclear β-­catenin expression and/or abnormal p53 staining (overexpression or global loss) (Fig. 91.28). For this reason, endocrine pathologists are increasingly using

B

Fig. 91.28  p53 overexpression and nuclear β-­catenin expression. p53 overexpression is seen in a subset of adrenocortical carcinomas that are often associated with high-­grade proliferative disease (A). Diffuse nuclear β-­catenin is also seen in some adrenocortical carcinomas, particularly in adverse molecular clusters (B).

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both p53 and β-­catenin immunohistochemistry in the workup of adrenocortical carcinomas.27,29,31,83

KEY POINTS  • Mitotic rate and proliferative activity as measured by Ki67 have been used in the grading and prognosis of adrenocortical carcinomas over time.81,83,102-­105

KEY POINTS  • IGF2 immunohistochemistry stands out as an important diagnostic biomarker of adrenocortical carcinomas.

19%, and greater than 20% are also suggested by the WHO classification.69 Given the critical value of Ki67 labeling index, eyeballing is less accurate. The use of pathologist-­driven automated image analysis nuclear algorithms or manual counting is helpful to more reliably determine the Ki67 proliferation index that can also be reproduced by other users27,106,107 (Fig. 91.31). SF1 immunohistochemistry not only confirms the cortical origin of an adrenal mass, but may also have prognostic impact.31,90 It has been reported that high expression of SF1 (N1665 antibody) positively correlated with high mitotic count, high Ki67 index, high stage, and decreased survival in a study of 75 adrenocortical carcinomas that also included myxoid in oncocytic variants.90 Immunohistochemistry for MMR proteins (MLH1, MSH2, MSH6, and PMS2) has been frequently used by endocrine pathologists,31 as adrenocortical carcinoma is one of the extracolonic manifestations of Lynch syndrome108,109 and may help determine eligibility for treatment options in affected patients.

Mete et al. demonstrated IGF2 immunohistochemistry optimized to detect paranuclear dot-­like reactivity in adrenocortical carcinomas in adults83 (Fig. 91.29). Proliferative activity in adrenocortical neoplasms is a continually important theme. The mitotic rate is present in numerous diagnostic algorithms and is considered by some as the most important criterion of malignancy.98 Proliferative activity as measured with a Ki67 labeling index of greater than 5% has been suggested as a sensitive and specific marker for malignancy83,99,100 (Fig. 91.30). A combination of Ki67 proliferative index and IGF2 expression has also been proposed to be useful in separating adrenocortical carcinomas from adenomas.83,100,101 The widely adopted Ki67 prognostic groups of less than 10%, 10% to

Molecular Immunohistochemistry in Pheochromocytoma In the evaluation of pheochromocytomas and paragangliomas, there are several biomarkers that can help predict disease pathogenesis and germline susceptibility. Commercially available antibodies against SDHB have revolutionized the screening process for more than a decade in endocrine pathology practice.110 Given the genotype–phenotype correlations, pheochromocytomas lacking a mature secretory phenotype (absence of adrenergic secretion) is usually assessed for SDHB immunohistochemistry.51 Germline DNA from patients with SDHdeficient tumors should be screened for all SDHx (SDHA, SDHB, SDHC, SDHD, and SDHF2) pathogenic variants, because a small fraction of SDH-deficient tumors may also be related to somatic/epigenetic alterations.111 Antibodies against SDHA (e.g., loss of SDHA expression suggests SDHA-­related pathogenesis) have also shown promise and are being used in clinical practice.110,111

Fig. 91.29  IGF2 immunohistochemistry in the distinction of adrenocortical carcinoma. Irrespective of tumor cytomorphology, proliferative grade, or functional status of the tumor, paranuclear IGF2 reactivity is a reliable diagnostic biomarker of adrenocortical carcinoma.

A

B

Fig. 91.30  Ki67 proliferation index in the distinction of adrenocortical carcinoma. Most adrenocortical adenomas have a Ki67 labeling index of less than 5% (A), whereas adrenocortical carcinomas tend to show higher labeling indices (B).

CHAPTER 91  Adrenal Pathology

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Fig. 91.31  Manual or automated counting of Ki67 labeling index. The current standard of practices requires meticulous assessment of the Ki67 labeling index in adrenocortical carcinomas. Eyeballing is no longer recommended. This photomicrograph illustrates a pathologist-­driven automated image analysis nuclear algorithm in a patient with adrenocortical carcinoma.

Fig. 91.32  SDHB immunohistochemistry in pheochromocytoma. This composite photomicrograph illustrates two pheochromocytomas. The tumor on the left shows normal SDHB expression, which argues against SDHx-­related pathogenesis. The tumor on the right tumor displays loss of cytoplasmic granular SDHB expression in the tumor, while the nontumorous endothelial cells still display preserved granular reactivity. This finding is consistent with a SDH-­deficient pheochromocytoma and requires germline testing of all SDH genes.

KEY POINTS  • The term SDH-deficient pheochromocytoma is applied to a pheochromocytoma that shows loss of cytoplasmic granular SDHB reactivity, while nontumorous elements preserve SDHB reactivity (Fig. 91.32).

KEY POINTS  • SDHB immunohistochemistry is also of clinical significance during the interpretation of SDHx variants of uncertain significance (VUS), because loss of SDHB expression by immunohistochemistry would favor a pathogenic process in the setting of VUS.51

Other molecular immunohistochemistry tools available to pathologists include antibodies against MAX and FH/2-­SC that correlates with MAX-­ and FH-­related pathogenesis.112,113 VHL-­related tumors often show membranous carbonic anhydrase IX staining93,114,115 (Fig. 91.21). Recently, alpha-­ inhibin immunohistochemistry has been shown to predict pseudohypoxia-­driven pathogenesis, including SDHx-­ and VHL-­related pheochromocytomas93 (Fig. 91.33).

SYNOPTIC REPORTING IN ADRENOCORTICAL CARCINOMA AND PHEOCHROMOCYTOMA The synoptic templates of the College of American Pathologists and International Collaboration on Cancer Reporting (ICCR)

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A

B

C

Fig. 91.33  Alpha-­inhibin expands biomarkers of pseudohypoxia-­driven pathogenesis. This composite photomicrograph illustrates a case of SDH-­deficient pheochromocytoma. By immunohistochemistry, the tumor is positive for tyrosine hydroxylase (A) and GATA3 (B), confirming the diagnosis. SDHB immunohistochemistry is not illustrated. This tumor is also variably positive for alpha-­inhibin (C). It is important to recognize this pitfall, as inhibin expression has been regarded as a marker of adrenal cortex, but recent evidence clearly confirms that this marker cannot be used in this distinction.

have been available for unified reporting of adrenocortical carcinomas.107,116 The synoptic reports often integrate the pathological tumor-­node-­metastasis (TNM) data at the time of diagnosis.

European Network for the Study of Adrenal Tumors (ENSAT) Stage I: Tumor confined to adrenal gland and 5 cm in diameter Stage III: Tumor involves surrounding tissue (not confined to the adrenal gland) or involvement of regional lymph node(s) or regional veins Stage IV: Distant metastases KEY POINTS  • Essential parameters that are reported in pheochromocytomas include (i) tumor size, (ii) presence/absence of extraadrenal invasion, (iii) presence/ absence of composite elements, (iv) vascular invasion, (v) lymphatic invasion, (vi) mitotic activity (mitoses per 2 mm2 based on 10 mm2 from hot spots), (vii) tumor necrosis, (viii) status of resection margins, (ix) status of adrenal medullary hyperplasia, (x) Ki67 proliferation index, (xi) histologically confirmed nodal metastasis, and (xii) molecular immunohistochemistry (at least SDHB immunohistochemistry in all tumors with no mature secretory phenotype). It is desirable to integrate the findings on biochemical and functional imaging studies into a pathological report.

Until the eight edition of the American Joint Committee on Cancer (AJCC) TNM staging system, there was no formal TNM staging for pheochromocytomas. The current AJCC TNM staging introduced three distinct pT stages based on size (the cut-off of 5 cm) and presence of extra-adrenal invasion into surrounding tissues (see https:// www.facs.org/quality-programs/cancer/ajcc for AJCC staging information). Because pheochromocytomas/paragangliomas are neuroendocrine neoplasms with metastatic potential, these tumors are now staged and are required to be reported using synoptic templates that are available to pathologists. The very first multidisciplinary international expert consensus synoptic template for pheochromocytomas and paragangliomas was introduced back in 2014110; and this was followed by additional reporting guides including but not limited to the ICCR template.117 KEY POINTS  • In adrenalectomy specimens, essential histological parameters that should be covered in adrenocortical carcinomas include (i) tumor size, (ii) presence of extraadrenal invasion, (iii) gross invasion into a large vein, (iv) microscopic vascular invasion, (v) microscopic lymphatic invasion, (vi) mitotic activity, reported as per number of mitoses per 50 high-­power fields, (vii) atypical mitoses, (viii)tumor mitotic grade (low or high grade, based on the cutoff of 20 per 50 high-­power fields), (ix) tumor necrosis, (x) Ki67 proliferation index, (xi) status of resection margins, and (xii) status of nodal disease (if lymph nodes are excised).

CHAPTER 91  Adrenal Pathology

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75. Hough AJ, Hollifield JW, Page DL, et al. Prognostic factors in adrenocortical tumors. A mathematical analysis of clinical and morphologic data. Am J Clin Pathol. 1979;72:390–399. 76. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol. 1984;8:163–169. 77. Weiss LM, Medeiros LJ, Vickery Jr AL. Pathologic features of prognostic significance in adrenocortical carcinoma. Am J Surg Pathol. 1989;13:202–206. 78. van Slooten H, Schaberg A, Smeenk D, et al. Morphologic characteristics of benign and malignant adrenocortical tumors. Cancer. 1985;55:766–773. 79. Weiss M, Ford VL. Differences in steroidogenesis by the subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus vulpecula). J Steroid Biochem. 1984;21:701–707. 80. Aubert S, Wacrenier A, Leroy X, et al. Weiss system revisited: a clinicopathologic and immunohistochemical study of 49 adrenocortical tumors. Am J Surg Pathol. 202;26:1612–1619. 81. Volante M, Bollito E, Sperone P, et al. Clinicopathological study of a series of 92 adrenocortical carcinomas: from a proposal of simplified diagnostic algorithm to prognostic stratification. Histopathology. 200;55:535–543. 82. Duregon E, Fassina A, Volante M, et al. The reticulin algorithm for adrenocortical tumor diagnosis: a multicentric validation study on 245 unpublished cases. Am J Surg Pathol. 2013;37:1433–1440. 83. Mete O, Gucer H, Kefeli M, et al. Diagnostic and prognostic biomarkers of adrenocortical carcinoma. Am J Surg Pathol. 2018;42:201–213. 84. Pennanen M, Heiskanen I, Sane T, et al. Helsinki score-­a novel model for prediction of metastases in adrenocortical carcinomas. Hum Pathol. 2015;46:404–410. 85. Duregon E, Cappellesso R, Maffeis V, et al. Validation of the prognostic role of the “Helsinki Score” in 225 cases of adrenocortical carcinoma. Hum Pathol. 2017;62:1–7. 86. Renaudin K, Smati S, Wargny M, et al. Clinicopathological description of 43 oncocytic adrenocortical tumors: importance of Ki-­67 in histoprognostic evaluation. Mod Pathol. 2018;31:1708–1716. 87. Brown FM, Gaffey TA, Wold LE, et al. Myxoid neoplasms of the adrenal cortex: a rare histologic variant. Am J Surg Pathol. 2000;24:396–401. 88. Wong DD, Spagnolo DV, Bisceglia M, et al. Oncocytic adrenocortical neoplasms–a clinicopathologic study of 13 new cases emphasizing the importance of their recognition. Hum Pathol. 2011;42:489–499. 89. Papathomas TG, Duregon E, Korpershoek E, et al. Sarcomatoid adrenocortical carcinoma: a comprehensive pathological, immunohistochemical, and targeted next-­generation sequencing analysis. Hum Pathol. 2016;58:113–122. 90. Duregon E, Volante M, Giorcelli J, et al. Diagnostic and prognostic role of steroidogenic factor 1 in adrenocortical carcinoma: a validation study focusing on clinical and pathologic correlates. Hum Pathol. 2013;44:822–828. 91. Renshaw AA, Granter SR. A comparison of A103 and inhibin reactivity in adrenocortical tumors: distinction from hepatocellular carcinoma and renal tumors. Mod Pathol. 1998;11:1160–1164. 92. Loy TS, Phillips RW, Linder CL. A103 immunostaining in the diagnosis of adrenocortical tumors: an immunohistochemical study of 316 cases. Arch Pathol Lab Med. 2002;126:170–172. 93. Mete O, Pakbaz S, Lerario AM, et al. The significance of alpha-­inhibin expression in pheochromocytomas and paragangliomas. Am J Surg Pathol. 2021 (in press). 94. Asa SL, Ezzat S, Mete O. The diagnosis and clinical significance of paragangliomas in unusual locations. J Clin Med. 2018;7:280. 95. Kimura N. Dopamine β-­hydroxylase: an essential and optimal immunohistochemical marker for pheochromocytoma and sympathetic paraganglioma. Endocr Pathol. 2021;32:258–261. 96. Kimura N, Miura Y, Nagatsu I, et al. Catecholamine synthesizing enzymes in 70 cases of functioning and non-­functioning phaeochromocytoma and extra-­adrenal paraganglioma. Virchows Arch A Pathol Anat Histopathol. 1992;421:25–32. 97. Powers JF, Tischler AS. Immunohistochemical staining for SOX10 and SDHB in SDH-­deficient paragangliomas indicates that sustentacular cells are not neoplastic. Endocr Pathol. 2020;31:307–309. 98. Blanes A, Diaz-­Cano SJ. Histologic criteria for adrenocortical proliferative lesions: value of mitotic figure variability. Am J Clin Pathol. 2007;127:398–408.

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ric patients with adrenocortical tumors carrying a germline mutation on TP53. Cancers (Basel). 2020;12:621. 110. Mete O, Tischler AS, de Krijger R, et al. Protocol for the examination of specimens from patients with pheochromocytomas and extra-­adrenal paragangliomas. Arch Pathol Lab Med. 2014;138:182–188. 111. Papathomas TG, Oudijk L, Persu A, et al. SDHB/SDHA immunohistochemistry in pheochromocytomas and paragangliomas: a multicenter interobserver variation analysis using virtual microscopy: a Multinational Study of the European Network for the Study of Adrenal Tumors (ENS@T). Mod Pathol. 2015;28:807–821. 112. NGS in PPGL (NGSnPPGL) Study Group, Toledo RA, Burnichon N, et al. Consensus statement on next-­generation-­sequencing-­based diagnostic testing of hereditary phaeochromocytomas and paragangliomas. Nat Rev Endocrinol. 2017;13:233–247. 1 13. Udager AM, Magers MJ, Goerke DM, et al. The utility of SDHB and FH immunohistochemistry in patients evaluated for hereditary paraganglioma-­ pheochromocytoma syndromes. Hum Pathol. 2018;71:47–54. 114. Pinato DJ, Ramachandran R, Toussi ST, et al. Immunohistochemical markers of the hypoxic response can identify malignancy in phaeochromocytomas and paragangliomas and optimize the detection of tumours with VHL germline mutations. Br J Cancer. 2013;108: 429–437. 115. Favier J, Meatchi T, Robidel E, et al. Carbonic anhydrase 9 immunohistochemistry as a tool to predict or validate germline and somatic VHL mutations in pheochromocytoma and paraganglioma-­a retrospective and prospective study. Mod Pathol. 2020;33:57–64. 116. Mete O, David JL, Rudzinski ER, et al. The College of American pathologists. Protocol for the examination of specimens from patients with carcinoma of the adrenal gland; 2020. Version: 4.1.0.0. Available at: https://www. cap.org/protocols-­and-­guidelines/cancer-­reporting-­tools. 117. Thompson LDR, Gill AJ, Asa SL, et al. Data set for the reporting of pheochromocytoma and paraganglioma: explanations and recommendations of the guidelines from the International Collaboration on Cancer Reporting. Hum Pathol. 2021;110:83–97.

92 Adrenal Gland Imaging David S. Lin, Ka Kit Wong, Elaine M. Caoili, James Shields, Anca M. Avram, and Benjamin L. Viglianti

OUTLINE Introduction, 1530 Adrenal Gland Anatomy, 1530 Adrenal Gland Imaging, 1530 Arteriography and Adrenal Vein Sampling, 1531 Clinical Utility, 1532 Adenoma or Metastases, 1532

Benign Adrenal Lesions, 1535 Neoplastic Adrenal Lesions, 1537 Interpretation of Adrenal Vein Sampling assays in Primary Hyperaldosteronism, 1547 Functional Imaging in Primary Hyperaldosteronism, 1548 Summary, 1548

 

INTRODUCTION A variety of different pathologies can affect the adrenal glands. High-­ resolution anatomic imaging with computed tomography (CT) and magnetic resonance imaging (MRI) can demonstrate subtle irregularities of the adrenal contours and can identify lesions millimeters in size. When combined with sensitive biochemical tests of adrenocortical and medullary hormones, diagnosis of adrenal gland dysfunction can be made at very early stages of disease. Certain adrenal pathologies can be confidently diagnosed based on imaging features alone and can potentially circumvent the need for percutaneous tissue sampling. Additionally, functional imaging with nuclear medicine scintigraphy and positron emission tomography (PET) add significant clinical utility, particularly in the evaluation of biochemically functional tumors as well as distant extra-­adrenal metastatic disease.

ADRENAL GLAND ANATOMY The adrenal glands are located in the retroperitoneum in the superior aspect of Gerota’s fascia in the perirenal space. These paired glands typically lie adjacent and lateral to the first lumbar vertebral body. The adrenal glands are suprarenal structures with the right gland lying more cranial relative to the left. They demonstrate an inverted-­Y or inverted-­V morphology with posteromedial and posterolateral limbs. The left gland tends to be slightly larger, with a width measuring up to 8 mm, whereas the width of the right gland measures up to approximately 6 mm (Fig 92.1).1 Histologically, there are two distinct components of the adrenal glands, an outer cortex and an inner medulla, which together can have a trilaminar appearance, although this is often not well depicted on imaging. The cortex is further subdivided into three zones, each with its own dedicated function: the zona glomerulosa, the zona fasciculata, and the zona reticularis. The zona glomerulosa is the outer most component of the cortex, secretes aldosterone, and is involved with electrolyte balance. The zona fasciculata, located just deep to the zona

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glomerulosa, synthesizes glucocorticoids and is involved with glucose metabolism. Lastly, the zona reticularis, the innermost component of the cortex, is involved with sex hormone production. The medulla is involved in the production of catecholamines, epinephrine, and norepinephrine, and regulates the stress response. The adrenal gland is surrounded by a collagenous capsule that is not seen on cross-­sectional imaging. The arterial supply to each adrenal gland comes from three sources: a superior adrenal artery arising from the inferior phrenic artery, the middle adrenal from the aorta, and the inferior adrenal from the renal artery. One or more central veins drain the cortex and the medulla and form the adrenal vein. The adrenal vein often freely communicates with one or more retroperitoneal and/or hepatic veins. The right adrenal vein is shorter than the left and usually enters the right posterolateral surface of the inferior vena cava (IVC) at a level between the eleventh and twelfth ribs. The central vein of the left adrenal gland joins the left inferior phrenic vein to form the phrenicoadrenal trunk, which then enters the superior aspect of the left renal vein. Collateral communications with renal capsular, azygous, and retroperitoneal veins are common. These descriptions are of classic venous anatomy and there are numerous described anatomic variants. KEY POINTS  • The adrenal gland is made of a cortex and medulla, each having specific endocrine function and related pathology.

ADRENAL GLAND IMAGING Ultrasound, while it is widely available and does not involve ionizing radiation, has limited diagnostic utility in the evaluation of the adrenal glands in adult patients due to size and location. It is, however, the imaging modality of choice for adrenal gland

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Fig. 92.1  Computed tomography appearance of normal adrenal glands. A, The cephalad portion of the right adrenal (arrow) is seen. B, The three limbs of the left adrenal (arrow) are shown at this level. (From Korobkin M, Francis IR. Adrenal imaging. Semin Ultrasound CT MR. 1995;16:317–330.)

evaluation in the pediatric population and is particularly useful for the assessment of adrenal hemorrhage and masses such as neuroblastoma (NB). The adrenal glands are normally hypoechoic or have echogenicity that is similar to that of adjacent perirenal fat. A hyperechoic center can sometimes be seen, corresponding with the medulla. Ultrasound may also have a role in percutaneous image-­guided biopsies of adrenal masses in both pediatric and adult patients. CT is currently the primary method of anatomic imaging of the adrenal gland and can visualize the glands in virtually all cases. The attenuation and enhancement of the adrenal glands are similar to soft tissue. Adrenal abnormalities are often identified incidentally (e.g., “incidentaloma”) during evaluation of a separate indication other than adrenal mass detection. Density measurements can be helpful for lesion analysis. A dedicated adrenal protocol CT can be especially beneficial for further characterization, particularly in distinguishing between a benign adenoma from metastatic disease. MRI, in comparison with CT, demonstrates improved tissue contrast and utility in patients with renal insufficiency or iodinated contrast allergy. On MRI, the adrenal glands are isointense to the spleen on T1-­weighted sequences and iso-­to hypointense to the spleen on T2-­ weighted sequences. Fat-­saturation sequences to detect macroscopic fat and in-­and out-­of-­phase sequences to detect intracellular lipid provide additional utility for lesion characterization. Recent advances in MRI technology have made MRI a competitive method with CT for imaging suspected adrenal disease. Positron emission tomography (PET)/CT using (18F)-­fluoro-­ deoxyglucose (FDG) is now widely used for imaging of hematological and solid malignancies that have characteristic overexpression of insulin-­independent glucose transporters (GLUT1, GLUT3) leading to uptake and intracellular trapping of FDG in cancer cells. FDG-­PET/ CT allows characterization of the “molecular signature” of an adrenal mass and can identify adrenal metastases with excellent sensitivity and high specificity.2,3 Radiopharmaceuticals for adrenocortical imaging have been available for more than 30 years, since the mid 1970s, with the radiocholesterol analog 131I-­6β-­iodomethylnorcholesterol (NP-­59) being the first in commercial use4 and newer radiopharmaceutical inhibitors of 11β-­hydroxylase, 11C-­etomidate and 11C-­, 18F-­, and 123I-­metomidate developed for imaging the normal adrenal cortex and various adrenocortical tumors.5 The first clinically successful adrenal medulla imaging agent, metaiodobenzylguanidine (MIBG), labelled with 123I

or 131I, was developed in the early 1980s and continues to be used clinically for imaging pheochromocytoma, paraganglioma, and NB.6 MIBG accumulation in neuronal tissues involves the norepinephrine transporter reuptake mechanism, and subsequent packaging by the vesicular monoamine transporter into catecholamine storage vesicles of adrenergic nerves and adrenomedullary cells. Other PET radiotracers for adrenomedullary imaging of pheochromocytoma are being investigated, including 18F-­6-­fluorodopamine (18F-­DA), 18F-­fluorodihydroxyphenylalanine (18F-­ DOPA), and 11C-­hydroxyephedrine (HED).7-­9 68Ga-­ 1,4,7,10–tetraazacyclododecane–1,4,7,10–tetraacetic acid (DOTA)– labeled somatostatin analogs have been developed for PET imaging of neuroendocrine tumors (NETs), affording image acquisition within sixty minutes postinjection and higher spatial resolution than radiotracers labeled for single-­ photon emission computed tomography (SPECT), supplanting the 111In-­DTPA-­octreotide scintiscan. 68Ga-­ DOTA peptide PET has been used to depict pheochromocytoma and paraganglioma successfully.10 KEY POINTS  • Several modalities are used to image the adrenal glands based on anatomy or function, the most common of which is computed tomography.

Arteriography and Adrenal Vein Sampling Arteriography is used only in a few selected adrenal lesions or in the case of extraadrenal pheochromocytoma, when additional information about tumor resectability and vascular anatomy is needed. Adrenal venous sampling (AVS) is performed in patients with proven primary hyperaldosteronism to distinguish hypersecretory unilateral adenoma from bilateral adrenal hyperplasia (Fig 92.2). Rarely, AVS is performed to determine whether autonomous hormone excess is unilateral or bilateral in Cushing syndrome. Indications, specificity, and sensitivity, as well as data interpretations and treatment recommendations based on AVS, are discussed in another chapter in this textbook. The specific risk with adrenal arteriography is rupture of the adrenal arteries with contrast extravasation. Potential complications of adrenal vein catheterization include intraadrenal extravasation of contrast medium, bleeding, and adrenal vein thrombosis. Adrenal hemorrhage can cause severe pain and fever, as well as destruction of adrenal glandular function and hypoadrenalism if both adrenal glands are damaged.

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Fig. 92.2  Bilateral adrenal venograms in a patient with primary hyperaldosteronism. A, Right adrenal venogram in the left anterior oblique projection. The intrarenal branches are filled poorly. The main vein enters the posterolateral aspect of the inferior vena cava. B, Left adrenal venogram in the anteroposterior projection with the injection of contrast medium via the 3F microcatheter introduced through the 5F reverse hook catheter positioned near the junction of the main adrenal vein and the left renal vein.

KEY POINTS  • Adrenal vein sampling (AVS) allows direct measure of adrenal hormone synthesis. • AVS is invasive and technically challenging.

CLINICAL UTILITY Adenoma or Metastases The increasing availability and use of anatomic imaging, particularly CT, performed for indications unrelated to the adrenal gland have led to a growing number of incidentally found adrenal lesions, also termed incidentalomas. The discovery of an adrenal mass presents a diagnostic and, at times, a therapeutic challenge. The evaluation of these incidentalomas could require identification of malignancy and would subsequently impact clinical management. The clinical history of a preexisting cancer diagnosis is important due to significant differences in prevalence of adrenal malignancy in oncologic versus nononcologic patients. In addition, it is necessary to distinguish if the adrenal mass is functional or hormonally active by biochemical testing of secretory function (screening for mineralocorticoid, glucocorticoid, and catecholamine secretion). Because the overwhelming majority of incidentalomas are benign, an aggressive approach is often not indicated. The prevalence of adrenal adenomas is high, ranging from 4% to 10% of patients studied with CT or MRI for indications other than suspected adrenal disease.11 Small, homogeneous adrenal masses discovered incidentally are likely to be adenomas. Unenhanced CT densitometry can be used to accurately differentiate adrenal adenomas from metastases. Most adenomas, which are lipid-­rich, have unenhanced CT attenuation values lower than those of metastases. In a histologic/radiologic study of a small number of resected adrenal adenomas that had undergone presurgical unenhanced CT, chemical shift MRI, or both, a good inverse linear correlation was seen between the estimated number of lipid-­rich cells and the unenhanced CT attenuation values, and a good linear correlation was noted with the relative change in signal intensity on opposed-­phase MRI (Figs. 92.3 and 92.4).12

In the scenario of an oncologic patient with an unsuspected adrenal mass and no other evidence of metastatic disease, the goal of noninvasive diagnostic adrenal imaging is to identify an adrenal adenoma with high specificity, and thus exclude adrenal metastases. With the use of pooled data from multiple published studies, it has been shown that the most optimal combination of a sensitivity (71%) and specificity (98%) for the diagnosis of lipid-­rich adrenal adenoma is achieved when a threshold attenuation value of 10 HU or lesions identified on a routine contrast-­ enhanced CT), a dedicated adrenal protocol CT is beneficial for distinguishing between benign adenomas from metastases. Adrenal adenomas, whether lipid-­rich or lipid-­poor, demonstrate rapid washout of contrast. A dedicated adrenal protocol CT typically includes precontrast, 60 to 75-­second postcontrast, and 15-­minute delayed imaging. Absolute washout is calculated as follows: [(postcontrast HU – delayed HU)/(postcontrast HU – precontrast HU)] x 100. In cases where precontrast imaging is not obtained, a relative washout can be calculated as follows: [(postcontrast HU – delayed HU)/postcontrast HU)] x 100. Adenoma can be diagnosed with absolute washout greater than or equal to 60% or relative washout greater than or equal to 40% (Fig. 92.6). In a prospective study of 166 adrenal masses with unenhanced CT, those with attenuation values greater than 10 HU underwent contrast-­enhanced and 15-­minute delayed enhanced CT. This protocol correctly characterized 160 of the 166 masses (96%). Exclusion of primary adrenal neoplasms increased sensitivity and specificity for characterizing a mass as adenoma versus metastasis to 98% (124/127) and 97% (33/34), respectively.14 Similar results were subsequently confirmed with similar protocols.15,16 Therefore, a dedicated adrenal protocol CT to calculate washout is being used in many centers to characterize incidentally discovered adrenal masses.

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C Fig. 92.3  Histologic specimens from resected adrenal adenomas. A, Primarily of lipid-­rich clear cells. B, Primarily of lipid-­poor clear cells. C, Of an admixture of clear and compact cortical cells. (Hematoxylin and eosin stain, original magnification ×200.) (From Korobkin M, Giordano TJ, Brodeur FJ, et al. Adrenal adenomas: relationship between histologic lipid and CT and MR findings. Radiology. 1996;200:743–747.) 40

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Lipid-rich cells (%) Fig. 92.4  Plot of unenhanced computed tomography attenuation number versus the percentage of lipid-­rich cells in 13 surgically resected adrenal adenomas. HU, Hounsfield units. (From Korobkin M, Giordano TJ, Brodeur FJ, et al. Adrenal adenomas: Relationship between histologic lipid and CT and MR findings. Radiology. 1996;200:743–747.)

Notably, the assessment of enhancement washout curves for adrenal masses is valid only for lesions with relatively homogeneous attenuation after contrast enhancement; the diagnosis of adrenal adenoma cannot be made in masses that contain prominent regions of necrosis or hemorrhage.

25 20 15 10 5 0 −5 −10 −15 −20 −25 −30

Adenomas Nonadenomas Fig. 92.5  Scattergram of attenuation values on unenhanced compute tomography of adrenal adenomas and nonadenomas. All masses with H values of less than 18 were adenomas. ●, Nonhyperfunctioning adenomas; O, Cushing adenomas; ●, primary aldosteronism adenomas; ▲, metastases; Δ, pheochromocytomas; ▲, cortical carcinomas. (From Korobkin M, Brodeur FJ, Yutzy GG, et al. Differentiation of adrenal adenomas from nonadenomas using CT attenuation values. Am J Roentgenol. 1996;166:531–536.)

On MRI, chemical shift sequences can be used to identify intracytoplasmic lipid and can therefore distinguish adenomas.17,18 Taking advantage of the different precession rates for hydrogen atoms in water

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Fig. 92.6  Bar graph shows mean computed tomography attenuation value plus 1 standard deviation for adrenal adenomas (black) and nonadenomas (white) on unenhanced, enhanced, and delayed enhanced scans. (From Korobkin M, Brodeur FJ, Francis IR, et al. CT-­time-­attenuation washout curves of adrenal adenomas and nonadenomas. Am J Roentgenol. 1998;170:747–752.)

and lipid molecules, chemical shift MRI results in a decrease in signal intensity on opposed-­phase versus in-­phase imaging in tissue containing intracellular lipid that is often present in adenomas and absent in most metastases. Assessment of the chemical shift change can be made via simple visual analysis or by quantitative methods using standard region-­of-­interest measurements of the mass (Fig 92.7). The two most commonly used formulae for quantitative analysis of chemical shift change are the signal intensity index (SII) and adrenal-­to-­spleen chemical shift ratio (ASR). SII is calculated by [(IPIa – OPIa)/IPIa] x 100 and ASR is calculated by [(OPIa/OPIs)/(IPIa/IPIs)] x 100, where IPIa represents in-­phase intensity in the adrenal, OPIa represents opposed-­ phase intensity in the adrenal, IPIs represents in-­phase intensity in the spleen, and OPIs represents opposed-­phase intensity in the spleen. An SII of 16.5% (or 20%) and an ASR of less than 0.71 are considered to be highly accurate for the characterization of lipid-­containing adrenal adenomas.18-­21 The advent of FDG-­PET/CT for determination of the metabolic signature of adrenal masses to predict malignant potential has replaced earlier work using functional adrenal imaging such as NP-­59 or MIBG scintigraphy to characterize specific molecular processes of adrenal tumors.22 A metaanalysis of 1217 patients from 21 studies reported that FDG-­PET had a pooled sensitivity of 97% and specificity of 91% for distinguishing malignant from benign adrenal masses.23 Another metaanalysis reported similar results, with pooled sensitivity 91% and specificity 91% for both FDG-­PET and PET/CT, although significant heterogeneity of the included studies was noted.24 In subjects without preexisting cancer history, FDG-­PET/CT had sensitivity 86.7% and specificity 86.1% to distinguish malignant from benign etiology of adrenal incidentaloma.25 The accuracy of FDG-­PET/CT appears to be similar using either qualitative or quantitative assessment of the adrenal mass uptake compared with adjacent liver background. In a recent study of metabolic parameters for characterization of adrenal incidentaloma in noncancer patients, a standardized uptake value (SUV) max cutoff greater than 3.1 and adrenal:liver ratio greater than 1.8 had 100% sensitivity and 67% specificity for identifying cancer.26 Similar findings were reported in 59 patients (malignant lesions in 52 using histological gold standard),

that adrenal tumor SUV threshold greater than 3.5 had an accuracy of 87% for predicting malignancy.27 FDG-­PET/CT metabolic measurements have been combined with CT histograms and unenhanced CT parameters (HU, size) on hybrid PET/CT cameras to improve diagnostic accuracy.28,29 Current evidence suggests a complementary role for FDG-­PET/CT to CT for the assessment of indeterminate adrenal masses characterized by CT or MRI. In a systematic review of 353 indeterminate adrenal lesions measuring greater than 10 HU, CT showed a sensitivity 100% with a specificity of 33%, while 89 patients with FDG-­PET/CT having a tumor:liver ratio greater than 1.8 had a sensitivity of 87% and a specificity 84% for malignancy. Therefore, FDG-­PET is helpful in further characterization of indeterminate lesions, improving specificity.30 Despite excellent sensitivity, FDG-­ PET/CT false negatives can occur with small tumors (87 mls), TLG (>229), or SUVmax (>8.8) had worse outcomes for both progression-­ free and overall survival.80 Overexpression of chemokine receptor 4 (CXCR4) in ACC is a potential diagnostic and therapeutic receptor target.81 FDG-­PET was compared to 68Ga-­pentixafor PET, with both studies performed within a week of each other. In 30 patients with disease, the PET studies in five (17%) were complementary and in 10 (33%) were comparable. In 13 (43%), more lesions were found with FDG-­PET than with 68Ga-­ pentixafor PET. Overall, 70% of patients with advanced metastases from the ACC may be suitable for CXCR4-­directed therapy (radiotherapy or pharmacotherapy). CXCR4 positivity in ACC correlates with expression and quantification of metastases.82

Pheochromocytoma. Adrenal pheochromocytomas and paragangliomas, arising from extraadrenal tissues, are rare tumors derived from the sympathomedullary cells of postganglionic sympathetic neurons. The majority arise within the adrenal medulla or in an extraadrenal location near the celiac axis or aortic bifurcation (organ of Zuckerkandl). Those that are catecholamine-­secreting result in the classic signs and symptoms of hypertension, tachycardia, headaches, palpitations, diaphoresis, and chest pain. Although many patients present with some or all of these manifestations of excess catecholamine production, approximately 8% to 10% of tumors are silent and incidentally detected by imaging studies. A significant proportion of these tumors are found to be malignant once evidence of metastatic spread is documented, usually with imaging. Although pheochromocytomas were traditionally taught as following the rule of 10s (10% being multiple, 10% being inherited, 10% being extraadrenal in location, and 10% being malignant), more

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Fig. 92.16  Adrenal cortical carcinoma. A, Contrast-­enhanced computed tomography examination shows a 9-­cm right adrenal mass. Irregular wall and low-­density center indicate necrosis. B, More cephalad image in same examination reveals tumor extension into inferior vena cava. (From Dunnick NR, Korobkin M. Imaging of adrenal incidentalomas: current status. Am J Roentgenol. 2002;179:559–568.)

Fig. 92.17 Intravenous contrast-­ enhanced computed tomography shows a right adrenal pheochromocytoma (P) with areas of necrosis.

contemporary understanding of genetic predisposition has identified closer to 40% of cases as being inherited and associated with disorders including multiple endocrine neoplasia (MEN) syndromes, neurofibromatosis, von Hippel–Lindau (VHL), or familial pheochromocytoma.83 The diagnosis of pheochromocytoma can be confirmed by elevated blood or urine levels of the catecholamines epinephrine and norepinephrine or their metabolites. Localization of a pheochromocytoma is essential, because surgical resection of the tumor can be curative. On CT, pheochromocytomas typically range from 2 to 5 cm and demonstrate heterogeneous attenuation (usually >10 HU on unenhanced images) due to necrosis or hemorrhage, particularly when they are larger in size. Imaging of pheochromocytomas with contrast-­ enhanced CT has historically been associated with contrast-­induced hypertensive crisis; however, use of nonionic contrast agents has eliminated this concern. On contrast-­enhanced CT, pheochromocytomas typically demonstrate heterogeneous enhancement similar to that seen in ACC or metastases (Fig. 92.17). Oral contrast opacification of the gastrointestinal tract can be occasionally helpful for the detection of extraadrenal pheochromocytomas/paragangliomas, because unopacified bowel may rarely be mistaken for a “mass” and may mimic these tumors (Fig. 92.18).84 Unusual locations of paragangliomas have also

Fig. 92.18  Retroperitoneal extraadrenal pheochromocytoma. Enhanced computed tomography shows an inhomogeneous enhancing mass medial to the left kidney. A normal left adrenal was demonstrated on scans cephalad to this mass.

been reported, such as the rare, intrapericardial paraganglioma adjacent to or involving the left atrium (Fig. 92.19). On MRI, most pheochromocytomas are hypointense on T1-­ weighted images and markedly hyperintense on T2-­weighted images. There is considerable overlap of signal on T2-­weighted images with other neoplasms, including ACC, in up to 33% of cases. This overlap on T2-­weighted imaging may be related to necrotic or cystic areas that are commonly seen in these tumors. Contrast-­enhanced MRI is useful for demonstrating the presence or absence of intracaval extension of adrenal pheochromocytoma. MRI is also useful for the evaluation of extraadrenal paragangliomas that can be seen from the neck to the bladder. 123I-­MIBG can be used to image pheochromocytoma and paraganglioma with high specificity, thus confirming their origin from the neural crest, with the additional benefit of whole-­body screening to identify multifocal and/or remote metastatic disease (Fig. 92.20). A metaanalysis found MIBG scintigraphy to have high sensitivity (94%) and specificity (95%) for imaging pheochromocytoma/paraganglioma.85 MIBG scintigraphy is clinically useful in depicting diagnostically elusive and

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often small paragangliomas because of their close relationship to other structures and remote locations (extraadrenal sites), screening from the base of the skull to the pelvis (Fig. 92.21). In malignant pheochromocytoma/paraganglioma studied with MIBG, the most common sites of metastatic disease are the skeleton, lymph nodes, lung, and peritoneum. Importantly, MIBG imaging is not particularly useful in head and neck paragangliomas, as these are tumors are of parasympathetic origin that lack chromaffin cells and are nonsecretory; these neoplasms are better imaged with 68Ga-­DOTAtate or 18F-­DOPA PET.86 A range of PET radioisotopes such as 18F-­FDG, 18F-­DA, 18F-­DOPA, and 11C-­HED (Fig. 92.24) have been successfully used to image pheochromocytoma and paraganglioma and can be useful in cases when

Fig. 92.19  Intrapericardial extraadrenal pheochromocytoma. Enhanced computed tomography shows an inhomogeneous mass in the expected location of the left atrium. Metaiodobenzylguanidine scan was positive in this location. (From Hamilton BH, Francis IR, Gross BH, et al. Intrapericardial paragangliomas [pheochromocytomas]: Imaging features. Am J Roentgenol. 1997;168:109–113.)

the disease does not show strong MIBG uptake.87 Most pheochromocytomas, whether benign or malignant, can be imaged with FDG-­PET, although focal FDG uptake is found in greater percentage of malignant than benign pheochromocytomas. Shulkin et al. reported that 22 of 29 patients with pheochromocytoma could be identified with FDG-­ PET, and that MIBG-­negative pheochromocytomas were depicted with FDG, a finding that has been confirmed by others.88 Timmers et al. found that FDG-­PET had a sensitivity of 77% and a specificity of 90% in 216 patients with suspected pheochromocytoma/paraganglioma and outperformed both MIBG and CT/MRI.89 In one study, the combination of FDG-­PET and apparent diffusion coefficient on MRI was useful to separate 11 pheochromocytomas from 22 other benign tumors.90 Preoperative PET may find extra lesions prior to surgical resection of pheochromocytoma.91 Metabolic subtyping of pheochromocytoma with FDG-­PET has been reported.92 Metabolic uptake is higher for malignant than benign pheochromocytoma and for extraadrenal paraganglioma than adrenal sites. The degree of FDG uptake in pheochromocytoma depends on genetic mutation. In genetic syndromes, cluster 1 genes (SDHx, VHL) have higher FDG uptake than cluster 2 genes (RET, NF1).93 The use of FDG-­PET for screening for SDHx mutation carriers has high sensitivity and intermediate specificity,94 and FDG-­PET use has been explored for patients with VHL, patients with MEN2, and for screening for pheochromocytoma in NF1 patients.95 Fottner et al. reported an 18F-­ DOPA-­ PET sensitivity of 98% (62/64 lesions) and specificity of 100%, much higher compared to an MIBG sensitivity of only 53% (34/64 lesions) and specificity of 91%.96 18F-­DOPA-­PET was reported to be markedly avid in a more recently described genetic predisposition due to MYC-­ associated factor X (MAX) pheochromocytoma.97 Studies of 18F-­DA-­PET for imaging pheochromocytoma/paraganglioma show high sensitivity and specificity.98 Discordant results with MIBG, 18F-­FDG, 18F-­DA, and 18F-­DOPA imaging appear to depend on tumor phenotype and are most likely related to the specific underlying mutation. In one comparative study of different molecular imaging

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Fig. 92.20  Left adrenal pheochromocytoma demonstrated by 123I-­metaiodobenzylguanidine (MIBG) and magnetic resonance imaging (MRI). A 50-­year-­old female with hypertension, a history of neurofibromatosis, elevated plasma catecholamines, and a 3-­cm left adrenal mass on MRI. A, Abdominal MRI, transverse section, with left adrenal pheochromocytoma (white arrow). B, Anterior abdominal 123I-­MIBG scan with normal liver (L) uptake. The black arrow indicates intense, focal tracer uptake in the region of the left adrenal gland. C, Posterior abdominal 123I-­MIBG scan. The black arrow indicates left adrenal pheochromocytoma. (From Rubello D, Bui C, Casara D, et al. Functional scintigraphy of the adrenal gland. Eur J Endocrinol. 2002;l47:13–28.)

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Fig. 92.21  A 53-­year-­old man with uncontrolled hypertension and catecholamine hypersecretion, suspected for pheochromocytoma. 123I-­metaiodobenzylguanidine (MIBG) composite anterior (A) and posterior (B) whole-­body images at 24 hours postinjection show focal intense activity in the region of the left adrenal gland (arrow). C, Axial computed tomography (CT), and fused single-­photon emission CT (SPECT)/CT images confirm a left adrenal pheochromocytoma with intense 123I-­MIBG uptake (arrowheads). There is also preaortic soft tissue nodule with intense 123I-­MIBG uptake (arrows) on the SPECT/CT that may represent an extraadrenal paraganglioma or, less likely, a metastatic retroperitoneal lymph node indicating malignant disease. (From Youssef E, Wong KK. Hybrid SPECT-­CT endocrine scintigraphy: a pictorial review. AUR, Annual Meeting in San Antonio, Texas, 2011.)

radiotracers, Timmers et al. investigated 30 patients with the succinate dehydrogenase subunit B (SDHB) germline mutation and metastatic pheochromocytoma/paraganglioma.89 They found that 18F-­FDG-­ PET had a sensitivity of 100% that exceeded 18F-­DOPA (88%), MIBG (80%), and somatostatin receptor scintigraphy (SRS) (81%). At least 90% of the lesions that were negative on 18F-­DOPA and MIBG imaging were depicted with 18F-­FDG, making it the imaging agent of choice in patients with SDHB germline mutations. PET imaging of 68Ga-­ DOTA-­ conjugate peptides such as 68Ga-­ DOTAtoc (DOTA0-­1NaI3-­octreotide), 68Ga-­DOTAnoc (DOTA0-­ Tyr3-­octreotide) and 68Ga-­DOTAtate (DOTA0-­Tyr3-­octreotate) has brought dramatic improvement in sensitivity and spatial resolution and is preferred for NET localization and staging studies. Further advantages are same-­day imaging protocols and availability of whole-­ body nondiagnostic CT component, use of newer-­generation DOTA chelator with improved affinity for SSTR2, and ability to predict efficacy of 177Lu or 90Y-­radionuclide labeled somatostatin analogs. 68Ga-­ DOTANOC-­PET imaging of pheochromocytoma has been reported

and can successfully image metastatic pheochromocytoma/paraganglioma in MIBG-­negative patients (Fig. 92.22).10,99 Widespread availability of 68Ga-­DOTANOC-­PET since 2016 in North America has resulted in it largely replacing 111In-­octreotide for imaging of NETs. It has high sensitivity for pheochromocytoma/paraganglioma; however, specificity is limited, as many nonendocrine tumors (e.g., breast cancer, meningioma, lymphoma), granulomatous and inflammatory diseases (e.g., sarcoidosis, tuberculosis), and autoimmune diseases (rheumatoid arthritis) express type 2 somatostatin receptors. Somatostatin receptor PET imaging is most useful when MIBG scanning is negative in metastatic pheochromocytoma/paraganglioma or when other neuroendocrine neoplasms are suspected (Fig 92.23).95,100,101

Lymphoma. Primary lymphoma of the adrenal glands is rare, and secondary involvement of the adrenals is seen more often in patients with non-­Hodgkin lymphoma than in Hodgkin lymphoma.102 In secondary lymphoma, adrenal involvement is often bilateral, and other retroperitoneal disease is usually present. On CT, there is homogeneous

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Fig. 92.22  68Ga-­DOTA-­tate positron emission tomography/computed tomography scan in a patient with elevated catecholamines and a right-­sided adrenal mass. Pheochromocytoma was confirmed on adrenalectomy specimen.

enlargement of the adrenal gland with variable enhancement.103 On MRI, lymphoma is characterized by hypointense signal on T1-­weighted images and heterogeneous hyperintensity on T2-­weighted images with minimal postcontrast enhancement. FDG-­PET/CT is a useful imaging modality for both secondary and primary adrenal lymphoma.104-­108 In secondary lymphoma, the adrenal masses typically have limited washout and high SUV values.109 They are most often due to diffuse large B cell lymphoma and are well-­ defined masses with preserved adreniform shape, having mean SUV 18.6 and size greater than 3 cm. FDG-­PET was reported in 28 patients

with primary adrenal lymphoma, with disease usually manifesting as intensely hypermetabolic adrenal masses. FDG-­PET provides prognostic information for primary adrenal lymphoma,110 with poor overall survival of 61.9% at 2 years despite chemotherapy (Fig 92.25).111

Metastases. The adrenal glands are a common site of metastatic disease, which is found in approximately 27% of postmortem examinations of patients with malignant neoplasms of epithelial origin.112 The most common neoplasms that metastasize to the adrenal glands are carcinomas of the lung and breast, and melanoma. Small metastases

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Fig. 92.24  11C-­Hydroxyephedrine (11C-­ HED) positron emission tomography scan in a patient with malignant pheochromocytoma. Multiple areas of increased uptake are seen in the liver, abdomen, and spleen (arrows). (From Gross MD, Shapiro B. Adrenal scintigraphy. In: Khalkhali I, Maublant J, Goldsmith S, eds. Nuclear Oncology. Philadelphia: Lippincott, Williams & Wilkins; 2000:472.)

Fig. 92.23  68Ga-­DOTA-­tate positron emission tomography maximum intensity projection images in a 25-­year-­old man with metastatic paraganglioma, demonstrated intensely avid uptake in multiple osseous lesions scattered in the skeleton.

(Figs. 92.26 and 92.27) are often homogenous on contrast-­enhanced CT or MRI, whereas larger metastases often appear heterogeneous due to necrosis and/or hemorrhage. Calcification is rare in adrenal metastases. Intracellular lipid can be seen in metastases from clear cell renal cell carcinoma and hepatocellular carcinoma. In addition, adrenal washout characteristics need to be interpreted with caution in patients with suspected metastases from hepatocellular carcinoma, renal cell carcinoma, and NET, as these lesions may demonstrate washout characteristics similar to that of benign adrenal adenomas.113 In a metaanalysis for staging/restaging lung cancer that included 707 patients from nine studies, metabolic imaging with FDG-­PET had a pooled sensitivity of 88% and specificity of 90.8% for detection of metastatic disease.114 It is recognized that in cancer patients with oligometastatic disease to the adrenal (i.e., solitary metastasis to the adrenal) adrenalectomy can have a survival advantage.115,116 In a retrospective study of 289 patients undergoing adrenalectomy, 39 (13%) had suspected oligometastatic disease due to a positive adrenal FDG-­PET.117 In 28/39 (71.8%) of these patients, the oligometastasis was confirmed at adrenalectomy; history of primary lung cancer and SUVmax greater than 2.65 increased the odds ratio of metastatic disease. However, notably in this series, ten benign cortical adenomas and one nonfunctioning pheochromocytoma had positive PET findings, leading to a 28.2% false-­positive rate in this setting (Figs. 92.28 and Fig 92.29).

Neuroblastoma. Neuroblastoma (NB), a malignant tumor composed of primitive neuroblasts, is the most common extracranial solid malignancy of children, representing 8% to 10% of pediatric cancers. The mean age of diagnosis is 15 months, and the majority occur in infancy and early childhood.118 While these tumors may occur anywhere along the sympathetic plexus, the most common site for primary NB is the

adrenal gland, accounting for approximately 35% to 40% of cases.119 Unfortunately, metastases are present in over half of pediatric patients at diagnosis. NB in adults, though rare, usually have imaging findings that are similar to those in children, with disseminated disease at presentation being common. On CT, NB appears as a large, irregular, and heterogeneous mass due to necrosis and/or hemorrhage. Calcification is common and can be seen in 80% to 90% of cases.120 On MRI, NB typically demonstrates heterogeneous hypointense signal on T1-­weighted images and hyperintense signal on T2-­weighted images with variable and heterogeneous enhancement. NB can be aggressive and infiltrative, causing narrowing and encasement of adjacent vessels. Invasion of adjacent soft tissues and organs can also occur.121 123I-­MIBG diagnostic imaging is an established modality for diagnosis, staging, and treatment response assessment of NB, as well as for determining suitability of targeting of metastatic disease for subsequent 131I-­MIBG treatment.122 123I-­MIBG is more accurate than bone scan for detection of osseous metastatic disease in NB.123 More recently, 68Ga-­DOTATATE-­PET/CT has been found to be positive in just over two thirds of patients with refractory NB, in six of eight patients in each of two small pilot studies.124,125 Furthermore, 68Ga-­DOTATATE-­ PET/CT detected additional sites of disease compared with 123I-­MIBG in three out of eight patients.125 68Ga-­DOTATATE-­PET/CT imaging has that potential to inform on suitable targeting of radionuclide treatment of NB with 177Lu-­DOTA peptides.124

Cushing Syndrome. Cushing syndrome results from excessive glucocorticoids, resulting in characteristic clinical signs and symptoms. Cushing syndrome refers to the clinical and metabolic disorder regardless of the underlying cause. The most common etiology is iatrogenic steroid administration. Endogenous Cushing syndrome is due to overproduction of cortisol by the adrenal cortex, caused by (1) excess production of ACTH by a pituitary tumor; (2) benign or malignant steroid-­producing adrenocortical neoplasm; or (3) bilateral adrenal hyperplasia secondary to an ectopic source of ACTH production. Very rare causes of ACTH-­independent bilateral hyperplasia have also been described. Strictly speaking, Cushing disease refers only to

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Fig. 92.25  (FDG) positron emission tomography/computed tomography (PET/ CT) in a 53-­year-­old woman with chronic myeloid leukemia, status post–bone marrow transplantation. Maximum intensity projection image (A), axial PET (B), and fusion PET/CT (C) demonstrate a 6.5 x 5.5 cm right suprarenal mass (arrows) with intensely increased FDG uptake (maximum standard uptake value = 17.1) above liver background (liver mean standard uptake value = 2.8). Histopathology specimen of the resected mass was compatible with a hematolymphoid malignancy, an extramedullary myeloid tumor. (From Wong KK, Arabi M, Bou-­Assaly W, et al. Evaluation of incidentally discovered adrenal masses with PET and PET/CT. Eur J Radiol. 2012;81:441–450.)

bilateral adrenal hyperplasia due to ACTH hypersecretion by a pituitary adenoma. Most cases of Cushing syndrome (up to 85%) are due to excess ACTH production from pituitary or ectopic sources. The adrenal glands may be normal or show diffuse bilateral gland thickening and nodularity on CT, a finding indicative of hyperplasia. A small percentage of patients (12%–15%) with Cushing disease demonstrate multiple or, less commonly, single macronodules, varying in size from several millimeters to as much as 7 cm in diameter.126 If macronodular adrenal hyperplasia is characterized by a single dominant nodule, this entity may be confused with a unilateral autonomous adrenal adenoma, leading to inappropriate unilateral adrenalectomy. Additional small nodules or diffuse overall enlargement of both glands are usually present; however, correlation with the biochemical findings allows the correct diagnosis to be made. A much rarer form of macronodular adrenal hyperplasia is ACTH-­ independent disease. Two unique CT and MRI features of this disorder are the large mass of cortical tissue and the size of individual nodules (Fig. 92.30).127 The size of the nodules can suggest alternate diagnoses such as bilateral metastases or bilateral adenomas, but in the presence

of Cushing syndrome, the appearance is practically pathognomonic of ACTH-­independent macronodular adrenal hyperplasia, and bilateral adrenalectomy is indicated as optimal therapy, on the basis of clinical and CT findings. Approximately 15% of cases of ACTH-­dependent Cushing syndrome are due to ectopic ACTH secretion from a nonpituitary source. The most common sources of ectopic ACTH-­producing tumors are small cell lung carcinomas and bronchial carcinoids. However, the diagnosis of patients with occult sources of ACTH secretion presents a difficult diagnostic challenge. In one study, five of eight bronchial carcinoid tumors measured between 4 and 10 mm in diameter, and thin-­section CT was required to detect these small tumors. Less common sources of ectopic ACTH production include elusive islet cell tumors of the pancreas, pheochromocytomas, medullary thyroid carcinomas, and thymic carcinoids. Another study that included molecular imaging found that the source of ectopic ACTH-­secreting tumors in descending order was the lung (55.3%), pancreas (8.5%), mediastinum thymus (7.9%), adrenals (6.4%), and gastrointestinal tract (5.4%). Bronchial NETs were the most common (54.8%) etiology. However, imaging was only moderately successful in localizing

CHAPTER 92  Adrenal Gland Imaging

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Fig. 92.27  Necrotic metastasis from adenocarcinoma of the lung in a 34-­year-­old woman. Bilateral adrenal masses with areas of central necrosis are seen on this contrast-­enhanced computed tomography examination. (From Dunnick NR, Korobkin M. Imaging of adrenal incidentalomas: current status. Am J Roentgenol. 2002;179:559–568.) Fig. 92.26  Metastasis from renal carcinoma in a 31-­year-­old woman. Small, homogeneous left adrenal mass is seen on this contrast-­enhanced computed tomography examination. (From Dunnick NR, Korobkin M. Imaging of adrenal incidentalomas: current status. Am J Roentgenol. 2002;179:559–568.)

the tumor site (CT 66.2%, MRI 51.5%, FDG-­PET 51.7%, 18F-­DOPA 57.1%, and MIBG 30.8%).128 The best imaging modality for bronchial carcinoid and other NETs causing ectopic ACTH secretion was 68Ga-­ DOTATATE-­PET, with a sensitivity of 81.8%,129 whereas FDG-­PET performs better for detection of small cell lung cancer.130 Up to 30% of cases of Cushing syndrome are due to an ACTH-­ independent adrenocortical neoplasm; approximately two-thirds of these are due to adrenal adenomas, and the other one-third to ACCs. These tumors are easily detectable on both CT and MRI. Adrenocortical adenomas are nearly always less than 5 cm in diameter, typically 2 to 2.5 cm in size, and have a nonspecific morphologic appearance (Fig. 92.31).

Primary Aldosteronism. Primary aldosteronism (PA) is characterized by moderate to severe hypertension caused by unregulated secretion of aldosterone with elevated levels of serum and urinary aldosterone, hypokalemia, and suppressed plasma renin activity. A solitary aldosterone-­producing adenoma (APA) is present in approximately 70% of patients, and surgical or laparoscopic adrenalectomy corrects hypertension and hypokalemia in 75% to 90% of cases (Fig. 92.32).131 The majority of the remainder of patients have idiopathic hyperaldosteronism (IHA) from bilateral adrenal hyperplasia. Unlike in patients with unilateral APA, adrenalectomy rarely cures hypertension and biochemical abnormalities, and as a result these patients are managed medically. It is essential, therefore, to distinguish APA from IHA at the outset.132 CT evaluation is often problematic, because unilateral aldosteronomas may be associated with ipsilateral or contralateral nonfunctioning adenomas, resulting in misdiagnosis of “nodular” adrenal hyperplasia.133 In addition, very rarely, bilateral hyperplasia may have a predominant unilateral macronodule and contralateral gland thickening that may lead to an erroneous diagnosis of a unilateral aldosteronoma. Doppman and colleagues134 demonstrated that CT cannot reliably differentiate APA from IHA whenever bilateral adrenal nodules are identified. In their series, six of 21 patients with APA were

incorrectly diagnosed on CT as having IHA due to the presence of non-aldosterone-­ secreting nodules (nonfunctioning adenomas), in addition to an aldosteronoma. Sam and colleagues demonstrated similar results in 342 patients: upwards of 22% were not correctly identified by CT.135 Most patients with PA and a unilateral adrenal mass can proceed to unilateral adrenalectomy with successful treatment of PA. Patients with bilateral adrenal nodules, as well as those with confounding CT and biochemical evaluations, should undergo bilateral, selective AVS for aldosterone levels to identify lateralization of, and thus the source or sources of, aldosterone secretion.136 Although most cross-­sectional studies of patients with PA have used CT, smaller series have suggested that MRI may be able to differentiate APA from IHA.137 Among 20 patients who underwent MRI, ten (50%) had APA, and ten (50%) had IHA. In the detection of APA, MRI had a sensitivity (70%), specificity (100%), and accuracy (85%) comparable to CT.138,139 Traditionally, diagnosis of IHA by CT or MRI has been made by excluding the presence of an “adenoma.” One CT-­based study suggested that diagnosis of hyperplasia can be made by measuring adrenal gland limb width, because adrenal glands were significantly larger in bilateral hyperplasia than in APA or in normal subjects. A sensitivity for identification of IHA of 100% was achieved when a mean limb width exceeded 3 mm and a specificity of 100% when the mean limb width was greater than 5 mm. The authors proposed restricting adrenal venous sampling to situations in which mean adrenal limb width fell between 3 and 5 mm.132 Catheterization for venography and AVS can help identify a cause for primary hyperaldosteronism. AVS is usually performed with a percutaneous right transfemoral venous approach. Tandem sheaths are utilized, with one large enough to allow for simultaneous aspiration of iliac venous blood that represents IVC sampling. Shaped catheters are directed into right and left adrenal veins to allow for simultaneous sampling. Aliquots are collected and sent for cortisol and aldosterone assays. If there is doubt about the catheter position, rapid cortisol assay may be performed. Rapid aldosterone assay is not yet available in the United States. Gentle hand injection into the adrenal vein with simultaneous digital subtraction imaging is usually sufficient to confirm successful adrenal venous catheterization. Samples are obtained from both adrenal veins and the iliac venous or IVC sheath. After baseline samples are obtained, adrenal cortical hormonal stimulation with Cortrosyn (0.25 mg) (synthetic analogue

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Fig. 92.28  (18F)-­ fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) study for staging of lung cancer. Maximum intensity projection (A), axial PET (B), and fused PET/ CT (C) images show thickening of the right adrenal gland (arrows) with an increased FDG uptake of similar intensity to the primary left lower lobe tumor. The adrenal maximum standard uptake value (SUV) of 28 compared with the liver background SUV mean of 2.6 is compatible with metastatic disease to the adrenal gland, in the presence of widespread metastases. (From Wong KK, Arabi M, Zerizer I, et al. Role of PET/ CT in adrenal and neuroendocrine tumors: FDG and non-­FDG tracers. Nuc Med Comm. 2011;32:764–781.)

C

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Fig. 92.29  positron emission tomography/computed tomography (PET/CT) study in a 74-­year-­old man with lung cancer. Restaging PET/CT images show a 3-­cm right adrenal lesion (arrows) with maximum standard uptake value (SUV) of 4.4 as compared with the normal liver background SUV mean of 2.4 with lesion/liver maximum SUV ratio of 1.5, meeting the metabolic criteria for adrenal nonadenoma. However, this lesion displayed low CT attenuation with 10 H) in patients without cancer: is further imaging necessary? Follow-­up of 321 consecutive indeterminate adrenal masses. AJR Am J Roentgenol. 2007;189:1119–1123. 17. Mayo-­Smith WW, Lee MJ, McNicholas MM, et al. Characterization of adrenal masses (< 5 cm) by use of chemical shift MR imaging: observer performance versus quantitative measures. AJR Am J Roentgenol. 1995;165:91–95. 18. Mitchell DG, Crovello M, Matteucci T, et al. Benign adrenocortical masses: diagnosis with chemical shift MR imaging. Radiology. 1992;185:345– 351.

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39. Otomi Y, Shinya T, Otsuka H, et al. Increased (18)F-­fluorodeoxyglucose accumulation in bilateral adrenal glands of the patients suffering from vasovagal reaction due to blood vessel puncture. Ann Nucl Med. 2016;30:501–505. 40. De Leo A, Mosconi C, Zavatta G, et al. Radiologically defined lipid-­poor adrenal adenomas: histopathological characteristics. J Endocrinol Invest. 2020;43(9):1197-1204. 41. Patel D, Gara SK, Ellis RJ, et al. FDG PET/CT scan and functional adrenal tumors: a pilot study for lateralization. World J Surg. 2016;40:683– 689. 42. Murayama R, Nishie A, Hida T, et al. Uptake of 18F-­FDG in adrenal adenomas is associated with unenhanced CT value and constituent cells. Clin Nucl Med. 2019;44:943–948. 43. Goldman HB, Howard RC, Patterson AL. Spontaneous retroperitoneal hemorrhage from a giant adrenal myelolipoma. J Urol. 1996;155:639. 44. Russell C, Goodacre BW, vanSonnenberg E, et al. Spontaneous rupture of adrenal myelolipoma: spiral CT appearance. Abdom Imaging. 2000;25:431–434. 45. Rao P, Kenney PJ, Wagner BJ, et al. Imaging and pathologic features of myelolipoma. Radiographics. 1997;17:1373–1385. 46. Sneiders A, Zhang G, Gordon BE. Extra-­adrenal perirenal myelolipoma. J Urol. 1993;150:1496–1497. 47. Rowe SP, Javadi MS, Solnes LB, et al. Appearance of adrenal myelolipomas on 2-­deoxy-­2-­((18)F) fluoro-­D-­glucose positron emission tomography-­computed tomography. World J Nucl Med. 2017;16:271– 274. 48. Burks DW, Mirvis SE, Shanmuganathan K. Acute adrenal injury after blunt abdominal trauma: CT findings. AJR Am J Roentgenol. 1992;158:503–507. 49. Bookstein JJ, Conn J, Reuter SR. Intra-­adrenal hemorrhage as a complication of adrenal venography in primary aldosteronism. Radiology. 1968;90:778–779. 50. Ling D, Korobkin M, Silverman PM, et al. CT demonstration of bilateral adrenal hemorrhage. AJR Am J Roentgenol. 1983;141:307–308. 51. Kawashima A, Sandler CM, Ernst RD, et al. Imaging of nontraumatic hemorrhage of the adrenal gland. Radiographics. 1999;19:949–963. 52. Cheema P, Cartagena R, Staubitz W. Adrenal cysts: diagnosis and treatment. J Urol. 1981;126:396–399. 53. Rozenblit A, Morehouse HT, Amis Jr ES. Cystic adrenal lesions: CT features. Radiology. 1996;201:541–548. 54. Mendiratta-­Lala M, Avram A, Turcu AF, et al. Adrenal imaging. Endocrinol Metab Clin North Am. 2017;46:741–759. 55. Wilson DA, Muchmore HG, Tisdal RG, et al. Histoplasmosis of the adrenal glands studied by CT. Radiology. 1984;150:779–783. 56. Zhou J, Lv J, Pan Y, et al. Unilateral adrenal cryptococcosis on FDG PET/ CT. Clin Nucl Med. 2017;42:565–566. 57. Papadakis GZ, Holland SM, Quezado M, et al. Adrenal cryptococcosis in an immunosuppressed patient showing intensely increased metabolic activity on (18)F-­FDG PET/CT. Endocrine. 2016;54:834–836. 58. Gajendra S, Sharma R, Goel S, et al. Adrenal histoplasmosis in immunocompetent patients presenting as adrenal insufficiency. Turk patoloji dergisi. 2016;32:105–111. 59. Kalathoorakath RR, Sharma A, Sood A, et al. 18)F-­FDG PET/CT imaging and PET-­guided biopsy in evaluation and treatment decision in adrenal histoplasmosis. BJR Case Rep. 2016;2:20150451. 60. Arambewela M, Ross R, Pirzada O, et al. Tuberculosis as a differential for bilateral adrenal masses in the UK. BMJ Case Rep. 2019;12(5). 61. Gorla AK, Gupta K, Sood A, et al. Adrenal tuberculosis masquerading as disseminated malignancy: a pitfall of (18)F-­FDG PET/CT Imaging. Rev Esp Med Nucl Imagen Mol. 2016;35:257–259. 62. Koh SA. Addison’s disease due to bilateral adrenal tuberculosis on 18F-­ fluorodeoxyglucose positron emission tomography computed tomography. Infect Dis Rep. 2018;10:7773. 63. Altinmakas E, Guo M, Kundu UR, et al. Computed tomography and (18) F-­fluorodeoxyglucose positron emission tomography/computed tomography findings in adrenal candidiasis and histoplasmosis: two cases. Clin Imaging. 2015;39:1115–1118.

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64. Honig SC, Klavans MS, Hyde C, et al. Adrenal hemangioma: an unusual adrenal mass delineated with magnetic resonance imaging. J Urol. 1991;146:400–402. 65. Krebs TL, Wagner BJ. MR imaging of the adrenal gland: radiologic-­ pathologic correlation. Radiographics. 1998;18:1425–1440. 66. Wilson B, Becker A, Estes T, et al. Adrenal hemangioma definite diagnosis on CT, MRI, and FDG PET in a patient with primary lung cancer. Clin Nucl Med. 2018;43:e192–e194. 67. Radin R, David CL, Goldfarb H, et al. Adrenal and extra-­adrenal retroperitoneal ganglioneuroma: imaging findings in 13 adults. Radiology. 1997;202:703–707. 68. Mylonas KS, Schizas D, Economopoulos KP. Adrenal ganglioneuroma: What you need to know. World J Clin Cases. 2017;5(10):373–377. 69. McMurry Jr JF, Long D, McClure R, et al. Addison’s disease with adrenal enlargement on computed tomographic scanning. Report on two cases of tuberculosis and review of the literature. Am J Med. 1984;77:365–368. 70. Caron P, Chabannier MH, Cambus JP, et al. Definitive adrenal insufficiency due to bilateral adrenal hemorrhage and primary antiphospholipid syndrome. J Clin Endocrinol Metab. 1998;83:1437–1439. 71. Piédrola G, Casado JL, López E, et al. Clinical features of adrenal insufficiency in patients with acquired immunodeficiency syndrome. Clin Endocrinol. 1996;45:97–101. 72. Dunnick NR, Heaston D, Halvorsen R, et al. CT appearance of adrenal cortical carcinoma. J Comp Assist Tomogr. 1982;6:978–982. 73. Hedican SP, Marshall FF. Adrenocortical carcinoma with intracaval extension. J Urol. 1997;158:2056–2061. 74. Schlund JF, Kenney PJ, Brown ED, et al. Adrenocortical carcinoma: MR imaging appearance with current techniques. JMRI. 1995;5:171–174. 75. Szolar DH, Korobkin M, Reittner P, et al. Adrenocortical carcinomas and adrenal pheochromocytomas: mass and enhancement loss evaluation at delayed contrast-­enhanced CT. Radiology. 2005;234:479–485. 76. Dunnick NR, Doppman JL, Geelhoed GW. Intravenous extension of endocrine tumors. AJR Am J Roentgenol. 1980;135:471–476. 77. Cistaro A, Niccoli Asabella A, Coppolino P, et al. Diagnostic and prognostic value of 18F-­FDG PET/CT in comparison with morphological imaging in primary adrenal gland malignancies -­a multicenter experience. Hellenic J Nucl Med. 2015;18:97–102. 78. Ardito A, Massaglia C, Pelosi E, et al. 18F-­FDG PET/CT in the post-­ operative monitoring of patients with adrenocortical carcinoma. Eur J Endocrinol. 2015;173:749–756. 79. Shekhar S, Gubbi S, Papadakis GZ, et al. Adrenocortical carcinoma and pulmonary embolism from tumoral extension. Endocrinol Diabet Metab Case Rep. 2019;19(95):1-4. 80. Satoh K, Patel D, Dieckmann W, et al. Whole body metabolic tumor volume and total lesion glycolysis predict survival in patients with adrenocortical carcinoma. Ann Surg Oncol. 2015;22:S714–S720. 81. Bluemel C, Hahner S, Heinze B, et al. Investigating the chemokine receptor 4 as potential theranostic target in adrenocortical cancer patients. Clin Nucl Med. 2017;42:e29–e34. 82. Weiss ID, Huff LM, Evbuomwan MO, et al. Screening of cancer tissue arrays identifies CXCR4 on adrenocortical carcinoma: correlates with expression and quantification on metastases using (64)Cu-­plerixafor PET. Oncotarget. 2017;8:73387–73406. 83. Crona J, Taïeb D, Pacak K. New perspectives on pheochromocytoma and paraganglioma: toward a molecular classification. Endocr Rev. 2017;38(6):489–515. 84. Lucon AM, Pereira MA, Mendonça BB, et al. Pheochromocytoma: study of 50 cases. J Urol. 1997;157:1208–1212. 85. Jacobson AF, Deng H, Lombard J, et al. 123I-­meta-­iodobenzylguanidine scintigraphy for the detection of neuroblastoma and pheochromocytoma: results of a meta-­analysis. J Clin Endocrinol Metab. 2010;95:2596– 2606. 86. Gabriel S, Blanchet EM, Sebag F, et al. Functional characterization of nonmetastatic paraganglioma and pheochromocytoma by (18) F-­ FDOPA PET: focus on missed lesions. Clin Endocrinol. 2013;79:170–177. 87. Ilias I, Chen CC, Carrasquillo JA, et al. Comparison of 6-­18F-­fluorodopamine PET with 123I-­metaiodobenzylguanidine and

111in-­pentetreotide scintigraphy in localization of nonmetastatic and metastatic pheochromocytoma. J Nucl Med. 2008;49:1613–1619. 88. Shulkin BL, Koeppe RA, Francis IR, et al. Pheochromocytomas that do not accumulate metaiodobenzylguanidine: localization with PET and administration of FDG. Radiology. 1993;186:711–715. 89. Timmers HJ, Kozupa A, Chen CC, et al. Superiority of fluorodeoxyglucose positron emission tomography to other functional imaging techniques in the evaluation of metastatic SDHB-­associated pheochromocytoma and paraganglioma. J Clin Oncol. 2007;25:2262–2269. 90. Nakajo M, Nakajo M, Fukukura Y, et al. Diagnostic performances of FDG-­PET/CT and diffusion-­weighted imaging indices for differentiating benign pheochromocytoma from other benign adrenal tumors. Abdom Imaging. 2015;40:1655–1665. 91. Nockel P, El Lakis M, Gaitanidis A, et al. Preoperative 18F-­FDG PET/CT in pheochromocytomas and paragangliomas allows for precision surgery. Ann Surg. 2019;269:741–747. 92. van Berkel A, Vriens D, Visser EP, et al. Metabolic subtyping of pheochromocytoma and paraganglioma by (18)F-­FDG pharmacokinetics using dynamic PET/CT scanning. J Nucl Med. 2019;60:745–751. 93. Tiwari A, Shah N, Sarathi V, et al. Genetic status determines (18) F-­FDG uptake in pheochromocytoma/paraganglioma. J Med Imaging Radiat Oncol. 2017;61:745–752. 94. Lepoutre-­Lussey C, Caramella C, Bidault F, et al. Screening in asymptomatic SDHx mutation carriers: added value of 1⁸F-­FDG PET/CT at initial diagnosis and 1-­year follow-­up. Eur J Nucl Med Mol Imaging. 2015;42:868–876. 95. Képénékian L, Mognetti T, Lifante JC, et al. Interest of systematic screening of pheochromocytoma in patients with neurofibromatosis type 1. Eur J Endocrinol. 2016;175:335–344. 96. Fottner C, Helisch A, Anlauf M, et  al. 6-­18F-­fluoro-­L-­dihydroxy phenylalanine positron emission tomography is superior to 123I-­ metaiodobenzyl-­guanidine scintigraphy in the detection of extraadrenal and hereditary pheochromocytomas and paragangliomas: correlation with vesicular monoamine transporter expression. J Clin Endocrinol Metab. 2010;95:2800–2810. 97. Taïeb D, Jha A, Guerin C, et al. 18F-­FDOPA PET/CT imaging of MAX-­ related pheochromocytoma. J Clin Endocrinol Metab. 2018;103:1574–1582. 98. Timmers HJ, Chen CC, Carrasquillo JA, et al. Comparison of 18F-­ fluoro-­L-­DOPA, 18F-­fluoro-­deoxyglucose, and 18F-­fluorodopamine PET and 123I-­MIBG scintigraphy in the localization of pheochromocytoma and paraganglioma. J Clin Endocrinol Metab. 2009;94:4757–4767. 99. Kroiss A, Putzer D, Uprimny C, et al. Functional imaging in phaeochromocytoma and neuroblastoma with 68Ga-­DOTA-­Tyr 3-­octreotide positron emission tomography and 123I-­metaiodobenzylguanidine. Eur J Nucl Med Mol Imaging. 2011;38:865–873. 100. Shell J, Tirosh A, Millo C, et al. The utility of (68)Gallium-­DOTATATE PET/CT in the detection of von Hippel-­Lindau disease associated tumors. Eur J Radiol. 2019;112:130–135. 101. Tuzcu SA, Pekkolay Z. Multiple endocrine neoplasia type 2A syndrome (MEN2A) and usefulness of 68Ga-­DOTATATE PET/CT in this syndrome. Ann Ital Chir. 2019;90:497–503. 102. Paling MR, Williamson BR. Adrenal involvement in non-­Hodgkin lymphoma. AJR Am J Roentgenol. 1983;141:303–305. 103. Falchook FS, Allard JC. CT of primary adrenal lymphoma. J Comput Ass Tomogr. 1991;15:1048–1050. 104. An P, Chen K, Yang GQ, et al. Diffuse large B cell lymphoma with bilateral adrenal and hypothalamic involvement: a case report and literature review. World J Clin Cases. 2019;7:4075–4083. 105. Dong P, Wang L, Shen G, et al. Primary adrenal extranasal NK/T cell lymphoma with subcutaneous involvement demonstrated on FDG PET/ CT: a clinical case report. Medicine. 2019;98:e14818. 106. Doroudinia A, Bakhshayesh Karam M, Ranjbar M, et al. Mantle cell lymphoma presenting as bilateral adrenal huge masses. BMJ Case Rep. 2018;bcr2017223247. 107. Li W, Lin W, Ma C, et al. A case of intravascular large B-­cell lymphoma in the left adrenal and another tumor in the right adrenal detected by (18)F-­FDG PET/CT. Hellenic J Nucl Med. 2016;19:57–59.

CHAPTER 92  Adrenal Gland Imaging 108. Zhou J, Zhao Y, Gou Z. High 18F-­fluorodeoxyglucose uptake in primary bilateral adrenal diffuse large B-­cell lymphomas with nongerminal center B-­cell phenotype: a case report. Medicine. 2018;97:e0480. 109. Altinmakas E, Üçışık-­Keser FE, Medeiros LJ, et al. CT and (18)F-­FDG-­ PET-­CT findings in secondary adrenal lymphoma with pathologic correlation. Acad Radiol. 2019;26:e108–e114. 110. Wang M, Xu H, Xiao L, et al. Prognostic value of functional parameters of (18)F-­FDG-­PET images in patients with primary renal/adrenal lymphoma. Contrast Media Mol Imaging. 2019;2019:2641627. 111. Laurent C, Casasnovas O, Martin L, et al. Adrenal lymphoma: presentation, management and prognosis. QJM. 2017;110:103–109. 112. Abrams HL, Spiro R, Goldstein N. Metastases in carcinoma; analysis of 1000 autopsied cases. Cancer. 1950;3:74–85. 113. Caoili EM, Korobkin M, Francis IR, et al. Delayed enhanced CT of lipid-­ poor adrenal adenomas. AJR Am J Roentgenol. 2000;175:1411–1415. 114. Wu Q, Luo W, Zhao Y, et al. The utility of 18F-­FDG PET/CT for the diagnosis of adrenal metastasis in lung cancer: a PRISMA-­compliant meta-­analysis. Nucl Med Comm. 2017;38:1117–1124. 115. Kanaya N, Noma K, Okada T, et al. A case of long-­term survival after surgical resection for solitary adrenal recurrence of esophageal squamous carcinoma. Surg Case Rep. 2017;3:61. 116. Tsuchida K, Watanabe H, Kameda Y, et al. [A case of solitary adrenal metastasis from Rectal cancer treated by adrenalectomy after preoperative chemotherapy]. Gan To Kagaku Ryoho Cancer Chemother. 2016;43:1751–1753. 117. Lang BH, Cowling BJ, Li JY, et al. High false positivity in positron emission tomography is a potential diagnostic pitfall in patients with suspected adrenal metastasis. World J Surg. 2015;39:1902–1908. 118. Carrasquillo JA, Pandit-­Taskar N, Chen CC. Radionuclide therapy of adrenal tumors. J Surg Oncol. 2012;106:632–642. 119. Papaioannou G, McHugh K. Neuroblastoma in childhood: review and radiological findings. Cancer Imag. 2005;5:116–127. 120. Abramson SJ. Adrenal neoplasms in children. Radiologic Clin North Am. 1997;35:1415–1453. 121. Lonergan GJ, Schwab CM, Suarez ES, et al. Neuroblastoma, ganglioneuroblastoma, and ganglioneuroma: radiologic-­pathologic correlation. Radiographics. 2002;22:911–934. 122. Shulkin BL, Shapiro B, Hutchinson RJ. Iodine-­131-­ metaiodobenzylguanidine and bone scintigraphy for the detection of neuroblastoma. J Nucl Med. 1992;33:1735–1740. 123. Sharp SE, Trout AT, Weiss BD, et al. MIBG in Neuroblastoma diagnostic imaging and therapy. Radiographics. 2016;36:258–278. 1 24. Gains JE, Bomanji JB, Fersht NL, et al. 177Lu-­DOTATATE molecular radiotherapy for childhood neuroblastoma. J Nucl Med. 2011;52:1041–1047. 125. Kong G, Hofman MS, Murray WK, et al. Initial experience with gallium-­68 DOTA-­octreotate PET/CT and peptide receptor radionuclide therapy for pediatric patients with refractory metastatic neuroblastoma. J Pediatr Hematology/Oncol. 2016;38:87–96. 126. Doppman JL, Miller DL, Dwyer AJ, et al. Macronodular adrenal hyperplasia in Cushing disease. Radiology. 1988;166:347–352. 127. Doppman JL, Chrousos GP, Papanicolaou DA, et al. Adrenocorticotropin-­independent macronodular adrenal hyperplasia: an uncommon cause of primary adrenal hypercortisolism. Radiology. 2000;216:797–802. 128. Isidori AM, Sbardella E, Zatelli MC, et al. Conventional and nuclear medicine imaging in ectopic Cushing’s syndrome: a systematic review. J Clin Endocrinol Metab. 2015;100:3231–3244. 129. Santhanam P, Taieb D, Giovanella L, et al. PET imaging in ectopic Cushing syndrome: a systematic review. Endocrine. 2015;50:297–305. 130. Parihar AS, Mittal BR, Vadi SK, et al. Ectopic Cushing Syndrome (ECS): 68Ga-­DOTANOC PET/CT localizes the site of ectopic adrenocorticotropic hormone production. Clin Nucl Med. 2018;43:769–770. 131. Korobkin M, Francis IR. Adrenal imaging. Semin Ultrasound CT MR. 1995;16:317–330. 132. Lingam RK, Sohaib SA, Vlahos I, et al. CT of primary hyperaldosteronism (Conn’s syndrome): the value of measuring the adrenal gland. AJR Am J Roentgenol. 2003;181:843–849.

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133. Hollak CE, Prummel MF, Tiel-­van Buul MM. Bilateral adrenal tumours in primary aldosteronism: localization of a unilateral aldosteronoma by dexamethasone suppression scan. J Int Med. 1991;229:545–548. 134. Doppman JL, Gill Jr JR, Miller DL, et al. Distinction between hyperaldosteronism due to bilateral hyperplasia and unilateral aldosteronoma: reliability of CT. Radiology. 1992;184:677–682. 135. Sam D, Kline GA, So B, et al. Discordance between imaging and adrenal vein sampling in primary aldosteronism irrespective of interpretation criteria. J Clin Endocrinol Metab. 2019;104:1900–1906. 136. Blevins Jr LS, Wand GS. Primary aldosteronism: an endocrine perspective. Radiology. 1992;184:599–600. 137. Sohaib SA, Peppercorn PD, Allan C, et al. Primary hyperaldosteronism (Conn syndrome): MR imaging findings. Radiology. 2000;214:527–531. 138. Dunnick NR, Leight Jr GS, Roubidoux MA, et al. CT in the diagnosis of primary aldosteronism: sensitivity in 29 patients. AJR Am J Roentgenol. 1993;160:321–324. 139. Sheaves R, Goldin J, Reznek RH, et al. Relative value of computed tomography scanning and venous sampling in establishing the cause of primary hyperaldosteronism. Eur J Endocrinol. 1996;134:308–313. 140. Betz MJ, Degenhart C, Fischer E, et al. Adrenal vein sampling using rapid cortisol assays in primary aldosteronism is useful in centers with low success rates. Eur J Endocrinol. 2011;165:301–306. 141. Daunt N. Adrenal vein sampling: how to make it quick, easy, and successful. Radiographics. 2005;25:S143–S158. 142. Bardet S, Chamontin B, Douillard C, et al. SFE/SFHTA/AFCE consensus on primary aldosteronism, part 4: Subtype diagnosis. Ann Endocrinol. 2016;77:208–213. 143. Naruse M, Umakoshi H, Tsuiki M, et al. The latest developments of functional molecular imaging in the diagnosis of primary aldosteronism. Horm Metab Res. 2017;49(12):929–935. doi: 10.1055/s-­0043-­. 144. Wong KK, Komissarova M, Avram AM, et al. Adrenal cortical imaging with I-­131 NP-­59 SPECT-­CT. Clin Nucl Med. 2010;35:865–869. 145. Yen RF, Wu VC, Liu KL, et  al. 131I-­6beta-­iodomethyl-­19-­norcholesterol SPECT/CT for primary aldosteronism patients with inconclusive adrenal venous sampling and CT results. J Nucl Med. 2009;50:1631–1637. 146. Wu MH, Liu FH, Lin KJ, et al. Diagnostic value of adrenal iodine-­131 6-­ beta-­iodomethyl-­19-­norcholesterol scintigraphy for primary aldosteronism: a retrospective study at a medical center in North Taiwan. Nucl Med Comm. 2019;40:568–575. 147. Mendichovszky IA, Powlson AS, Manavaki R, et al. Targeted molecular imaging in adrenal disease-­an emerging role for metomidate PET-­CT. Diagnostics. 2016;6(4):42. 148. Minn H, Salonen A, Friberg J, et al. Imaging of adrenal incidentalomas with PET using (11)C-­metomidate and (18)F-­FDG. J Nucl Med. 2004;45:972–979. 149. Zettinig G, Mitterhauser M, Wadsak W, et al. Positron emission tomography imaging of adrenal masses: (18)F-­fluorodeoxyglucose and the 11beta-­hydroxylase tracer (11)C-­metomidate. Eur J Nucl Med Mol Imaging. 2004;31:1224–1230. 150. Hennings J, Lindhe O, Bergström M, et al. [11C]metomidate positron emission tomography of adrenocortical tumors in correlation with histopathological findings. J Clin Endocrinol Metab. 2006;91:1410– 1414. 151. Powlson AS, Gurnell M, Brown MJ. Nuclear imaging in the diagnosis of primary aldosteronism. Curr Opin Endocrinol Diabet Obesity. 2015;22:150–156. 152. Burton TJ, Mackenzie IS, Balan K, et al. Evaluation of the sensitivity and specificity of (11)C-­metomidate positron emission tomography (PET)-­ CT for lateralizing aldosterone secretion by Conn’s adenomas. J Clin Endocrinol Metab. 2012;97:100–109. 153. O’Shea PM, O’Donoghue D, Bashari W, et al. 11) C-­Metomidate PET/ CT is a useful adjunct for lateralization of primary aldosteronism in routine clinical practice. Clin Endocrinol. 2019;90:670–679. 154. Bongarzone S, Basagni F, Sementa T, et al. Development of [(18)F]FAMTO: a novel fluorine-­18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands. Nucl Med Bio. 2019;68–69:14–21.

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155. Ding J, Tong A, Zhang Y, et al. Intense 68Ga-­pentixafor activity in aldosterone-­producing adrenal adenomas. Clin Nucl Med. 2020;45:336– 339. 156. Heinze B, Fuss CT, Mulatero P, et al. Targeting CXCR4 (CXC chemokine receptor type 4) for molecular imaging of aldosterone-­producing adenoma. Hypertension. 2018;71:317–325.

157. Ding J, Zhang Y, Wen J, et al. Imaging CXCR4 expression in patients with suspected primary hyperaldosteronism. Eur J Nucl Med Mol Imaging. 2020;47(11):2656-2665.

93 Adrenal Insufficiency David J. Torpy, Irina Bancos, and Eystein Husebye

OUTLINE Brief Historical Perspective: the Changing Spectrum of Adrenal Insufficiency Etiology, 1553 Epidemiology of Adrenal Insufficiency, 1554 Pathogenesis of Adrenal Insufficiency, 1554 Clinical Features of Adrenal Insufficiency, 1554 Etiology, 1555 Primary Adrenal Insufficiency, 1555 Critical Illness-Related Corticosteroid Insufficiency, 1560 Diagnosis of Adrenal Insufficiency, 1560 Adrenocorticotropic Hormone Stimulation Testing, 1560 Overnight Metyrapone Test, 1561 Insulin Tolerance Test, 1561

Adrenal Autoantibody Tests, 1561 Serum 17-­Hydroxyprogesterone, 1561 Very Long Chain Fatty Acids, 1561 Adrenal Imaging, 1561 Treatment of Adrenal Insufficiency, 1561 Replacement of Dehydroepiandrosterone, 1562 Quality of Life in Adrenal Insufficiency, 1563 Adrenal Suppression, 1563 Adrenal Crisis, 1563 Treatment of Adrenal Crisis, 1563 Prevention of Adrenal Crises, 1564 The Need for Novel Treatments for Adrenal Insufficiency, 1566

 

BRIEF HISTORICAL PERSPECTIVE: THE CHANGING SPECTRUM OF ADRENAL INSUFFICIENCY ETIOLOGY Adrenal insufficiency (AI) is defined by inadequate secretion of cortisol and/or aldosterone by the adrenal cortex to maintain homeostasis. Bartolomeo Eustachi, in 1563, first recognized the adrenal glands as organs distinct from the kidneys.1 Addison disease, eponymously named after Thomas Addison, refers to primary AI (PAI), that is, resulting from direct adrenal pathology. Thomas Addison described many diseases through correlation of clinical presentation with autopsy examination. Addison’s description of AI remains as accurate today as it was in 1855: “anaemia, general languor and debility, remarkable weakness of the heart’s action, irritability of the stomach, and a peculiar change of colour in the skin.” Addison was studying pernicious anemia when he discovered adrenal pathology: tuberculous adrenal lesions in some patients with tuberculosis and adrenal atrophy in others, some of which had vitiligo and pernicious anemia. Of 11 patients described, five had bilateral adrenal tuberculosis, one had unilateral adrenal tuberculosis, three had carcinomatous adrenal involvement, one had adrenal hemorrhage, and one showed atrophy and fibrosis. Addison’s findings were quickly confirmed by Brown-­Sequard,2 who verified Addison’s hypothesis by showing that, in several laboratory animals, bilateral adrenalectomy was uniformly fatal. The clinical syndrome was named after Addison by Trousseau.3 Osler attempted unsuccessfully to treat a young patient with Addison disease, employing a glycerine extract of fresh porcine adrenals given orally,4-­6 and isolated and characterized cortisone and cortisol in the 1930s, and Sarett devised a partial synthesis for cortisone from deoxycholic acid in 1945. The clinical effects of cortisone soon were made apparent by the work of Hench and coworkers7 in the treatment of rheumatoid arthritis and

by Thorn and Forsham8 in the treatment of AI. Cushing largely clarified the role of the pituitary gland in regulating adrenal function, and Harris9 in the 1940s clarified the role of the hypothalamus in regulating pituitary function via the hypothalamo-­hypophyseal portal circulation. Adrenocorticotropic hormone (ACTH) was isolated and characterized by Li,10 and corticotrophin-­releasing hormone (CRH), in turn, was characterized by Vale11. Sampson, in 1961, first recognized the syndrome of acute AI in a surgical patient who had atrophic adrenal glands secondary to longstanding glucocorticoid treatment. The etiological spectrum of AI has evolved considerably. The majority of cases now result from the use of supraphysiologic, antiinflammatory doses of glucocorticoids in the many diseases that respond well. Among children, genetic disorders (e.g., developmental pituitary defects or congenital adrenal hyperplasia [CAH]) are frequent causes of AI, as well as childhood cerebral tumors or their treatment, now often successful. Autoimmunity is a common cause of PAI, but newer drugs, particularly those used in oncology to increase immune activity, the CTLA-­4, PD-­1, and PD-­L1 antibodies, may produce hypophysitis/secondary AI (SAI).12 In addition, opioids cause SAI, especially in high cumulative doses.13 Human immunodeficiency virus infection of the adrenals or attendant adrenal mycotic infections and/or antifungal inhibition of adrenal steroidogenesis may lead to AI, an outcome less frequent with antiretroviral therapies.14 Observations of salutory pressor effects of hydrocortisone in septic shock and their possible relation to impaired responsiveness of cortisol to ACTH stimulation have led to the notion of relative AI, now referred to as critical illness-related corticosteroid insufficiency (CIRCI), an area that requires better endocrine definition.15 These observations highlight the importance of considering clinical context in AI differential diagnosis and considering newer causes as they appear in the community.

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EPIDEMIOLOGY OF ADRENAL INSUFFICIENCY Primary AI has an annual incidence of 4 to 6 cases per million and a prevalence of 100 to 220 cases per million, which may be increasing SAI is considered to be twice as common as PAI. The exact prevalence of exogenous adrenal suppression is not known. Adrenal crises are more frequent in PAI than in SAI (estimated in one study to be 5.2 and 3.6 adrenal crisis (AC) per 100 person-­years (PY), respectively).16

PATHOGENESIS OF ADRENAL INSUFFICIENCY The hypothalamic-­pituitary-­adrenal (HPA) axis comprises a classic negative feedback loop (see Chapter 132). Many pathological processes can lead to loss of adrenal or hypothalamic-­pituitary function, and each of these have intrinsic implications.7 Endocrine consequences arise from loss of adrenal hormones, most importantly cortisol and aldosterone. Although not routinely replaced, there is a loss of production of adrenal androgens, DHEA(-­S) and androstenedione, which may result in reduced androgen-­dependent hair, especially in females, where adrenal androgens represent approximately 50% of circulating androgens. A lack of adrenal cortisol secretion markedly reduces adrenomedullary epinephrine secretion through depriving the phenylethanolamine-­N-­methyltransferase enzyme of its stimulatory drive; the extent of practical pathological implications is unclear, but may theoretically include a blunted response to hypoglycemia and an altered sympathoadrenal (epinephrine) response to stress. Low plasma epinephrine and metanephrine levels and low urinary epinephrine excretion can be observed CAH patients with 21-­hydroxylase deficiency, which, when severe, can result in frequent hospitalizations for hypoglycemia and hypotension.18 Primary AI, most often autoimmune where tuberculosis is not endemic, results in loss of glomerulosa cells and fasciculata cells, leading to lack of aldosterone and cortisol. Typical sequential hormone deficits include increased serum renin, reduced serum aldosterone, and increased serum ACTH, followed by reduced serum cortisol.19 Usually, AI patients are diagnosed in the most symptomatic low-­cortisol state. Isolated loss of cortisol secretion may be seen in autoimmune disease but more commonly follows other insults such as adrenal hemorrhage. In general, PAI involves deficiency of aldosterone and cortisol, as well as ACTH hypersecretion. The loss of aldosterone secretion and ACTH hypersecretion are not seen in SAI; hence, only PAI patients typically have hyperkalemia from aldosterone deficiency and hyperpigmentation from ACTH and other proopiomelanocortin (POMC) peptides such as α-­MSH that have melanocyte-­stimulating properties via melanocortin-­1 receptors. SAI is caused by deficits in ACTH secretion, because of loss of hypothalamic CRH production or direct pituitary pathology. The result is low cortisol secretion and normal or low serum ACTH concentration. Common pituitary/hypothalamic pathologies are pituitary adenomas, suprasellar or hypothalamic tumors such as craniopharyngioma, inflammatory disease of either organ, or iatrogenic damage from surgery or radiotherapy. These pathologies may have distinct clinical features or influence the rate of onset of AI. Hypothalamic-­pituitary lesions produce cortisol deficiency, but aldosterone is normal because it is regulated primarily by the renin-­angiotensin system and serum potassium. The actions and mechanisms of action of glucocorticoids and mineralocorticoids are discussed in detail elsewhere in this text (see Chapters 88-89). Cortisol has protean effects, including: immunomodulation with an initial immune enhancing effect on infection or injury followed by immune response containment to prevent healthy tissue damage; substrate mobilization increasing circulating glucose, fatty acids, and amino acids to maintain nutrition of key tissues in times of stress or starvation; cardiovascular support acting to maintain cardiac contractility;

modulation of the vascular response to the β-­adrenoceptor agonists; and neurocognitive effects so as to enhance attention and awareness, thereby facilitating an optimum response to stress. Aldosterone regulates exchange of sodium, potassium, and hydrogen ions at the renal tubule, the gut, and the salivary glands, promoting sodium reabsorption at the expense of potassium and hydrogen excretion.

CLINICAL FEATURES OF ADRENAL INSUFFICIENCY Primary adrenal glucocorticoid insufficiency is manifest by weakness, fatigue, nausea, loss of appetite, vomiting, and weight loss, ACTH-­ mediated hyperpigmentation (typically of the palmar creases, scars, knuckles, and oral mucosa) (Fig. 93.1), hypotension, often postural, with tachycardia, because of reduced stroke volume, and decreased peripheral vascular resistance. Some cases may present with hypoglycemia. Depressed mood and reduced ability to concentrate are common. Back and leg pains may develop. Concomitant mineralocorticoid deficiency is manifest by hyponatremia, hyperkalemia, and metabolic acidosis. Hyperpigmentation is evident in palmar creases, scars, knuckles, and oral mucosa. In SAI, isolated glucocorticoid insufficiency lead to the effects listed earlier for PAI. Although hyperkalemia does not occur, hyponatremia develops because of arginine vasopressin antidiuretic hormone ADHmediated water retention as a physiological response to hypotension/ volume depletion with exacerbation from the vasopressin-­stimulating effect of nausea. Clinical features of AI may arise acutely or chronically. The insidious, nonspecific evolution of symptoms in most patients leads to delayed diagnosis, sometimes in excess of 1 year. Adrenal crisis may supervene before diagnosis of AI, with an attendant mortality risk. Prediagnosis considerations sometimes include mood disorders, anorexia nervosa, or malignancy. Cortisol deficiency leads to derepression of inflammatory cytokines, leading to increased body temperature with various patterns of pyrexia and the sickness syndrome.20 In females, decreased axillary and pubic hair, loss of libido because of loss of adrenal androgens, and amenorrhea may occur because of weight loss. Routine laboratory tests may show hyponatremia, mild metabolic acidosis, and prerenal azotemia, but hyperkalemia (in PAI) is more specific and facilitates diagnosis. Although hyperkalemia is typical, many patients have normal potassium at least in part because of frequent vomiting. Less common laboratory abnormalities include hypoglycemia (more frequent in children) and hypercalcemia. Hematologic abnormalities associated with AI include normochromic, normocytic anemia, relative lymphocytosis, and eosinophilia. Adrenal crisis is a medical emergency with generally severe features of AI and an acute deterioration in health associated with absolute hypotension (systolic blood pressure 480 stimulated) Variable 17OHP response, variable cortisol response (often inadequate) Minimal to no response NC: variable response, ↓cortisol common

Other Testing

Early-­morning follicular phase serum 17OHP 100 ng/dL [>3 nmol/L]) and corticosterone (>4000 ng/dL [>116 nmol/L]) with low cortisol (60 years), in whom the prevalence reaches up to 9%.1 Most neoplasms are benign adrenocortical tumors like adrenocortical adenomas (ACA); exceedingly rare forms of adrenal neoplasia include malignant adrenocortical carcinomas (ACC), adrenal medullary pheochromocytomas (Pheo), and extraadrenal paragangliomas (PGL; often associated with inherited/syndromic Pheo).1 Benign adrenal neoplasia also encompasses rare forms of diffuse and multinodular bilateral hyperplasia.1 Hormonally active adrenocortical tumors can produce any class of steroid hormones or precursors, including glucocorticoids, androgens, mineralocorticoids, and estrogens. While ACA and hyperplasia usually produce a single class of steroid hormones (typically aldosterone or cortisol), ACC commonly secrete several types of steroids and precursors.2,3 Hormonally active Pheo is also heterogeneous; epinephrine production is an exclusive feature of adrenal Pheo, norepinephrine production is more common in PGL, and elevated dopamine and production of its metabolite methoxytyramine is a feature of malignant Pheo.4,5 In terms of the genetic context, unilateral and sporadic forms of ACA, ACC, and Pheo are more common.6-­8 However, pediatric/early-­onset, bilateral, and multifocal tumors occur and are clinically suggestive of inherited forms.6,7 Consistent with the extraordinary clinical heterogeneity of adrenal neoplasia, the genetics of these diseases is complex. To date, approximately 20 genes are associated with inherited forms of adrenocortical neoplasia, including different types of bilateral adrenocortical hyperplasia, ACA, and ACC.9-­11 Likewise, approximately 20 genes are currently associated with inherited forms of Pheo/PGL.7 Interestingly, these genes converge on a few signaling pathways. Genes associated with inherited

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adrenocortical neoplasia primarily converge on programs coordinating cell proliferation, stemness, and zone-­specific hormone production. Specifically, frequently altered genes coordinate cell cycle machinery itself or signaling through IGF, Wnt/β-­catenin, protein kinase A (PKA), and calcium/calmodulin signaling pathways. Genes associated with inherited Pheo/PGL primarily converge on the mitogen-­activated protein kinase (MAPK) pathway, energy metabolism, and hypoxia-­sensing pathways. Clinical aspects of adrenal tumor syndromes including manifestations, diagnosis, and management are discussed in other chapters. The genetics of sporadic adrenal tumors is also complex. Candidate gene approaches revealed that genes and pathways targeted in inherited adrenal tumors are also recurrently altered in different subtypes of sporadic tumors.9 More recently, pangenomic approaches have illuminated a spectrum of novel recurrent somatic events converging on the same signaling pathways spanned by genes associated with inherited syndromes.12-­14 Furthermore, integration of somatic alteration profiles with other “omics” such as transcriptomics, methylomics, miRnomics, and metabolomics has enabled identification of core molecular subtypes. This large array of somatic alterations has added complexity to our understanding of the molecular basis of primary adrenal tumors, while simultaneously reconciling the broad spectrum of clinical manifestations. In this chapter, we will briefly summarize the genomics of inherited and sporadic primary adrenal tumors from a pangenomic perspective.

GENETIC CAUSES OF ADRENAL NEOPLASIA Adrenocortical Tumors KEY POINTS  • Genetic events in benign adrenocortical neoplasia target endocrine and paracrine signaling pathways that serve to integrate homeostatic maintenance and organ function. • Affected pathways include the Wnt/β-­catenin signaling pathway, the protein kinase A pathway, and the calcium/calmodulin signaling pathway, which regulate the stem/progenitor cell compartment of the adrenal cortex, cortisol secretion in response to adrenocorticotropic hormone, and aldosterone secretion, respectively. • Inherited and sporadic adrenocortical carcinomas possess genetic alterations in these pathways, in addition to recurrent alterations in growth factor signaling, cell cycle checkpoints, DNA mismatch-­repair genes, and epigenetic regulators.

CHAPTER 95  Adrenal Genomics II: Familial and Sporadic Neoplasia

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Zona glomerulosa Na+ Na+ Ca2+ +

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-ATP

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+

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+

+

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Fig. 95.1  Calcium/calmodulin signaling pathway of the adrenocortical zona glomerulosa. High levels of serum potassium and/or angiotensin II (Ang II) binding to its receptor (ATR, e.g., the receptor encoded by AGTR1) initiates zG membrane depolarization by inhibiting the actions of Na/K ATPase ATP1A1 and decreasing permeability of potassium channel GIRK4 (encoded by KCNJ5). Consequent changes in transmembrane potential trigger opening of voltage-­gated chlorine channels (CLCN2) and voltage-­gated calcium channels (CaV, such as those encoded by CACNA1D and CACNA1H), calcium influx and cytoplasmic accumulation, and activation of calcium/calmodulin-­dependent protein kinase (CAMK). Active CAMK directly or indirectly phosphorylates and/or upregulates immediate early response transcription factors (TFs) such as the NR4A, ATF, or CREB family members, leading to transcription of aldosterone synthase (encoded by CYP11B2). Resting membrane potential is restored by GIRK4, ATP1A1, and calcium efflux pump ATP2B3. Inherited or somatic mutations in several components of this pathway, leading to constitutive membrane depolarization, cytoplasmic calcium accumulation, and/or transcription of CYP11B2, cause primary aldosteronism.

Inherited disorders associated with adrenal hyperfunction and/or overgrowth may affect the cortex or medulla. These rare diseases are typically inherited in an autosomal dominant manner and are caused by alterations in genes coordinating developmental and homeostatic pathways. Frequently altered adrenocortical signaling pathways include the Wnt/β-­catenin signaling pathway, the PKA pathway, and the calcium/calmodulin signaling pathway. Disease-­causing mutations in these pathways disrupt adrenocortical-­specific mechanisms of integration of endocrine-­paracrine signals that govern adrenal homeostasis. In addition, other pathways that are associated with broader mechanisms of tumorigenesis, including epigenetic regulation, fetal growth, and cell cycle control, are also targeted by mutations that cause adrenal cortex tumors. Calcium/calmodulin signaling pathway: zG aldosterone production is controlled by angiotensin II (AngII) through its receptor, the angiotensin II receptor (ATR), such as the G protein-coupled receptor (GPCR) encoded by AGTR1. AngII binding to ATR triggers membrane depolarization, release of intracellular calcium stores, and activation of the calcium/calmodulin signaling pathway. This pathway is also activated in an ATR-­independent manner, by membrane depolarization initiated by increased serum potassium. Pathway activation culminates in the rapid transcription of target genes essential for aldosterone production, including CYP11B2.15-­17 Germline mutations in genes that affect zG cell membrane polarization, including CLCN2 and KCNJ5, and genes that regulate intracellular calcium homeostasis, including CACNA1D and CACNA1H, cause familial hyperaldosteronism type II, III, and IV and PASNA (primary aldosteronism, seizures,

and neurological abnormalities). These syndromes are characterized by early-­onset primary hyperaldosteronism and bilateral zG hyperplasia. Sporadic aldosterone-­producing ACA bear recurrent mutations in the same classes of genes, including KCNJ5, CLCN2, CACNA1D, and CACNA1H.10 Another class of genes recurrently mutated in sporadic aldosterone-­producing ACA encode ion-­transporting ATPases, including ATP1A1 and ATP2B3, also enabling constitutive zG cell membrane depolarization.10 The aforementioned mutations are collectively present in approximately 90% of aldosterone-­ producing ACA.18 This pathway is depicted in Fig. 95.1. Wnt/β-­catenin signaling pathway: The Wnt/β-­catenin signaling pathway is required for adrenocortical development and homeostasis. It is a key regulator of the progenitor cell pool and shapes adrenocortical functional and anatomic zonation, presumably through tissue-­specific roles.19 The Wnt/β-­catenin signaling pathway is tightly regulated by cell-­ autonomous and nonautonomous mechanisms (Fig. 95.2). Inactivating germline mutations in APC, encoding a negative regulator of β-­catenin stability, causes familial adenomatous polyposis (FAP). FAP is an autosomal dominant disorder characterized by predisposition to colon cancer and other tumors such as lipomas, connective tissue sarcomas, osteomas, and bilateral adrenal tumors.9 Subsequent studies identified somatic activating mutations in CTNNB1, encoding β-­catenin, in up to a third of sporadic ACC and ACA, and somatic APC inactivation in a small subset of sporadic ACC.9 More recently, whole-­exome sequencing (WES) studies on large cohorts of ACC identified frequent (∼20%) recurrent somatic biallelic deletions in ZNRF3, encoding an E3 membrane-­bound ubiquitin ligase that also functions

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WNT OFF (-Rspo)

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Fig. 95.2  Wnt/β-­catenin signaling pathway. The Wnt/β-­catenin signaling pathway is required for stem cell maintenance and homeostasis of a variety of organs, including the adrenal gland. The final effect of pathway activation by secreted Wnt ligands is to turn on expression of Wnt target genes through β-­catenindependent coactivation of TCF/LEF-­driven transcription. Pathway activation is controlled at several levels, primarily through regulation of stability/compartmentalization of Wnt receptors (Frizzled receptors [FZD]) and β-­catenin. Membrane localization of FZDs is controlled by an additional secreted ligand, R-­spondin (Rspo). Shown in the upper left (“WNT OFF (-­Rspo)”), in the absence of Rspo, FZDs are targeted for degradation by membrane ubiquitin ligase RNF43 or ZNRF3. Shown in bottom left (“WNT OFF (-­Wnt)”), when Rspo binds to its receptor LGR4/5, the Rspo/LGR/RNF43 or ZNRF3 complex is internalized, increasing FZD availability at the membrane. In the absence of Wnt ligands, however, β-­catenin stability is regulated by a large, cytoplasmic multiprotein complex termed the “destruction complex,” which includes the protein APC. In the “WNT OFF” state (as depicted bottom left), this complex binds β-­catenin, phosphorylates it, and targets it for ubiquitination and proteasome-­mediated destruction. Finally, when Wnt ligands bind to FZD receptors and Wnt signaling is turned on (“WNT ON,” right), the destruction complex is sequestered at the cell membrane and incapable of targeting β-­catenin for degradation. This allows β-­catenin to accumulate in the cytoplasm and translocate to the nucleus to turn on TCF/LEF-­dependent transcription. Benign and malignant adrenocortical lesions frequently harbor activating mutations in CTNNB1, encoding β-­catenin. These mutations are often located at key residues for phosphorylation of β-­catenin by the destruction complex, promoting constitutive accumulation of the protein and TCF/LEF-­dependent transcription. Adrenocortical carcinomas also harbor inactivating mutations/deletions in APC and ZNRF3.

as a negative regulator of Wnt/β-­catenin signaling.12,13,20 Importantly, somatic alterations in Wnt pathway components are prevalent (nearly 40% of all ACC) and mutually exclusive, demonstrating the selective advantage conferred by constitutive Wnt/β-­catenin signaling as a core driver mechanism of adrenocortical tumorigenesis. The mechanisms by which Wnt/β-­catenin signaling induces adrenocortical tumorigenesis are not fully understood. Observations from mouse models and the clinical spectrum of these tumors suggest that this mechanism is highly context-­dependent. In human patients, germline APC mutations are associated with nonfunctional hyperplasia, and more rarely, ACA and ACC.9 CTNNB1 mutations are prevalent in up to 20% of adenomas and carcinomas.9 In ACA, tumors bearing CTNNB1 mutations are typically large and nonfunctional, exhibit mild autonomous cortisol secretion, or, rarely, exhibit aldosterone production.21,22 In ACC, tumors bearing CTNNB1 mutations are associated with cortisol production.13 Pathological mutations and deletions in

ZNRF3 are exclusive to ACC. In mouse models, constitutive Wnt/β-­ catenin pathway activation through biallelic deletion of Apc or deletion of exon 3 of Ctnnb1 leads to disrupted zonation, progressive zG hyperplasia, and coincident aldosterone overproduction.23-­25 In contrast, biallelic adrenal deletion of Znrf3 leads to zF-­like hyperplasia with massive adrenal enlargement.26 These observations suggest constitutive Wnt/β-­catenin pathway activation confers a context-­dependent selective advantage to either zG or zF cells of the adrenal cortex. Combined constitutive Wnt/β-­catenin pathway activation with other alterations targeting Igf2 (encoding IGF2) or Trp53 (encoding p53) leads to ACC and glucocorticoid excess.23,25 Protein kinase A: While Wnt/β-­catenin signaling is an essential part of a paracrine relay that establishes and maintains the adrenocortical stem cell compartment, the PKA pathway is the effector arm of the endocrine system (adrenocorticotropic hormone [ACTH]) that induces differentiation and steroid production in the zF and zR. In

CHAPTER 95  Adrenal Genomics II: Familial and Sporadic Neoplasia

Zona fasciculata MC2R

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αs β γ

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Fig. 95.3  Adrenocorticotropic hormone (ACTH)/protein kinase A (PKA) signaling pathway of the adrenocortical zona fasciculata. ACTH binds to its cell surface receptor, G protein-coupled receptor (GPCR) MC2R to activate the protein kinase A (PKA) signaling pathway. ACTH binding to MC2R triggers dissociation of the Gα,s subunit from the associated Gs protein. Gα,s binds to and activates adenylyl cyclase. Adenylyl cyclase then catalyzes the conversion of cellular ATP to cyclic AMP (cAMP). PKA is a heterotetramer, comprised of two regulatory subunits (R) and two catalytic subunits (C). cAMP binds the regulatory subunits, liberating the catalytic subunits of PKA which then catalyze phosphorylation and nuclear translocation of transcription factor CREB. CREB drives transcription of a variety of genes, including CYP11B1, required for glucocorticoid synthesis in the zF. Pathway activation is extinguished by intracellular phosphodiesterases (PDEs), which catalyze the conversion of cAMP to AMP, enabling reassembly of the PKA tetramer. Genetic alterations in components of this pathway leading to constitutive PKA activation and/ or CYP11B1 transcription are major causes of primary hypercortisolism in benign lesions of the adrenal cortex. Recurrent loss of function mutations in genes encoding the R subunit of PKA (e.g., PRKAR1A) have also been identified in adrenocortical carcinoma.

the adrenal, ACTH binding to its receptor, the GPCR MC2R, leads to cAMP accumulation and PKA activation. Catalytically active PKA in turn phosphorylates CREB family transcription factors, which translocate into the nucleus to drive expression of genes required for glucocorticoid production (e.g., CYP11B1). ACTH-­driven PKA activation results in cell proliferation and zF differentiation with increased output of cortisol. PKA signaling is rapidly terminated by the actions of phosphodiestarases, which dephosphorylate the PKA regulatory subunits, favoring inhibitory binding to the catalytic subunit11,27,28 (Fig. 95.3). Several genetic syndromes presenting with adrenocortical hyperplasia and hyperfunction are caused by germline defects in genes that encode members of the PKA pathway, including GNAS, PRKAR1A, PDE11A, PDE8B, and PRKACA.11 These genetic defects lead to constitutive PKA activation, rendering adrenocortical cells in a permanent state of activation characterized by cortisol overproduction and hyperplasia. Somatic mutations in PKA pathway members, including GNAS,

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PRKACA, PRKAR1A, PRKAR1B, and PDE11A, are commonly found in cortisol-­producing adenomas.29 Recurrent somatic mutations of PRKAR1A have been recently identified in ACC, implicating constitutive PKA signaling in malignant programming.13 Growth factor signaling: IGF2 is a growth factor that is widely expressed during fetal life and is one of the major regulators of intra-­ uterine growth. Growth-­promoting effects of IGF2 are mediated by the insulin-­like growth factor 1 receptor (IGF1R), a receptor tyrosine kinase that activates the MAPK pathway. In physiological somatic tissues, the IGF2 locus on 11p15.5 is imprinted and exclusively expressed by the paternal allele. The imprinting of IGF2 is regulated by the H19/ IGF2-­imprinting control region (ICR1), located between H19, a long noncoding RNA with growth inhibitory functions expressed exclusively from the maternal allele, and IGF2. Patients with germline mutations or deletions in imprinted genes located on 11p15.5, including CDKN1C, H19, and KCNQ1OT1, and hypermethylation of ICR1 develop Beckwith–Wiedemann syndrome, an overgrowth disorder characterized by abdominal wall defects, abnormal growth, macroglossia, visceromegaly (including adrenocortical cytomegaly), and embryonal malignancies such as Wilms tumor, hepatoblastoma, and ACC. Interestingly, IGF2 overexpression is present in 90% of sporadic ACC, usually associated with loss of heterozygosity (LOH) at 11p15.5 (and consequent loss of ICR1), suggesting a critical role for dysregulation of the MAPK pathway in ACC tumorigenesis.9 Cell cycle regulation: Germline mutations in TP53 cause Li– Fraumeni syndrome (LFS). The prevalence of ACC in patients with LFS is approximately 5%.30 Interestingly, the prevalence of germline TP53 mutations among pediatric ACC cases is 50% to 90%. Somatic mutations and frequent LOH of 17p13 have often been described in sporadic ACC. The prevalence of somatic TP53 mutations is approximately 20%. In addition to alterations in TP53, recurrent somatic events targeting other checkpoint regulators include inactivating mutations/deletions of CDKN2A and RB1 and amplification of CDK4 and CCNE1.9,12,13,31 DNA mismatch repair: Germline mutations in mismatch-­repair genes cause Lynch syndrome. This disease is characterized by predisposition to several types of neoplasia, including gastrointestinal and gynecological tumors. Characteristically, these tumors present with microsatellite instability. ACC has also been described as part of the spectrum of Lynch syndrome.32 Somatic mutations in mismatch-­ repair genes have been recently described in ACC by The Cancer Genome Atlas (TCGA), and up to 30% of ACC exhibit a mismatch repair-deficient signature, but their role in tumorigenesis is unclear.9,33 Beyond Lynch syndrome genes, variants in other genes implicated in DNA repair, such as MutYH, seem to predispose to ACC, with a specific mutational signature.34 Other mechanisms: Germline and somatic alterations targeting other general cellular intrinsic regulatory mechanisms such as chromatin remodeling and telomere maintenance may also play a role in ACC tumorigenesis. Germline mutations in MEN1 cause multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder characterized by multiple endocrine tumors, including gastrinomas, insulinomas, and parathyroid and pituitary adenomas. MEN1 encodes menin, a protein best characterized as a scaffold protein for chromatin remodelers. Precisely how menin contributes to tissue-­specific tumorigenesis is still not understood. Furthermore, up to 40% of patients with MEN1 develop nonfunctional bilateral adrenal macronodular hyperplasia that may rarely evolve to ACC.9 Recently, recurrent somatic mutations in MEN1 have been described in ACC, suggesting a driver role for mutations in this gene in adrenocortical carcinogenesis.13 In addition to bearing mutations in MEN1, sporadic ACC may also possess alterations in genes encoding other chromatin remodeling and

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associated proteins, including MLL family members, ATRX, DAXX, TET1, SETD2, and SMARCA4.12,13 KEY POINTS  • Integrative multiplatform analyses of adrenocortical tumors have enabled the identification of distinct molecular subtypes, characterized by the coocurrence of specific molecular features such as somatic mutations targeting a given signaling pathway, activation of specific transcriptional programs, DNA methylation patterns, and chromosome copy-­number alteration profiles. • In adrenocortical carcinoma, at least three molecular subtypes have been identified, and they are strongly associate with clinical and histological metrics such as hormone secretion, mitotic counts, metastatic potential, and clinical outcomes.

Integrated molecular classification of adrenocortical adenomas: By integrating tumor genome sequencing data with gene expression (microarray, RNA-­seq, and miRNA-­seq), DNA methylation (Illumina arrays), chromosome alteration profiles (single nucleotide polymorphism [SNP] arrays), and proteomics, unbiased and multiplatform studies further characterized the heterogeneity of adrenal tumors and helped illuminate the molecular consequences of driver events for tumorigenesis.12,13 Furthermore, while these studies have limited immediate translational or clinical applicability, they offer an excellent opportunity for identification of prognostic and predictive biomarkers, providing the rationale for preclinical studies directing new agents and existing targeted therapies to specific molecular subtypes. Few studies have performed pangenomic analyses of ACA. Di Dalmazi et al.21 performed RNA-­seq in a large cohort of ACA, including samples from patients with overt Cushing syndrome, mild autonomous cortisol secretion, endocrine inactive adenomas, and low-­grade low-­stage ACC. Unsupervised analyses have identified three different types of ACA that presented with specific clinical and demographical features, as well as distinct somatic mutational profiles. Samples bearing somatic mutations in genes encoding PKA components including PRKACA and GNAS formed an individual cluster of tumors characterized by overt Cushing syndrome presentation, increased frequency of female sex, and smaller size. On the other hand, ACA with CTNNB1 mutations formed another cluster of tumors characterized by mild autonomous cortisol secretion or endocrine silent tumors, older age at presentation, and no gender predilection. Finally, a small subgroup of ACA with CTNNB1 mutations formed a cluster that exhibit transcriptional features resembling low-­grade ACC. These results are consistent with other transcriptome studies of non-aldosterone-­ producing ACA, which identified two subtypes of tumors by unsupervised analysis that segregated cortisol-­producing adenomas from those that are endocrine-­silent or exhibit mild cortisol production.22,35 Two integrated multiplatform studies have performed molecular profiling of a large number of ACC samples.12,13 These studies have identified discrete molecular subtypes of ACC that exhibit distinct clinical features and prognosis. De Reynies et al. identified two major transcriptional classes of ACC, C1A and C1B,36 with striking differences in prognosis (C1A is associated with poor prognosis, and C1B is associated with good prognosis, often cured by surgical resection). Using a multiplatform approach that included microarray, methylation, microRNA-­seq, SNP-­array, and WES, Assie et al. expanded the characterization of the two previously identified molecular subtypes of ACC. C1A ACC exhibited a higher mutation burden, as well as frequent somatic events in driver genes, including CTNNB1, ZNRF3, APC, TP53, RB1, CDKN2A, and DAXX.12 In addition, approximately two-thirds of C1A tumors exhibited abnormal CpG island hypermethylation (CIMP), a phenomenon previously associated with poor

outcomes in ACC.37 On the other hand, C1B exhibited a lower mutation burden, and several tumors in this class did not bear an identifiable somatic “driver.” However, approximately two thirds of C1B tumors exhibited a profound deregulation of a cluster of microRNAs located in 14q32 (DLK1-­MEG3 cluster), coupled with LOH of this region, suggesting this may be the molecular event driving these tumors. As part of the TCGA consortium, Zheng et al.13 expanded on these studies to provide a broader multiplatform classification of ACC (ACC-­ TCGA). ACC-­TCGA identified three unique classes of ACC (COC1, COC2, COC3), driven by differential regulation of steroidogenesis, cell cycle, and immune infiltration. Notably, each type of ACC also possessed a unique somatic copy number alteration (SCNA) profile and DNA methylation landscape. Strikingly, each molecular subtype of ACC is associated with a distinct prognostic outcome: COC1 tumors exhibited good prognosis (progression rate [PR] of 7%; median event-­ free survival [EFS] not reached), COC2 tumors exhibited intermediate prognosis (PR 56%; EFS 38 months), and COC3 tumors exhibited dismal prognosis (PR 96%; EFS 8 months).13 The vast majority of ACC is “pure,” that is, free of stromal and other infiltrating immune cells, with average purity approaching 90%; however, while COC1 tumors still exhibit high purity levels in comparison to other TCGA cancers, they are characterized by low expression of steroidogenic enzymes and high expression of immune genes consistent with a higher degree of immune infiltration.13 Like C1B tumors identified by de Reynies et al., COC1 tumors also did not possess recurrent somatic alterations. COC1 tumors had low levels of genome-­ wide CpG island hypermethylation (CIMP-­low) and an SCNA profile termed “chromosomal,” characterized by whole-­ chromosomal gains and losses and copy-­neutral LOH, sometimes accompanied by whole-­genome doubling. COC2 tumors were characterized by largely absent immune infiltration, high expression of steroidogenic enzymes, and recurrent somatic alterations targeting the Wnt/β-­catenin pathway. COC2 ACC had intermediate levels of genome-­wide CpG island hypermethylation (CIMP-­intermediate) and a chromosomal SCNA profile. Finally, COC3 tumors were characterized by largely absent immune infiltration, high expression of steroidogenic enzymes and cell cycle machinery, and recurrent somatic alterations targeting the Wnt/β-­catenin pathway and cell cycle. COC3 tumors had high levels of genome-­wide CpG island hypermethylation (CIMP-­high) and a SCNA profile termed “noisy,” characterized by focal arm-­level gains and losses and numerous breakpoints, sometimes accompanied by whole-­genome doubling (Fig. 95.4). The completion of ACC-­TCGA enabled incorporation of ACC into larger pancancer pangenomic datasets. These studies have identified several unique characteristics of ACC, with therapeutic implications. Up to a third of ACC exhibit a somatic mismatch repair-deficient signature,33 and most ACC is immune-­poor, with minimal lymphocyte infiltration,38 suggesting these tumors are as a majority resistant to immunotherapy, consistent with recent clinical studies.39 On an epigenetic level, the accessible chromatin landscape of ACC is largely dominated by the adrenocortical transcription factor SF1,40 suggesting that potentially efficacious epigenetic therapies would need to unwire this highly tissue-­specific program.

Pheochromocytoma Up to 40% of Pheo/PGL are associated with inherited syndromes, and to date there are approximately 20 well-­documented susceptibility genes.7 Clinical aspects of these syndromes and strategies for genetic testing and clinical management are beyond the scope of this chapter and are discussed elsewhere. Broadly, two signaling pathways are most affected by these germline mutations: cellular hypoxia and activated kinase signaling.41,42 Activation of the cellular hypoxia pathway

CHAPTER 95  Adrenal Genomics II: Familial and Sporadic Neoplasia C1B

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Fig. 95.4  Molecular subtyping of adrenocortical carcinoma (ACC). Recent pangenomic studies (namely, The Cancer Genome Atlas study on ACC [ACC-TCGA]) have revealed that ACC is comprised of three distinct molecular subtypes, COC1, COC2, and COC3. Not shown in this figure, each subtype is associated with a unique clinical outcome where patients with COC1 tumors have the best outcomes (longer progression-­free survival), patients with COC2 tumors have intermediate outcomes, and patients with COC3 tumors have the worst outcomes (shortened progression-­free survival with near uniform likelihood of relapse within 2 years). Strikingly, ACC-­TCGA revealed that each class of tumors is defined by a combination of core transcriptional programs, DNA methylation profiles, and somatic alterations. All ACC possess high levels of IGF2 expression, secondary to loss of imprinting of this locus. COC1 tumors possess the lowest expression of steroidogenic enzymes, the lowest degree of cell cycle activation, the lowest levels of promoter CpG island (CpGi) methylation (CIMP-­low), and few to no recurrent somatic alterations. COC1 tumors bear a somatic copy number alteration profile characterized by whole arm/whole chromosome loss of heterozygosity (LOH), called “chromosomal.” COC1 tumors also possess a moderate degree of immune infiltration (approaching 50% in some tumors). In contrast, COC2 and COC3 tumors bear high expression of steroidogenic enzymes, higher levels of CpGi methylation (CIMP-­intermediate and CIMP-­high), and frequent Wnt/β-­catenin pathway alterations. These tumors are also characterized by a low to near-­ absent immune infiltration. COC3 tumors possess the highest expression of steroidogenic enzymes, highest degree of cell cycle activation, and highest degree of CpGi methylation (CIMP-­high). In contrast to COC1–2 tumors, COC3 possess a “noisy” somatic copy number alteration profile, characterized by numerous focal arm-­level copy number gains and losses throughout the genome. Finally, as shown at the top of the plot, the transcriptional programs driving COC1 and COC2–3 tumors resemble the C1B and C1A signatures originally identified by de Reynies et al. in a seminal unbiased study using microarray to characterize transcriptional programs driving adrenocortical tumors.

is driven by the hypoxia-­inducible factor (HIF) family of transcription factors. Physiologically, this pathway is activated in response to cellular hypoxia, a state that is recognized by hypoxia-­sensing proteins, which trigger a downstream signaling cascade that leads to the accumulation of HIF family members. HIF induces the expression of a broad spectrum of genes that promote erythropoiesis, angiogenesis, cell survival, cell proliferation, and glycolysis. A central protein in this pathway is the von Hippel–Lindau protein (VHL), encoded by VHL. Under normal oxygen concentrations, VHL is a member of a multiprotein complex that promotes ubiquitin/proteasome-­mediated degradation of HIF transcription factors. Hydroxylation of proline residues in HIF by the prolyl-­hydroxylase PHD2 (EGLN1) is critical for VHL docking, a step crucial for ubiquitination.43

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Inactivating germline mutations in VHL cause von Hippel– Lindau syndrome, an autosomal dominant disorder characterized by increased risk for several tumor types, including retinal, brain, and spinal cord hemangioblastomas, pancreatic neuroendocrine tumors, clear cell renal carcinomas, angiolymphatic sac tumors, and Pheo/ PGL. Complete loss of VHL is usually a consequence of a somatic LOH event at 3p25.3. Consistent with increased angiogenesis, the tumors associated with VHL are rich in blood vessels. Interestingly, rare germline mutations in VHL that lead to mild defects in the protein function cause an autosomal recessive form of congenital erythrocytosis. This is presumably through global low-­level activation of HIF-­induced transcription, which leads to inappropriately high levels of erythropoietin, thereby inducing otherwise hypoxia-­dependent erythrocytosis.43 Recurrent somatic mutations in VHL have also been described in Pheo/PGL.14 In addition to VHL, mutations targeting another gene of the hypoxia pathway have been described in patients with syndromic forms of Pheo/PGL syndromes, including the polycythemia-­paraganglioma-­ somatostatinoma syndrome. Genetically, this syndrome is characterized by a somatic mosaicism involving activating mutations of EPAS1, also known as HIF2A. These mutations also lead to constitutive activation of HIF-­dependent transcriptional programs, leading to erythropoietin overproduction and tumor formation in target tissues.44 Recurrent somatic mutations in EPAS1 have also been described in sporadic Pheo/PGL.14 Finally, mutations in genes encoding energy metabolism enzymes, including SDH family members (SDHx, encompassing SDHA, SDHAF2, SDHB, SDHC, and SDHD), FH, and MDH2 lead to a metabolic switch that results in abnormal accumulation of metabolites (succinate or fumarate), which disrupt cell metabolism through the TCA cycle by inhibiting iron(II)-­and α-­ketoglutarate-­ dependent (α-­KG) dioxygenases. These enzymes coordinate a broad spectrum of cellular programs. In particular, prolyl hydroxylases such as EGLN1, which are negative regulators of HIF stability, are inhibited by succinate and fumarate accumulation. TET enzymes, which coordinate DNA demethylation, are also inhibited by abnormal metabolite accumulation. TET inhibition leads to global epigenetic remodeling, including acquisition of the CIMP signature, thought to play a major role in tumorigenesis and in malignant transformation.45,46 At the somatic level, the hotspot IDH1 p.R132C mutation, which is known to dysregulate the TCA cycle through oncometabolite accumulation,47 has also been described in a subset of sporadic Pheo/PGL.14,48 The other broad signaling pathway that is targeted by germline mutations in Pheo/PGL, activated kinase signaling,41,42 is a growth-­ promoting pathway that promotes activation of translation and cell proliferation through MAPK. Transcriptional effectors of this signaling pathway include the MYC/MAX complex.49 In Pheo/PGL, germline mutations associated with increased kinase activity include activating mutations of RET, inactivating mutations of NF1, and inactivating mutations in TMEM127, which cause multiple endocrine neoplasia type 2, neurofibromatosis type 1, and familial pheochromocytoma, respectively.42 Mutations in RET occur at specific hotspots, including C634 and M918, and lead to the constitutive activation of its tyrosine kinase activity. Mutations in NF1 are scattered throughout the gene and include large deletions. The protein NF1 is a negative regulator of the MAPK signaling pathway. Recurrent somatic mutations in RET and NF1 have also been described in spontaneous Pheo/PGL, with RET mutations occurring at restricted hotspots.14,50,51 Finally, germline mutations in MAX, which encodes MYC-­associated factor X (MAX protein), are another cause of familial Pheo/PGL. Disruption of MAX promotes oncogenic effects of MYC. Recurrent somatic mutations in MAX have also been described in sporadic Pheo/PGL.52 Constitutive activation of the MAPK pathway can also be triggered by mutations

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PART 7  Adrenal

Ligand RET

TMEM 127

PIP3

Hras

Hras

PI3K

GDP

GTP

PIP2 NF1 MD

H2

Malate FH

mTOR

Fumarate

MAPK

TCA Oxaloacetate cycle

SDH

MAML

β-catenin

Succinate

EGLN1 OH

HIF

VHL

β-catenin TCF/LEF

OH Ub

Wnt target genes

HIF

Ub Ub Ub

Myc Max

Kinase program genes

HIF

Hypoxia program genes

Proteasome

Fig. 95.5  Recurrent genetic alterations driving pheochromocytomas and paragangliomas (Pheo/PGL). Recent pangenomic studies of Pheo /PGL have revealed that these tumors are defined by activation of key cellular programs: cellular hypoxia signaling and activated kinase signaling. This is accomplished through alterations in genes encoding components of the TCA cycle (SDHx, FH, MDH2), effectors/putative modulators of receptor tyrosine kinase and mTOR/MAPK signaling (RET, NF1, HRAS, TMEM127, MAX), and critical regulators of cellular hypoxia (genes encoding hypoxia-­inducible transcription factors, von Hippel–Lindau, and EGLN1). Sporadic Pheo also possess recurrent fusions targeting the MAML3 locus, leading to expression of a chimeric fusion transcript and upregulation of Wnt/β-­catenin–dependent transcription. How these different pathway components coordinate signaling cascades believed to drive expression of oncogenic programs is depicted in this figure. Notably, mutations in enzymes coordinating TCA cycle are frequently loss of function, driving the hypoxia program through aberrant accumulation of TCA metabolites (succinate and fumarate). Partially depicted, accumulation of succinate and fumarate inhibit several classes of enzymes including EGLN1 and TET enzymes that erase DNA methylation. Consequently, tumors bearing TCA cycle mutations also possess a DNA hypermethylation signature.

that only occur somatically in sporadic Pheo/PGL, such as hotspot mutations in HRAS.14,53 More recently, a study that performed multiplatform molecular profiling of a large number of Pheo/PGL identified novel recurrent somatic events, including mutations in CSDE1 and gene fusions involving MAML3.14 CSDE1 encodes cold shock domain-containing E1, an RNA-­binding protein involved in RNA stability, translation initiation, and neuronal development. Somatic mutations in CSDE1 were present in 2% of the samples, usually affecting splicing sites, resulting in loss of function. Fusions involving MAML3 were present in 5% of Pheo/PGL and involve the promoter regions of either UBTF or TCF4 and the coding region of MAML3, resulting in in-­frame chimeric transcripts. Because these resultant chimeric transcripts are overexpressed, MAML3 fusions are presumably gain-­of-­function. Additional fusions were identified involving other genes, such as KIAA1737-­NGFR, RUNDC1-­BRAF, and NF1-­RAB11F1P4. The first two result in overexpression of the kinase transcripts, and therefore are thought to be gain-­of-­function; the last results in disruption of NF1 expression.

Other recurrent somatic mutations that reached statistical significance, according to this study, were the previously identified HRAS, NF1, EPAS1, and RET (present in 10%, 9%, 5%, and 3% of samples, respectively). In addition to these, somatic hotspot and cancer-­relevant genes mutations in BRAF, IDH1 (p.R132C), FGFR1, VHL, ATRX, TP53, SETD2, and ARNT were also detected. Importantly, unlike other tumor types that may have mutations in more than one driver gene, a striking mutual exclusivity was detected among somatic mutations and pathogenic germline variants detected in this study, indicating that tumors typically require only one mutation in this class of genes, except for ATRX mutations which usually cooccur with germline SDHB mutations, a previously described association.54 Pathways relevant for Pheo/PGL biology are depicted in Fig. 95.5. Integrated molecular classification of pheochromocytomas/ para­gangliomas: Multiomics profiling of Pheo/PGL have confirmed previous observations that prominent somatic and germline driver alterations converge on specific molecular subtypes.41,42,51 Unsupervised consensus clustering analysis on the transcriptome

CHAPTER 95  Adrenal Genomics II: Familial and Sporadic Neoplasia data revealed that Pheo/PGL consists of four molecular subtypes, designated “kinase signaling,” “pseudohypoxia,” “Wnt-­altered,” and “cortical-­admixture.”14 Consistent with previous studies, the “kinase-­ signaling” subtype was observed predominantly in epinephrine-­ producing adrenal pheochromocytoma of the “adrenergic” phenotype (see other chapters for a detailed discussion). This subtype was enriched for germline and somatic mutations targeting NF1, RET, TMEM127, and HRAS, as well as rarer fusion events involving NF1, HRAS, and NGFR. Furthermore, specific arm-­level chromosomal deletions involving 1p, 3q, and 17q were also enriched in this subtype. Also consistent with previous observations, the “pseudohypoxia” subtype is enriched for germline mutations and somatic mutations in SDHx, VHL, EPAS1, and EGLN1 (which were exclusive of this subtype) and non-epinephrine-­producing Pheo/PGL. Other distinctive molecular features of the “pseudohypoxia” cluster include increased DNA methylation on CpG islands secondary to metabolite-­dependent inhibition of DNA demethylases. Finally, whole-­genome doubling events, characterized by extra copies of all chromosomes, were significantly enriched in the “pseudohypoxia” subtype, frequently in association with VHL and EPAS1 mutations. The previously unappreciated “Wnt-­altered” cluster exclusively consisted of sporadic adrenal pheochromocytomas, and was significantly enriched for MAML3-­containing fusion transcripts and CSDE1 mutations. This subtype features increased expression of targets of Wnt/β-­catenin and Sonic Hedgehog pathways. Finally, the “cortical-­admixture” subtype is characterized by increased expression of several adrenocortical genes, including STAR, CYP11B2, and CYP21A2, consistent with significant infiltration with cortical tissue. The presence of MAX mutations in this group, which is often associated with multifocal local disease, suggests that coalescing multiple tumor foci may “trap” cortical cells interspersed within the tumor.

SUMMARY AND FUTURE DIRECTIONS Adrenal tumors are a heterogeneous group of diseases with diverse clinical phenotypes. This complexity is mirrored at the molecular level. A large number of genetic drivers targeting recurrent biological processes or molecular pathways have been described in sporadic and familial forms of adrenal tumors and are usually shared between the inherited and equivalent sporadic form. Within a similar tumor type, these genetic alterations cluster around molecular subtypes, which are biologically correlated groups of tumors based on unbiased multiomics classification. These molecular subtypes closely explain important aspects of the clinical variability, such as the endocrine manifestations, growth patterns, and disease prognosis. However, classifying a new tumor sample in a given molecular subtype outside the scope of a comprehensive multiomics study can be challenging. This task can be accomplished by the use of a biomarker, which is a single measurement of an analyte that can be used as a surrogate to identify a specific disease state. In adrenal tumors, these biomarkers can be hormones, mutations, or other molecular features, such as patterns of expression of specific genes, and targeted DNA methylation.2,3,36,55-­57 Examples of biomarkers for adrenal tumors include catecholamine/metanephrine levels to establish a diagnosis of Pheo/PGL, steroid profiling and IGF2 expression to distinguish between ACA and ACC, steroid profiling and catecholamine/metanephrine levels to diagnose recurrence, expression levels of BUB1B, PINK1, and G0S2, and methylation levels of G0S2 to diagnose highly recurrent/aggressive (C1A and ACC-­TCGA COC3) subtypes of ACC. However, implementing biomarkers into clinical practice to aid into the decision-­making process requires extensive validation, which is the goal of ongoing clinical studies. Molecular subtypes, in particular, offer several meaningful insights that can be exploited therapeutically under the umbrella of personalized

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medicine as an alternative to the prevailing blanket therapy approaches. Molecular subtypes are particularly relevant for targeted therapies, such as kinase inhibitors, cell cycle inhibitors, epigenetic drugs, and immunotherapy. Early clinical trials using targeted therapies for adrenal tumors were disappointing.58,59 However, these trials were performed at a time when the molecular subtypes were not fully characterized. In fact, small subgroups of patients exhibited measurable and long-­term benefits from “failed” agents. Prospective identification of the potential responders could have changed the history of these trials, as molecular subtyping may more effectively narrow down likely responders according to the therapy’s mechanism of action. In this setting, the use of molecular biomarkers is essential. However, the potential advantages should be extensively demonstrated and validated in preclinical studies and clinical trials in order to support this paradigmatic shift and justify the increase in cost and complexity of healthcare.

REFERENCES 1. Sherlock M, Scarsbrook A, Abbas A, et al. Adrenal incidentaloma. Endocr Rev. 2020;41(6):775–820. 2. Wajchenberg BL, Albergaria Pereira MA, Medonca BB, et al. Adrenocortical carcinoma: clinical and laboratory observations. Cancer. 2000;88:711– 736. 3. Arlt W, Biehl M, Taylor AE, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors. J Clin Endocrinol Metab. 2011;96:3775–3784. 4. Eisenhofer G, Lenders JW, Siegert G, et al. Plasma methoxytyramine: a novel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status. Eur J Cancer. 2012;48:1739–1749. 5. Crona J, Lamarca A, Ghosal S, et al. Genotype-­phenotype correlations in pheochromocytoma and paraganglioma: a systematic review and individual patient meta-­analysis. Endocr Relat Cancer. 2019;26:539–550. 6. Else T, Kim A, Sabolch A, et al. Adrenocortical carcinoma. Endocr Rev. 2014;35(2):282–326. 7. Neumann HPH, Young WF, Eng C. Pheochromocytoma and paraganglioma. N Engl J Med. 2019;381:552–565. 8. Lenders JW, Eisenhofer G, Mannelli M, et al. Phaeochromocytoma. Lancet. 2005;366:665–675. 9. Lerario AM, Moraitis A, Hammer GD. Genetics and epigenetics of adrenocortical tumors. Mol Cell Endocrinol. 2014;386:67–84. 10. Boulkroun S, Fernandes-­Rosa FL, Zennaro MC. Old and new genes in primary aldosteronism. Best Pract Res Clin Endocrinol Metab. 2020;34:101375. 11. Berthon A, Bertherat J. Update of genetic and molecular causes of adrenocortical hyperplasias causing Cushing syndrome. Horm Metab Res. 2020;52:598–606. 12. Assié G, Letouzé E, Fassnacht M, et al. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet. 2014;46(6):607–612. 13. Zheng S, Cherniack AD, Dewal N, et al. Comprehensive pan-­genomic characterization of adrenocortical carcinoma. Cancer Cell. 2016;30:363. 14. Fishbein L, Leshchiner I, Walter V, et al. Comprehensive molecular characterization of pheochromocytoma and paraganglioma. Cancer Cell. 2017;31:181–193. 15. Bandulik S, Penton D, Barhanin J, et al. TASK1 and TASK3 potassium channels: determinants of aldosterone secretion and adrenocortical zonation. Horm Metab Res. 2010;42:450–457. 16. Bassett MH, White PC, Rainey WE. The regulation of aldosterone synthase expression. Mol Cell Endocrinol. 2004;217:67–74. 17. Szekeres M, Turu G, Orient A, et al. Mechanisms of angiotensin II-­ mediated regulation of aldosterone synthase expression in H295R human adrenocortical and rat adrenal glomerulosa cells. Mol Cell Endocrinol. 2009;302:244–253. 18. Nanba K, Omata K, Else T, et al. Targeted molecular characterization of aldosterone-­producing adenomas in white Americans. J Clin Endocrinol Metab. 2018;103:3869–3876.

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19. Little DW, Dumontet T, LaPensee CR, et al. β-­catenin in adrenal zonation and disease. Mol Cell Endocrinol. 2021;522:111120. 20. Juhlin CC, Goh G, Healy JM, et al. Whole-­exome sequencing characterizes the landscape of somatic mutations and copy number alterations in adrenocortical carcinoma. J Clin Endocrinol Metab. 2015;100:E493–E502. 21. Di Dalmazi G, Altieri B, Scholz C, et al. RNA sequencing and somatic mutation status of adrenocortical tumors: novel pathogenetic insights. J Clin Endocrinol Metab. 2020;105(12):e4459–e4473. 22. Faillot S, Foulonneau T, Néou M, et al. Genomic classification of benign adrenocortical lesions. Endocr Relat Cancer. 2021;28:79–95. 23. Heaton JH, Wood MA, Kim AC, et al. Progression to adrenocortical tumorigenesis in mice and humans through insulin-­like growth factor 2 and beta-­catenin. Am J Pathol. 2012;181:1017–1033. 24. Berthon A, Sahut-­Barnola I, Lambert-­Langlais S, et al. Constitutive beta-­catenin activation induces adrenal hyperplasia and promotes adrenal cancer development. Human Mol Genet. 2010;19(8):1561–1576. 25. Borges KS, Pignatti E, Leng S, et al. Wnt/β-­catenin activation cooperates with loss of p53 to cause adrenocortical carcinoma in mice. Oncogene. 2020;39:5282–5291. 26. Basham KJ, Rodriguez S, Turcu AF, et al. A ZNRF3-­dependent Wnt/β-­ catenin signaling gradient is required for adrenal homeostasis. Genes Dev. 2019;33:209–220. 27. Ruggiero C, Lalli E. Impact of ACTH signaling on transcriptional regulation of steroidogenic genes. Front Endocrinol (Lausanne). 2016;7:24. 28. Azevedo MF, Faucz FR, Bimpaki E, et al. Clinical and molecular genetics of the phosphodiesterases (PDEs). Endocr Rev. 2014;35:195–233. 29. Zennaro M-­C, Boulkroun S, Fernandes-­Rosa F. Genetic causes of functional adrenocortical adenomas. Endocr Rev. 2017;38:516–537. 30. Raymond VM, Else T, Everett JN, et al. Prevalence of germline TP53 mutations in a prospective series of unselected patients with adrenocortical carcinoma. J Clin Endocrinol Metab. 2013;98:E119–E125. 31. Ragazzon B, Libé R, Assié G, et al. Mass-­array screening of frequent mutations in cancers reveals RB1 alterations in aggressive adrenocortical carcinomas. Eur J Endocrinol. 2014;170:385–391. 32. Raymond VM, Everett JN, Furtado LV, et al. Adrenocortical carcinoma is a lynch syndrome-­associated cancer. J Clin Oncol. 2013;31:3012–3018. 33. Knijnenburg TA, Wang L, Zimmermann MT, et al. Genomic and molecular landscape of DNA damage repair deficiency across the Cancer Genome Atlas. Cell Rep. 2018;23:239–254.e6. 34. Pilati C, Shinde J, Alexandrov LB, et al. Mutational signature analysis identifies MUTYH deficiency in colorectal cancers and adrenocortical carcinomas. J Pathol. 2017;242:10–15. 35. Wilmot Roussel H, Vezzosi D, Rizk-­Rabin M, et al. Identification of gene expression profiles associated with cortisol secretion in adrenocortical adenomas. J Clin Endocrinol Metab. 2013;98:E1109–E1121. 36. de Reyniès A, Assié G, Rickman DS, et al. Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol. 2009;27:1108– 1115. 37. Barreau O, Assié G, Wilmot-­Roussel H, et al. Identification of a CpG island methylator phenotype in adrenocortical carcinomas. J Clin Endocrinol Metab. 2013;98:E174–E184. 38. Thorsson V, Gibbs DL, Brown SD, et al. The immune landscape of cancer. Immunity. 2019;51:411–412. 39. Raj N, Zheng Y, Kelly V, et al. PD-­1 blockade in advanced adrenocortical carcinoma. J Clin Oncol. 2020;38(1):71–80. 40. Corces MR, Granja JM, Shams S, et al. The chromatin accessibility landscape of primary human cancers. Science. 2018;362(6413):eaav1898. 41. Dahia PL, Ross KN, Wright ME, et al. A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas. PLoS Genet. 2005;1:72–80.

42. Castro-­Vega LJ, Letouzé E, Burnichon N, et al. Multi-­omics analysis defines core genomic alterations in pheochromocytomas and paragangliomas. Nat Commun. 2015;6:6044. 43. Kaelin WG. The von Hippel-­Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer. 2008;8:865–873. 44. Yang C, Hong CS, Prchal JT, et al. Somatic mosaicism of EPAS1 mutations in the syndrome of paraganglioma and somatostatinoma associated with polycythemia. Hum Genome Var. 2015;2:15053. 45. Letouzé E, Martinelli C, Loriot C, et al. SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer Cell. 2013;23:739–752. 46. Xiao M, Yang H, Xu W, et al. Inhibition of α-­KG-­dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors. Genes Dev. 2012;26:1326– 1338. 47. Dang L, White DW, Gross S, et al. Cancer-­associated IDH1 mutations produce 2-­hydroxyglutarate. Nature. 2009;462:739–744. 48. Gaal J, Burnichon N, Korpershoek E, et al. Isocitrate dehydrogenase mutations are rare in pheochromocytomas and paragangliomas. J Clin Endocrinol Metab. 2010;95:1274–1278. 49. Zhu J, Blenis J, Yuan J. Activation of PI3K/Akt and MAPK pathways regulates Myc-­mediated transcription by phosphorylating and promoting the degradation of Mad1. Proc Natl Acad Sci USA. 2008;105:6584–6589. 50. Burnichon N, Buffet A, Parfait B, et al. Somatic NF1 inactivation is a frequent event in sporadic pheochromocytoma. Hum Mol Genet. 2012;21:5397–5405. 51. Burnichon N, Vescovo L, Amar L, et al. Integrative genomic analysis reveals somatic mutations in pheochromocytoma and paraganglioma. Hum Mol Genet. 2011;20:3974–3985. 52. Burnichon N, Cascón A, Schiavi F, et al. MAX mutations cause hereditary and sporadic pheochromocytoma and paraganglioma. Clin Cancer Res. 2012;18:2828–2837. 53. Stenman A, Welander J, Gustavsson I, et al. HRAS mutation prevalence and associated expression patterns in pheochromocytoma. Genes Chromosomes Cancer. 2016;55:452–459. 54. Fishbein L, Khare S, Wubbenhorst B, et al. Whole-­exome sequencing identifies somatic ATRX mutations in pheochromocytomas and paragangliomas. Nat Commun. 2015;6:6140. 55. Garinet S, Néou M, de La Villéon B, et al. Calling chromosome alterations, DNA methylation statuses, and mutations in tumors by simple targeted next-­generation sequencing: a solution for transferring integrated pangenomic studies into routine practice? J Mol Diagn. 2017;19:776–787. 56. Mohan DR, Lerario AM, Else T, et al. Targeted assessment of G0S2 methylation identifies a rapidly recurrent, routinely fatal molecular subtype of adrenocortical carcinoma. Clin Cancer Res. 2019;25:3276–3288. 57. Gicquel C, Bertagna X, Gaston V, et al. Molecular markers and long-­term recurrences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res. 2001;61:6762–6767. 58. Lerario AM, Worden FP, Ramm CA, et al. The combination of insulin-­ like growth factor receptor 1 (IGF1R) antibody cixutumumab and mitotane as a first-­line therapy for patients with recurrent/metastatic adrenocortical carcinoma: a multi-­institutional NCI-­sponsored trial. Horm Cancer. 2014;5:232–239. 59. Fassnacht M, Berruti A, Baudin E, et al. Linsitinib (OSI-­906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-­blind, randomised, phase 3 study. Lancet Oncol. 2015;16:426–435.

96 Primary Aldosteronism Anand Vaidya, Maria-­Christina Zennaro, and Michael Stowasser

OUTLINE Pathophysiology, 1599 Pathogenesis And Genetics, 1599 Familial Forms of Primary Aldosteronism, 1600 Somatic Mutations in Unilateral Primary Aldosteronism, 1601 Somatic Mutations in Bilateral Adrenal Hyperplasia, 1602 New Diagnostic and Therapeutic Opportunities Associated with Genetic Knowledge, 1603

Prevalence, 1603 Clinical Diagnosis, 1603 Symptoms and Signs of Primary Aldosteronism, 1603 Assays Used in the Diagnostic Workup of Primary Aldosteronism, 1604 Diagnostic Workup for Primary Aldosteronism, 1604 Treatment, 1608

 

PATHOPHYSIOLOGY Primary aldosteronism is a syndrome of inappropriate and relatively nonsuppressible renin-­independent aldosterone production that persists despite expansion of effective arterial circulating volume and hypokalemia. This autonomous production of aldosterone results in excessive activation of the mineralocorticoid receptor (MR) in the principal cell in the collecting duct, thereby inducing increased epithelial sodium channel mediated sodium reabsorption and consequent volume expansion. To maintain electroneutrality, the reabsorption of sodium in the distal nephron is paired with an obligate excretion of either potassium or hydrogen ions. This pathophysiology begets a vicious cycle: increased distal nephron sodium reabsorption contributes to increases in effective arterial circulating volume expansion, in turn increasing blood pressure and glomerular filtration, thereby resulting in suppression of renin and angiotensin II, which begets further increases in distal nephron sodium delivery, hence further increasing distal nephron sodium reabsorption.1,2 The cardinal features of primary aldosteronism (PA) include a suppression of baseline renin secretion, an inability to stimulate renin normally (despite provocation by upright posture, volume contraction or sodium depletion, or angiotensin-­converting enzyme [ACE] inhibition), and inappropriate and nonsuppressible aldosterone production despite renin suppression.3,4 Importantly, hypertension and hypokalemia are not obligate characteristics of PA; rather, they are dependent features that occur depending on the degree of intraarterial volume expansion and distal nephron sodium delivery. The pathophysiology of PA extends beyond mechanisms in the kidney alone. The MR is expressed in the myocardium, vascular endothelium, smooth muscle, and other tissues. Animal experiments have elegantly demonstrated that, in contrast to the renin-­dependent aldosteronism that is vital for the physiologic response to volume depletion, activation of the cardiovascular MR in the context of volume expansion and/or sodium-­avid conditions results in myocardial fibrosis, necrosis, and clinical features of cardiovascular disease.5-­10 The molecular mechanism for this paradox remains unresolved: why is activation of the MR by aldosterone in volume/sodium-­depleted states

physiologic, whereas activation of the MR by aldosterone in volume/ sodium-­expanded states is pathophysiologic? However, observations from human studies have echoed these basic findings. Clinical trials in patients with heart failure with reduced ejection fraction (a state of increased left ventricular volume/pressure) have shown that MR antagonists dramatically reduce adverse clinical events and mortality.11,12 Observational studies have suggested that patients with PA, compared with patients with essential hypertension, have a higher risk for coronary artery disease, congestive heart failure, left ventricular hypertrophy, atrial fibrillation, stroke, diabetes mellitus, metabolic syndrome, decreased bone density, and kidney disease.2 Importantly, many of these observational studies demonstrated that the increased cardiometabolic risk associated with untreated PA, compared with essential hypertension, persists even when patients are matched for blood pressure and duration of hypertension.

PATHOGENESIS AND GENETICS Primary aldosteronism results from autonomous production of aldosterone in the adrenal cortex. Under normal circumstances, aldosterone biosynthesis in the adrenal cortex is tightly regulated by the renin-­angiotensin system and extracellular potassium concentrations to maintain blood volume, sodium, potassium and hydrogen homeostasis. The zona glomerulosa expresses the enzyme aldosterone synthase, encoded by CYP11B2, which catalyzes the three final enzymatic reactions leading to aldosterone biosynthesis.13 Due to the expression of a large number of potassium channels14 and the Na+/K+-­ATPase, unstimulated zona glomerulosa cells show a strongly negative membrane potential, which parallels the potassium equilibrium potential. Angiotensin II and increased plasma potassium lead to cell membrane depolarization, followed by opening of voltage-­gated calcium channels and increased intracellular calcium concentrations. Angiotensin II also acts by binding to its membrane G protein-coupled angiotensin II type 1 receptor that activates signaling via the inositol triphosphate pathway, increasing intracellular calcium levels from intracellular calcium stores. The increase in cytosolic calcium activates calcium signaling via calmodulin and calcium/calmodulin-­dependent protein kinases,

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PART 7  Adrenal

TABLE 96.1  Genetic Forms of Primary Aldosteronism Disease

Age of Onset

Specific Features

Gene

Transmission

Specific Treatment

Familial hyperaldosteronism type I

In most cases before age 20 yrs

Chimeric CYP11B1/B2

AD

Glucocorticoids, MRA

Familial hyperaldosteronism type II Familial hyperaldosteronism type III

Variable, mostly in childhood Before age 20 yrs Variable in mild forms

CLCN2

AD

MRA

KCNJ5

AD

Familial hyperaldosteronism type IV PASNA

Variable, often in childhood Childhood

CACNA1H

AD

MRA Bilateral adrenalectomy in severe cases MRA

CACNA1D

de novo mutations

Familial hyperaldosteronism: genetic basis undefined*

Variable

Increased risk of cerebrovascular events before age 30 yrs; aldosterone suppressible by dexamethasone; increased urinary levels of hybrid steroids 18-­oxocortisol and 18-­hydroxycortisol Usually mild, bilateral hyperaldosteronism Massive bilateral adrenal hyperplasia in severe cases Developmental disorder in some cases Seizures and neurological abnormalities None; diagnosis is based on two or more affected family members

To be determined*

Variable

Calcium channel blockers, MRA Unknown

*The definition familial hyperaldosteronism reflects the clinical diagnosis. Genetic analysis allows identifying patients with chimeric CYP11B1/B2, CLCN2, KCNJ5, or CACNA1H mutations and reclassifying them as FH-­I, -­II, -­II, or -­IV, respectively. Other genes may be involved in cases without identified gene mutation. PASNA, Primary Aldosteronism, Seizures and Neurologic Abnormalities; AD, autosomal dominant; MRA, mineralocorticoid receptor antagonists.

which is the major regulator of CYP11B2 expression and aldosterone production.15 In PA, regulatory mechanisms of aldosterone biosynthesis are bypassed by the chronic activation of intracellular calcium signaling resulting from different genetic defects. Different genetic abnormalities have been identified in familial forms of PA and in aldosterone-­producing adenoma (APA). While they occur as somatic mutations in APA, they are germline in familial cases and transmitted as an autosomal dominant trait. It is worth noting however, that somatic mutations also occur in the normal adrenal cortex in so-­called aldosterone-­producing cell clusters (APCCs) and in secondary aldosterone-­producing nodules from adrenals with APA.

Familial Forms of Primary Aldosteronism At least four different Mendelian forms of familial hyperaldosteronism (FH) have been reported (Table 96.1). They are defined as FH-­I to FH-­IV, depending on the underlying genetic defect. Familial forms occur in approximately 6% of patients with PA16 and are transmitted as an autosomal dominant trait. Although these forms are rare, genetic screening should be offered to patients with early-­onset PA before the age of 20 years and/or with a family history of PA, given the low cost and the benefits of early diagnosis in affected family members,17 as deleterious cardiovascular effects due to aldosterone excess may occur well before patients become hypertensive.18 FH-­I, also called glucocorticoid-­remediable aldosteronism (OMIM #103900), is characterized by early and severe hypertension, usually before 20 years of age, biochemical abnormalities of PA, excessive production of the hybrid steroids 18-­hydroxycortisol and 18-­oxocortisol, and bilateral hyperplasia, in rare cases associated with adrenal nodules; remarkably, symptoms are relieved by treatment with dexamethasone.19 FH-­I is due to the presence of a chimeric gene resulting from an unequal crossing over between CYP11B2 and the highly homologous CYP11B1 gene coding 11β-­hydroxylase, which is responsible for the last steps of cortisol biosynthesis in the adrenal zona fasciculata. The chimeric gene contains the regulatory regions of CYP11B1 juxtaposed to

CYP11B2-­specific coding sequences. Therefore, aldosterone synthase is expressed ectopically throughout the adrenal cortex under the control of adrenocorticotropic hormone (ACTH) rather than angiotensin II.20 Exogenous glucocorticoids are effective in reducing aldosterone synthase expression and aldosterone production. Low-­dose glucocorticoids have been shown to be sufficient to normalize blood pressure and plasma potassium levels without affecting hormonal parameters, and may provide prolonged control of hypertension.21 Mineralocorticoid antagonists or amiloride may be added in order to lower doses of glucocorticoids if required. In children, eplerenone is preferable, to avoid the side effects of glucocorticoids (retarded growth) or spironolactone (antiandrogen effects).22 The prevalence of FH-­I has been estimated to be approximately 0.5% to 1.0% of PA in the hypertensive adult population,16,23 but its frequency appears to be higher in hypertensive children, where a prevalence of 3% of the chimeric CYP11B1/CYP11B2 gene has been reported.24 Although FH-­I is a rare disease, it is associated with high morbidity and mortality before age 40 years from early-­ onset hemorrhagic stroke and ruptured intracranial aneurysms,25 criteria that may orient diagnosis in affected patients. Abnormalities in cardiac structure and function may appear before the rise in blood pressure in carriers of the chimeric gene.18 The genetic diagnosis is usually made by long-­range polymerase chain reaction (PCR) or Southern blot, while urinary levels of 18-­oxocortisol and 18-­hydroxycortisol and the dexamethasone suppression test do not show as high sensitivity and specificity.17 FH-­II (OMIM #605635), first described in 1991,26 was originally defined as PA occurring in two or more family members (i.e., FH) that is not glucocorticoid-­remediable and is not associated with the hybrid gene mutation. By that definition, it was reported to be the most frequent form of FH, accounting for approximately 1.2% to 6% of cases,16,27 with a presentation undistinguishable from that of sporadic PA. Patients showed heterogeneous clinical presentations with varying aldosterone response on postural test and to angiotensin II and different subtypes of PA even within the same family.28 Recently,

CHAPTER 96  Primary Aldosteronism germline mutations in CLCN2, coding for the chloride channel ClC-­ 2, have been identified in a large Australian family with FH-­II and in early-­onset PA.29,30 A recurrent germline CLCN2 mutation was found in the first reported FH-­II family, as well as in four additional unrelated patients,29 while other CLCN2 mutations were found in unrelated patients with early-­onset PA.29,30 This has led to the term “FH-­II” being restricted to PA caused by germline CLCN2 mutations. CLCN2 encodes a voltage-­ gated chloride channel composed of 18 transmembrane helices and an intracellular N-­terminus and C-­terminus. Mutations are mostly located in particular regions of the protein and induce a loss of the voltage gating of the channel. Thus, the channel is constitutively open at the zona glomerulosa resting potential, leading to sustained chloride efflux, followed by cell membrane depolarization, activation of calcium signaling, and increased expression of aldosterone synthase and autonomous aldosterone production.29,30 In addition to CLCN2 mutations, some patients previously diagnosed with FH-­II harbor mutations in genes identified in other familial forms and are therefore reclassified after genetic diagnosis. In particular, germline KCNJ5 mutations are found in patients with a moderate phenotype resembling FH-­II,31,32 as well as mutations in CACNA1H.33 Somatic mutations of KCNJ5 were also reported in APA from patients diagnosed with FH-­II according to the clinical definition.32 Hence, the phenotype of FH includes different subtypes of FH, which, in the absence of discriminating clinical features, require genetic testing for classification. In addition, there may be some cases in which FH results from familial aggregation of sporadic PA, given the high frequency of the condition in patients with hypertension. FH-­III (OMIM #613677) is a rare form of FH characterized in most patients by severe hypertension early in childhood that is refractory to treatment and associated with severe hypokalemia and massive bilateral adrenal hyperplasia.34 The hybrid steroids 18-­ oxocortisol and 18-­hydroxycortisol in urine are increased, but aldosterone production is not suppressed by dexamethasone.34 FH-­III is due to recurrent mutations of the KCNJ5 gene coding for the G protein-activated inward rectifier potassium channel GIRK4.35 Some heterogeneity in the clinical presentation, particularly in the severity of the disease, has been described, which appears to be associated with the type of mutation.31,32 Some patients present a particularly mild form of PA, diagnosed in young adulthood, with or without hypokalemia with no evident signs of hyperplasia at imaging. This form responds well to pharmacological treatment with MR antagonists and had often been previously diagnosed as FH-­II.31,32 Almost all KCNJ5 mutations are located within or near the selectivity filter of the GIRK4 potassium channel. They induce a change in ion selectivity, with loss of potassium selectivity and increased sodium conductance. This leads to increased sodium influx into the cell, followed by opening of voltage-­gated Ca2+ channels, activation of Ca2+ signaling, and stimulation of aldosterone biosynthesis.35,36 Pathological examination of the adrenal gland in two affected individuals after adrenalectomy has shown adrenal enlargement with loss of zonation and expression of aldosterone synthase throughout the adrenal cortex.37 Coexpression of 11β-­hydroxylase or 17α-­hydroxylase with aldosterone synthase in some adrenal areas explains the increased levels of hybrid steroids in these patients. In one patient carrying a KCNJ5 mutation, cosecretion of aldosterone and cortisol has been reported.38 A fourth form of FH, FH-­IV (OMIM #617027), has been discovered by whole-­exome sequencing of patients with early-­onset PA before the age of 10 years.33,39 A recurrent mutation in the CACNA1H gene was identified in five patients, with autosomal dominant transmission in four families and a de novo mutation in one case.39 In parallel, different CACNA1H mutations were reported in patients with early-­onset PA or with mild PA diagnosed as FH-­II.33 In some cases, CACNA1H mutations are associated with developmental disorders or attention deficit.33,39 In most cases, no adrenal abnormalities are observed on imaging,

1601

but in one case, the pathological examination of the resected adrenal has shown micronodular adrenal hyperplasia.39 CACNA1H encodes the pore-­forming α1 subunit of the T-­type voltage-­dependent calcium channel Cav3.2. The channel is composed of four repeat domains (I–IV), each consisting of six transmembrane segments (S1–S6), and mediates voltage-­dependent calcium influx into the cell upon membrane depolarization. Cav3.2 mutations affect highly conserved amino acids of the protein, modifying important channel properties. In particular, they shift the voltage dependence of the channels towards more negative membrane potentials, inducing channel opening at less depolarized membrane potentials, and/or delay the voltage-­dependent inactivation of the channel. The functional consequence is sustained calcium influx into the cell, activating calcium signaling and aldosterone biosynthesis.33,39 A particular form of PA that is not inherited, but rather due to germline mutations affecting another calcium channel, is PASNA (Primary Aldosteronism, Seizures, and Neurologic Abnormalities; OMIM #615474). In this condition, mutations affect the CACNA1D gene, coding for the voltage-­dependent L-­type calcium channel subunit alpha-­1D.40 Those patients show early onset PA in the context of a complex neurological disorder, including cerebral palsy and epileptic seizures. Patients have severe hypertension and hypokalemia, without adrenal hyperplasia on imaging, and carry de novo germline mutations of the CACNA1D gene. The disease has been described in only four patients so far, in one case associated with congenital hyperinsulinism, and its prevalence is currently unknown.40-­42 The pathogenic mechanism whereby CACNA1D mutations lead to PA is similar to FH-­IV: the mutations affect the channel properties, leading to opening of the channel at less depolarized membrane potentials and activation of calcium signaling and aldosterone biosynthesis.

Somatic Mutations in Unilateral Primary Aldosteronism The majority of cases of PA occur sporadically and are due to a unilateral APA or bilateral adrenal hyperaplasia. From a clinical point of view, the distinction is made based on unilateral versus bilateral aldosterone secretion defined by adrenal venous sampling (AVS). From a pathological point of view, it has become apparent that unilateral forms encompass a large variety of morphological alterations, ranging from an isolated APA to multinodular adrenals. Recurrent somatic mutations are found in a majority of APAs, as well as aldosterone-­producing structures in adrenal glands from patients with PA.43-­46 They affect genes involved in familial forms of the disease (KCNJ5, CACNA1H, CLCN2), as well as genes coding for ATPases (ATP1A1, coding for the α1 subunit of the Na+,K+-­ATPase, and ATP2B3, coding for the plasma membrane calcium-­transporting ATPase 3 [PMCA3]) and CACNA1D, coding for the α1 subunit of the L-­type Ca2+ channel Cav1.3. In certain cases, somatic mutations have been identified in genes mainly involved in adrenal disorders associated with cortisol excess. Indeed, 2% to 5% of APAs carry somatic mutations in CTNNB1, coding for β-­catenin.47-­49 Somatic mutations affecting PRKACA (coding for catalytic alpha subunit of the cAMP-­dependent protein kinase) have been described in rare cases as well, and may be associated with cortisol excess.50 Somatic mutations affect specific conserved amino acids of the proteins and lead to major functional modifications. They can be classified in two major categories: those that lead to cell membrane depolarization by altering intracellular ion homeostasis (KCNJ5, ATP1A1, CLCN2), and those that directly increase intracellular calcium levels by affecting calcium channels and pumps (ATP2B3, CACNA1D, CACNA1H) (Fig. 96.1). As described for FH-­III, the most prevalent mutations in KCNJ5 lie within or near the selectivity filter of GIRK4 and modify the ion selectivity of the channel, rendering it permeable to sodium.35,36 Mutations in ATP1A1 decrease the activity of the sodium potassium ATPase and reduce the affinity for K+,51 while mutations in .

l

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AngII and K+ stimulate aldosterone production through cell membrane depolarization and increased intracellular calcium signaling Response to K+ or AngII stimulation + − +

K+ −

+

AngII +

−

−

K+

Ca

3Na+

2+

−

Na+ −

Mutated CIC-2

+

−

+

−

−

− (Cl−)I ↓ Cl

Depolarization

K+

−

(Na+)I ↑

Ca2+

(H+)I ↑ Mutated + + Na /K ATPase

(Ca2+)I↑ CYP11B2 2H+ Aldosterone ↑

−

+ +

+

+

Mutated GIRK channel

+ −

Depolarization

2K+ +

Mutations in driver genes lead to cell membrane depolarization (KCNJ5, ATP1A1, CLCN2) or directly increase intracellular calcium concentrations (ATP2B3, CACNA1D, CACNA1H)

Mutated Cav1.3 or Cav3.2

Ca2+ (Ca2+)I ↑

pH ↓ CYP11B2

Ca2+

+

2H+ Aldosterone ↑

Ca2+ Mutated PMCA3

Fig. 96.1  Different mechanisms lead to APA formation. Following increase of extracellular potassium or AngII binding to its AT1R receptor, inhibition of potassium channels leads to cell membrane depolarization and opening of voltage gated calcium channels. Increased intracellular calcium concentrations lead to activation of calcium signaling, which induces aldosterone biosynthesis. Binding of AngII to AT1R also inhibits Na+/ K+-ATPase, inducing cell membrane depolarization, while AT1R signaling through Gαq promotes the release of calcium from intracellular calcium stores (left panel). Mutations in KCNJ5, coding for the potassium channel GIRK4, change the channel’s ion selectivity, promoting sodium entry into the cell instead of potassium efflux, leading to cell membrane depolarization and opening of voltage gated calcium channels. Mutations in ATP1A1, coding for the α1 subunit of the Na+/K+-ATPase, lead to increased aldosterone production by increasing intracellular Na+ and H+ concentrations, resulting in cell membrane depolarization and decrease of intracellular pH. Mutations in CLCN2, coding for the chloride channel ClC-2, lead to increased chloride efflux from the cell, similarly leading to cell membrane depolarization. Mutations in CACNA1D, encoding the Cav1.3 calcium channel, CACNA1H, encoding the Cav3.2 calcium channel, and ATP2B3, coding for the Ca2+ pump PMCA3 directly affect intracellular calcium concentrations and thus aldosterone biosynthesis (right panel). Ca2+, calcium; K+, potassium; Na+, sodium; Cl-, chloride. (Modified from Zennaro, et al. Endocr Rev 38, 516-537 (2017).)

CLCN2 act by increasing chloride efflux as in FH-­II.52 On the other hand, ATP2B3 mutations decrease pump activity of the PMCA3, impairing calcium export. In addition, ATP2B3 mutations may also induce a sodium leak, inducing cell membrane depolarization as well as a calcium leak through mutated pumps.53 CACNA1D and CACNA1H mutations have similar functional consequences. They shift the voltage dependency of the mutated calcium channels towards more negative potentials, therefore leading to the channels opening at less depolarized potentials upon stimulation, or delay their voltage-­dependent inactivation.33,40,54 In fine, both these mechanisms increase intracellular calcium concentrations and lead to activation of calcium signaling, activating molecular cascades triggering increased expression of aldosterone synthase and autonomous aldosterone production. The most frequent somatic mutations found in APA are KCNJ5 mutations. They account for around 40% of adenomas,55,56 with higher frequencies in patients from East Asia.56 These figures were not changed by the introduction of CYP11B2 immunohistochemistry­guided next-­generation sequencing.43-­46 In contrast, the prevalence of somatic mutations in other genes increased significantly following next-­generation sequencing, which allows extended coverage of genes. CACNA1D mutations are now identified in 14% to 42% of APAs,

ATP1A1 mutations in 5% to 17% of APAs, and ATP2B3 mutations in 4% to 10% of patients, depending on the population.43-­46 CACNA1H and CLCN2 mutations are found in rare cases.52,57

Somatic Mutations in Bilateral Adrenal Hyperplasia Idiopathic hyperaldosteronism, or bilateral adrenal hyperplasia, is diagnosed on the basis of bilateral secretion of aldosterone at AVS, and treatment with MR antagonists is the recommended therapeutic approach. A recent study had the opportunity to investigate 15 adrenals from patients with idiopathic hyperaldosteronism who had undergone adrenalectomy for exceptional reasons, including difficult-­to-­control hypertension or hypokalemia and unwillingness for long-­term medical treatment. An increased number of so-­ called APCCs of the adrenal cortex was identified, which carried somatic mutations in CACNA1D, as well as a KCNJ5 mutation in a micronodule.58 APCCs are clusters of cells located in the zona glomerulosa, express CYP11B2, and are composed of an outer part of compact cells expressing disabled-­2, a marker of the zona glomerulosa, and an inner part of larger cells not expressing disabled-­2, suggesting that they may be intermediate structures between the zona glomerulosa and the zona fasciculata.59,60,70,72 In normal adrenals, the number of APCCs increases with age and progressively replaces

CHAPTER 96  Primary Aldosteronism the continuous zona glomerulosa observed in younger individuals; this is associated with an age-­related dysregulation of aldosterone production.61 The peculiar feature of APCCs is that they carry somatic mutations even in normal adrenal glands, and therefore have been suggested to represent a possible precursor of APA.60 Whether or not an accumulation of APCCs carrying somatic mutations underlies the development of idiopathic hyperaldosteronism in general, or rather is a contributor to autonomous aldosterone production in hyperplastic glands, requires further studies. Indeed, APCCs are also present in adrenals with APA and may carry different somatic mutations in the same adrenal gland.45

New Diagnostic and Therapeutic Opportunities Associated with Genetic Knowledge One important question is whether genetic knowledge may improve diagnosis and treatment of patients with PA. There is no doubt that genetic diagnosis in familial forms allows for earlier detection of affected family members and better prevention of cardiovascular complications that may occur before hypertension onset.18 On the other hand, identification of somatic mutations in resected adrenal glands has not provided benefit to patients so far. Identification of surrogate biomarkers of the presence of somatic mutations may eventually allow simplification of the diagnostic procedure by identifying patients likely to carry a unilateral APA to be selected for AVS. Some attempts have indicated that steroid profiling in peripheral vein samples may provide good information on the presence of somatic mutations. Indeed, levels of the hybrid steroids 18-­oxocortisol and 18-­hydroxycortisol are significantly higher in patients with KCNJ5 mutations compared with other mutational groups,62,63,45 and steroid fingerprints have been applied for distinguishing different genetic groups.62 Similarly, the pharmacological properties of mutated GIRK4 channels are currently used to test the hypothesis that macrolides may be used for diagnostic purposes.64 These approaches may be used together with peculiar imaging characteristics of APA carrying KCNJ5 mutations on computed tomography, which are characterized by larger tumor size and lower precontrast Hounsfield units,48 as well as the high prevalence in young patients and in women,55,56 to elaborate a decision tree guiding diagnostic assessment. It is therefore the purpose of ongoing genetic studies not only to understand the development, but also to allow for personalized approaches to diagnosis and management of patients with PA. KEY POINTS  • Mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H, CLCN2) and ATPases (ATP1A1, ATP2B3) are found in familial hyperaldosteronism and in aldosterone-­producing adenoma. They lead to cell membrane depolarization, followed by opening of voltage-­gated calcium channels, or directly increase intracellular calcium concentrations, with activation of calcium signaling leading to increased aldosterone biosynthesis.

PREVALENCE Iterative population-­based and convenience sample-based studies have shown that the prevalence of PA is high and largely underestimated. The challenge in establishing accurate prevalence estimates has been the lack of a histopathologic or other gold-­standard reference diagnostic. Rather, the diagnosis of PA is made using a variety of arbitrary screening and confirmatory diagnostic thresholds; variations in these diagnostic thresholds can easily modulate prevalence estimates. Population-­based studies that used the aldosterone-­to-­renin ratio (ARR) have suggested that 7% to 12% of the hypertensive population may have PA,65,66 and that higher aldosterone levels in the context of

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suppressed renin activity were associated with higher risk for incident coronary artery calcification and all-­cause mortality.67 Many international studies have used a two-­tiered approach to estimate prevalence, wherein the ARR is first used to identify a “positive” screen, which is then confirmed using a variety of physiologic or dynamic confirmatory tests designed to evaluate aldosterone suppressibility.68-­76 Collectively, these studies have shown that overt PA can be detected in 2% to 19% of hypertensive individuals, with the prevalence paralleling the severity of hypertension. In contrast, some studies have estimated prevalence by conducting dynamic aldosterone suppression tests in hypertensive individuals without antecedent ARR screening and have reported much higher prevalence estimates, presumably because many ARRs are considered to be falsely “negative” owing to the variability of aldosterone production and the arbitrary nature of screening thresholds.77 For example, 28% to 30% of participants with hypertension were confirmed to have PA when undergoing direct dexamethasone-­fludrocortisone suppression testing,78 29% of patients with resistant hypertension were confirmed to have PA when undergoing direct saline suppression testing,79 and 24% of patients with resistant hypertension were confirmed to have PA when undergoing direct oral sodium suppression testing.80 Beyond these crude prevalence estimates born out of reliance on arbitrary thresholds for diagnosis, studies have shown a continuum of renin-­independent aldosterone production in hypertension that is associated with the severity of hypertension and kaliuresis,80,81; thus, suggesting that the prevalence of PA captured using binary diagnostics likely underestimates the true burden of pathogenic aldosteronism. The clinical relevance of this high prevalence of PA and the continuum of renin-­independent aldosterone is the association of this biochemical phenotype with incident cardiometabolic disease that can be mitigated by MR antagonists82-­84 or, in patients with unilateral PA, unilateral adrenalectomy. Intervention studies have consistently shown that, the greater the magnitude of the renin-­independent aldosterone production, the greater the efficacy of MR antagonists in lowering blood pressure. Finally, although clinical indications for PA screening generally focus on individuals with hypertension, accruing evidence indicates that a PA phenotype can be detected even among those with normal blood pressure (sometimes referred to as subclinical or nonclassical PA85). Multiple studies that have used dynamic confirmatory testing have observed that approximately 9% to 14% of normotensive participants have nonsuppressible aldosterone production that meets conventional definitions for diagnosing PA.80,86-­90 In parallel with these observations, population-­based studies have shown that this biochemical phenotype of renin-­independent aldosterone production in normotensive people is associated with an increased risk for developing incident hypertension,65,91-­93 thereby suggesting a pathologic continuum of PA that may have its origins in normotension and may contribute to the pathology of hypertension.

CLINICAL DIAGNOSIS Symptoms and Signs of Primary Aldosteronism The great majority of patients with PA are hypertensive. Within some families with inheritable forms, blood pressure levels in members testing positive for the causative mutation and demonstrating biochemical evidence of PA have varied from normal to severely elevated.94,95 Occasional normotensive patients with apparently nonfamilial PA, usually heralded by hypokalemia rather than hypertension, have also been described. Hypokalemia is now known to present in a minority (approximately 20%–25%) of patients, and hence its absence does not exclude PA.96,97 When it is present, associated symptoms may include muscle weakness, cramping, tetany, paresthesias, palpitations, polyuria, and nocturia. Nocturia often occurs l

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PART 7  Adrenal

even when plasma potassium is normal and may reflect the rise in atrial natriuretic peptide levels that has been associated with PA or low intracellular renal levels of potassium resulting from increased release in an attempt to maintain normokalemia. Other common symptoms in patients with PA include lethargy, impaired mental concentration, and mood disturbances.98-­101 Hypertension and symptoms may be masked during pregnancy102,103 due to the high circulating levels of placental progesterone (which is an MR antagonist). In other women, development of PA due to APA during pregnancy has been linked to the presence of somatic activating mutations in CTNNB1 that encodes β-­catenin in the Wnt cell differentiation pathway (possibly explaining tumor development) and increased expression of LHCGR (encoding lutotropin-­ chorionogonadotropic hormone receptor) and GNRHR (encoding gonadotropin-­releasing hormone receptor), suggesting that high levels of human chorionic hormone during pregnancy may result in stimulation of aldosterone production by these APAs via aberrant receptor activation.47 Once thought not to occur in PA, malignant hypertension has been reported by several investigators.104,105 The intrarenal ischemia that occurs in this may lead to unsuppression of renin, which could result in the diagnosis of PA being missed.

Assays Used in the Diagnostic Workup of Primary Aldosteronism Highly reproducible assays are essential for all stages of the diagnostic workup of PA, including screening, confirmatory suppression testing, and AVS.

Aldosterone Assays. Accurate and reproducible measurement of plasma or serum aldosterone is a challenge for clinical laboratories, because it circulates at relatively low (picomolar) concentrations. Although radioimmunassays were until recently the most widely used method of measuring aldosterone, these have now been replaced in many laboratories by faster and less labor-­intensive automated platforms using immunometric assay technology. Concerns have been raised regarding the accuracy of immunoassays in general and the need for high-­ affinity, highly specific antibodies and meticulous laboratory technique. Lack of specificity probably explains why newer, highly accurate and reproducible methods of measuring aldosterone within the clinically relevant range using high-­performance liquid chromatography and tandem mass spectrometry (LC-­MS/MS) generate aldosterone levels that are substantially lower than those measured by immunoassays within the same plasma samples,106,107 necessitating adjustment of diagnostic thresholds such as the ARR and confirmatory suppression testing.108,109 These methods, previously laborious and time-­ consuming, now employ semiautomated technology, which allows high sample throughput and faster generation of assay results, making them suitable for the clinical setting. While running costs are relatively low, the initial cost outlay associated with purchasing the equipment represents a barrier to wide application of LC-­MS/MS, a situation that may change with time. Renin Assays. The most commonly utilized renin assay methods are the measurement of plasma renin activity (PRA) or of direct (or active) renin concentration (DRC). Each has its advantages and disadvantages. PRA measures (usually by radioimmunoassay, but in some laboratories by LC-­MS/MS) the amount of angiotensin I generated by the action of renin on its substrate, angiotensinogen, in a defined volume of plasma per unit of time, and hence is a measure of renin enzyme activity. For samples with levels less than 1 ng/mL/min, reproducibility of the assay can be maintained by extending the incubation time from the recommended 90 minutes to 18 hours.110

The principal advantage of DRC, which measures the plasma concentration of the active (cleaved) form of renin, is that it can be undertaken by automated immunometric assay,111 which is much faster and less labor-­intensive than PRA. Some laboratories offer combined aldosterone and renin measurement on the same sample using automated immunometric technology. However, concern has been raised over the lack of reliability and reproducibility of these assays at the lower end of the human reference range, which is especially pertinent for the diagnostic workup of PA, where renin levels are suppressed. In this situation, small changes in renin can result in large changes of the ARR,112 and it could therefore be argued that it is more important to measure renin accurately than aldosterone when screening for PA. A further concern for DRC measurement is that it may not reflect the true activity of the renin-­angiotensin system as well as PRA. Renin does not affect blood pressure or aldosterone synthesis directly, but only through angiotensin II, concentrations of which may be better reflected by PRA. For example, with administration of exogenous estrogen (in oral contraceptive or hormone replacement preparations), the concentration of angiotensinogen (substrate) increases, and DRC falls to maintain angiotensin II concentrations in the normal range, whereas PRA remains relatively unaffected.113,114 On the other hand, advocates for automated immunometric renin assays have reported that they appear to have better interlaboratory agreement.115 A potential alternative to the measurement of renin in the diagnostic workup of PA is recently reported mass spectrometric methods of assaying angiotensin I (generated from angiotensinogen as per the PRA approach) or, more promising still, angiotensin II (measured during a period of equilibrium of formation versus metabolism). Unlike renin, angiotensin II directly regulates aldosterone synthesis via AT1 receptors on adrenal zona glomerulosa cells, and its measurement is therefore arguably more physiologically relevant in clinically assessing patients for the presence of angiotensin II-independent aldosterone overproduction.116

Diagnostic Workup for Primary Aldosteronism This is divided into three main phases: screening, confirmatory testing, and determining the subtype of PA. All require expertise for optimal performance and interpretation of results and are the subject of considerable ongoing research efforts towards refinement.

Screening. Screening of at-­risk populations for PA is primarily geared towards identifying those patients who do not have this condition, and therefore are not required to undergo confirmatory testing and subtype differentiation (both of which involve procedures that are relatively invasive and expensive). It is therefore important that the screening test is highly sensitive, while retaining a reasonable degree of specificity. Most patients with hypertension, and especially those with moderate to severe forms, should be regarded as candidates for screening. In addition, screening is recommended for patients with hypertension and spontaneous or diuretic induced hypokalemia, adrenal incidentaloma, sleep apnea, or a family history of early-­onset hypertension or cerebrovascular accident at a young age (140 to >280 pmol/L).125,126 A major advantage of this approach over FST is that it can be performed as an outpatient procedure. However, concerns have been raised over its lack of sensitivity, especially in patients in whom plasma aldosterone demonstrates responsiveness to the assumption of upright posture, as their recumbent aldosterone levels are lower than those in posture-­unresponsive patients.127 A recently reported approach has been to perform SST in the seated position, resulting in substantially improved sensitivity for detecting PA while retaining high specificity.128 By this method, diagnostic cutoffs for normal

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TABLE 96.2  Factors Causing False-­Positive Aldosterone-­to-­Renin Ratios Effect on Aldosterone

Effect on Renin

Mechanism

β-­blockers

↓

↓↓

Blockade of JG cell beta-­adrenergic receptors

Clonidine α-­methyldopa NSAIDs

↓

↓↓

Suppression of CNS sympathetic output

↔

↓↓

Estrogen-­and progestogen-­ containing agents*

↑

↔

Premenopausal women in luteal ↑ menstrual phase*

↔

Tendency to Na+ retention and suppression of renal prostaglandins (which normally enhance release of renin) leads to renin suppression Tendency to K+ retention prevents fall in aldosterone Progestogenic component induces natriuresis, which stimulates renin and aldosterone Estrogenic component stimulates renin substrate (angiotensinogen) and thus angiotensin production, resulting in greater negative feedback on renin (and hence DRC), thereby opposing the stimulatory effects of the progestogenic component Progesterone induces natriuresis which stimulates renin and aldosterone Estrogen stimulates renin substrate and thus angiotensin production, resulting in greater negative feedback on renin Reducing nephron (and JG cell) number and tendency to Na+ retention leads to renin suppression Tendency to K+ retention prevents fall in aldosterone Reducing nephron number and tendency to Na+ retention leads to renin suppression Tendency to K+ retention prevents fall in aldosterone Suppression of renin (to a greater degree than aldosterone) Mutations in WNK1, WNK4, CUL3 or KLH3L cause upregulation of NCC. Resulting Na+ retention leads to renin suppression and K+ retention prevents fall in aldosterone

Advancing age

↔

↓↓

Chronic kidney disease

↔

↓↓

High dietary Na+ Familial hyperkalemic hypertension

↓ ↔

↓↓ ↓↓

*False positives only occur if renin is measured as direct renin concentration (and not plasma renin activity). JG, Juxtaglomerular; CNS, central nervous system; NSAIDs, nonsteroidal antiinflammatory drugs; NCC, sodium chloride cotransporter; DRC, direct renin concentration.

TABLE 96.3  Factors Causing False-­Negative Aldosterone-­To-­Renin Ratios Effect on Aldosterone

Effect on Renin

Mechanism

Diuretics (K+ wasting or sparing)

↑↔

↑↑

ACE inhibitors

↓

↑↑

ARBs

↓

↑↑

Dihydropyridine CCBs

↔↑

↑

SSRIs

↑

↑↑

Pregnancy Low dietary Na+ Vomiting and diarrhea Malignant hypertension Concomitant renovascular hypertension Hypokalemia

↑ ↑

↑↑ ↑↑

↑

↑↑

Volume contraction and sympathetic nervous system stimulation stimulate renin Fall in plasma K+ with K+ wasting diuretics lowers aldosterone Reduced production of angiotensin II relieves negative feedback on JG cells, leading to marked increase in renin while also reducing angiotensin II stimulation of aldosterone production by ZG cells Blockade of angiotensin II receptors relieves negative feedback on JG cells, leading to marked increase in renin while also reducing angiotensin II stimulation of aldosterone production by ZG cells Reflex sympathetic stimulation as BP falls, natriuretic effects, and direct stimulation of calcium-­dependent renin regulatory pathways Interference with aldosterone biosynthesis by blocking intracellular calcium-­dependent steps Uncertain––serotonin may stimulate renin via the 5-­HT2 subtype of 5-­HT receptors Progesterone induces natriuresis, which stimulates renin and aldosterone Volume contraction and sympathetic nervous system stimulation stimulate renin Intrarenal ischemia leads to renin production

↓

↔↑

Reduced stimulation of aldosterone production

ACE, Angiotensin-­converting enzyme; ARB, angiotensin II receptor blocker; ZG, zona glomerulosa; JG, juxtaglomerular; CCB, calcium channel blocker; SSRI, selective serotonin receptor inhibitor; BP, blood pressure.

CHAPTER 96  Primary Aldosteronism suppression of plasma aldosterone were reported to be less than 162 pmol/L if measured by mass spectroscopy128 and less than 171 pmol/L (but with a grey zone up to 217 pmol/L) if measured by chemiluminescence immunoassay.109 Unlike the above approaches, the captopril challenge test does not involve salt loading, but instead measures aldosterone and renin responses to 25 to 50 mg oral captopril, an ACE inhibitor, administered after sitting or standing for at least 1 h.129 Blood samples are drawn basally and at 1 or 2 h after challenge, with the patient remaining seated throughout. In subjects without PA, plasma aldosterone is suppressed (>30%) and PRA stimulated by captopril, and, as a result, the ARR falls. In patients with PA aldosterone fails to suppress, PRA remains suppressed, and the ARR remains elevated. In one commonly used protocol, the cutoff for normal suppression of plasma aldosterone is less than 8.5 ng/dL (240 pmol/L) and for the ARR is less than 30 (with aldosterone expressed as ng/dL and PRA as ng/mL/h) 2 h after receiving 50 mg of captopril. Because it does not involve salt loading, the captopril challenge testing avoids the potential for inducing fluid overload in patients at risk because of compromised cardiac or renal function and hypokalemia. However, it has been reported to have poor discriminatory power130,131 and may be associated with symptomatic hypotension due to the blood pressure-lowering effect of captopril in upright patients. Because of the enhanced risk of potentially dangerous fluid overload, it may be necessary to avoid confirmatory testing involving salt loading in patients with severe hypertension or compromised cardiac status (e.g., reduced left ventricular systolic function or marked left ventricular hypertrophy) or renal dysfunction. Alternatively, consideration may be given to either a period of medical treatment with an agent that blocks aldosterone action as an attempt to establish better hypertension control and improve cardiac function in order to reduce the risk of confirmatory testing to a more acceptable level, or to proceeding directly to subtype differentiation (including AVS). Confirmatory testing (followed, if positive by AVS) could also be avoided in favor of an empiric trial of medical treatment for PA in patients for whom unilateral adrenalectomy is not likely to be a viable treatment option, either because they are poor candidates for surgery or do not desire surgical intervention.

Subtype Differentiation. As described earlier in this chapter, the availability of immunohistochemical staining using antibodies directed against CYP11B2 has enabled investigators to identify the true source of aldosterone excess within adrenals removed from patients with PA, and this in turn has led to the realization that the morphology associated with PA is diverse and far more complex than previously suspected. From a clinical standpoint, however, rather than attempting to distinguish the many pathological variants from each other, subtype differentiation aims to assist selection of the optimal management approach. This is achieved by: -­ genetic testing when a familial form of PA is suspected; -­ imaging to enable detection of large adrenal lesions that may warrant consideration of removal because of their malignant potential; and -­ determining whether autonomous aldosterone production is unilateral (confined to one adrenal), and therefore potentially curable by unilateral adrenalectomy, or bilateral, in which case management usually involves treatment with medications that antagonize aldosterone action. Genetic testing for FH-­I involves the use of either Southern blotting or a faster and less expensive PCR-­based method to detect the causative hybrid CYP11B1/CYP11B2 mutation within peripheral blood DNA.132,133 The principal reasons for identifying individuals with this

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subtype of PA are that (1) patients with FH-­I demonstrate excellent clinical responses to glucocorticoid medications given in small doses that do not cause Cushingoid side effects, making them the treatment of choice for this (but no other) subtype of PA; and (2) it heralds the need for genetic testing of other family members to detect additional individuals with FH-­I similarly at risk of hypertension and early death from hemorrhagic stroke, but who will also benefit greatly from specific glucocorticoid treatment. The recommendation by the Endocrine Society is to test patients with onset of confirmed PA earlier than at 20 years of age and those who have a family history of PA or of strokes at young age (2.5 cm) mass lesions that have greater potential for malignant behavior and may warrant consideration for removal on that basis alone, or at the very least deserve careful follow-­up. While the above facets of subtype differentiation are relatively straightforward, distinguishing unilateral from bilateral PA is much more complex. It is nevertheless an important aspect of the diagnostic workup of PA, as the clinical responses to unilateral adrenalectomy in patients with unilateral forms of PA are generally quite marked, with cure of hypertension achieved in 40% to 70%, and greater reductions in pill burden, better cardiovascular outcomes, and greater improvements in quality of life compared with patients with PA treated medically with drugs that antagonize aldosterone action.99,101,134,135 For several decades, AVS has served as the mainstay of differentiating these two PA subtypes, as it is the most reliable. It is also the most invasive and expensive and requires considerable expertise. As a result, many alternative approaches have been explored, but with inefficient reliability to replace AVS. Although unilateral PA (including APA) is generally associated with more florid features of PA than bilateral, there is considerable overlap, and clinical prediction algorithms that have incorporated such parameters as plasma sodium and potassium, total carbon dioxide, aldosterone, renin, and the ARR have shown variable (and not very reproducible) performance characteristics.136 Adrenal imaging by CT or MRI frequently misses APAs and can be frankly misleading by detecting nonfunctioning adrenal lesions.97,137 Scintigraphy lacks sufficient resolution to detect smaller APAs. Posture stimulation studies (comparing recumbent with upright plasma aldosterone concentrations) more often showed posture unresponsiveness and plasma or urinary levels of “hybrid steroids” (18-­hydroxy-­and 18-­oxocortisol) are more often elevated in patients with APA compared with bilateral PA,138 but again, considerable overlap exists. The diversity in phenotype among patients with APA regarding these two parameters may in part be genetically determined: patients with APAs associated with somatic KCNJ5 mutations usually show posture-­ unresponsiveness and elevated hybrid steroid levels, whereas the reverse holds true for those with somatic CACNA1D (and other) mutations.139 Despite the shortcomings of these alternatives to AVS, they can nevertheless provide useful ancillary information that may help in the decision-­making process, especially when AVS has been declined for some reason or results have been inconclusive (e.g., because of failure to cannulate one

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or both adrenal veins). For example, the presence of an obvious unilateral mass lesion on CT in a young patients with florid, hypokalemic, posture-­unresponsive PA and elevated hybrid steroid levels (if available) would make APA highly likely. Nuclear imaging using positron emission tomography–CT with (11)C-­metomidate has been reported to be a viable alternative or adjunct to AVS in difficult cases.140,141 However, the short half-­life and lack of specificity of the isotope for CYP11B2 (being a ligand also for CYP11B1) limits the current widespread application and reliability of this technique, though alternatives are being investigated. The Endocrine Society guideline22 recommends that AVS be offered to all patients with PA who desire adrenalectomy and for whom surgery is not contraindicated, regardless of biochemical or CT findings. Some investigators advocate performing AVS in the morning following overnight recumbency in order to avoid the confounding effects of changes in posture on aldosterone concentrations and to capitalize on the higher endogenous ACTH levels (compared with later in the day), which ensure that samples are collected during maximal ACTH-­ induced stimulation of aldosterone production (unless stimulation with exogenous ACTH is employed).124 The likelihood of achieving successful cannulation of the adrenal veins is maximized when CT scanning is performed prior to AVS to assist in localizing the adrenal veins,142 a radiologist highly skilled in AVS performs the procedure, at least two samples are collected from each side, and rapid point-­of care cortisol testing is used to confirm correct catheter placement. Examination of the ratio or “gradient” between adrenal and peripheral venous cortisol concentrations permits confirmation (or otherwise) of cannulation success, with gradients of at least two or three (or at least five if exogenous ACTH stimulation is used) most commonly used to indicate adequacy of AVS. The right adrenal vein tends to be harder to cannulate than the left because it is usually smaller and shorter and, unlike the left adrenal vein, usually empties into the inferior vena cava rather than the renal vein.143 Calculating the aldosterone/cortisol ratio for each adrenal and peripheral venous sample corrects for differences in dilution with nonadrenal venous blood. Criteria for lateralization vary widely from one institution to another,144 with some comparing adrenal venous aldosterone/cortisol ratios on each side with the simultaneous peripheral venous ratio (with lateralization defined as a the ratio being at least 2.5 times peripheral on the affected side and no higher than peripheral on the other side), while others rely on the comparison of aldosterone/cortisol ratios on one side versus the other (the so-­called “lateralization index”), with lateralization defined as the ratio on the higher side being at least two to four times, depending on the center, that on the lower side. Several groups employ exogenous ACTH stimulation during AVS to (1) maximize adrenal/peripheral venous cortisol gradients, (2) reduce fluctuations in steroid secretion during AVS and (3) stimulate aldosterone production by APAs, and thus avoid sampling during a period of secretion “quiescence.”144,145 The risk of adrenal hemorrhage associated with AVS is low (less than 2% of all procedures) in experienced hands.142 Recent advances and changes in thinking regarding AVS have included: (1) “superselective” sampling of branch adrenal veins, enabling more precise localization of the source (or sources) of aldosterone excess within a gland, which is particularly relevant in situations where subtotal adrenalectomy is considered desirable (for example, if the patient has only one functioning adrenal or is thought to have bilateral APAs)146; (2) the development of point-­of-­care plasma cortisol measurement, which permits semiquantitative determination of adrenal venous cortisol levels, and thus an assessment of the success or otherwise of cannulation within minutes of collection147; and (3) normalization of aldosterone levels with hormones other than cortisol, either to improve apparent rates of successful cannulation or where the

patient is suspected of having an adrenal tumor that is concomitantly autonomously secreting both cortisol and aldosterone.107,148 KEY POINTS  • Detection and diagnostic workup for primary aldosteronism involves screening by measurement of the plasma aldosterone/renin ratio, confirmatory suppression testing, and determining the subtype by genetic testing (when familial forms are suspected), adrenal imaging, and adrenal venous sampling to differentiate unilateral from bilateral forms.

TREATMENT Early recognition of PA is paramount to facilitate the opportunity to initiate targeted treatments. The objective of targeted treatments is to reduce the risk for incident cardiovascular and kidney disease. The traditional dogma for decades has been to prescribe surgical adrenalectomy or lifelong MR antagonist therapy, depending on the subtype of PA. It is important to note that there have been no robust, randomized, and controlled trials to evaluate the validity of this dogma or the comparative efficacy of these options. For example, whether MR antagonists are superior to conventional (nontargeted) antihypertensive medications, or whether surgical adrenalectomy is superior to MR antagonist therapy, has not been validated in prospective and controlled studies. Rather, these approaches have been recommended based on their pathophysiologic mechanisms of action, their provenance, and supportive observational human studies. Herein, treatments for PA are discussed in two groupings: surgical adrenalectomy and medical therapy. Surgical adrenalectomy may involve curative unilateral adrenalectomy, noncurative unilateral adrenalectomy, and bilateral adrenalectomy. Medical therapy may involve MR antagonists, dietary sodium restriction, and epithelial sodium channel inhibitors.

Surgical Adrenalectomy. The treatment of choice for unilateral PA is curative unilateral laparoscopic adrenalectomy when patients are willing and able to undergo this operation. Laparoscopic and retroperitoneoscopic approaches are the gold standard and allow surgical resection with a minimally invasive approach. The unique attribute of adrenalectomy for unilateral PA is the opportunity to completely cure the disease by eliminating the source of inappropriate aldosterone production, curing hypokalemia, and either curing hypertension or dramatically improving blood pressure control. The definition of cure following unilateral adrenalectomy has been defined by the Primary Aldosteronism Surgery Outcomes (PASO) study,149 where a “complete clinical cure” was defined as postoperative normalization of blood pressure without the use of any antihypertensive medication use. A “complete biochemical cure” was defined by normalization of serum potassium without the need for supplements, along with a decrease in circulating aldosterone and/or an increase in renin such that there was normalization of the ARR. Notably, the PASO study highlighted that, whereas the majority of patients achieve a biochemical cure following adrenalectomy for unilateral PA (94%), only a minority (37%) of patients achieve a complete clinical cure, in part owing to the vasculopathy induced by longstanding hypertension and aldosteronism. Biochemical changes induced by curative unilateral adrenalectomy include normalization of serum potassium, an increase in serum creatinine and decrease in estimated glomerular filtration rate, a dramatic decline in plasma aldosterone, an increase in or normalization of renin (from suppressed to unsuppressed/detectable), and consequently a decline in the ARR.150 These biochemical changes all reflect the

CHAPTER 96  Primary Aldosteronism expected physiology induced by removing inappropriate and excessive aldosterone production: as intravascular volume contracts, renin and angiotensin II increase, glomerular hyperfiltration decreases, delivery of sodium ion to the distal nephron decreases, and excretion of urinary potassium and hydrogen ion decrease. The increase in serum creatinine following curative adrenalectomy is usually not a marker of kidney injury, but rather represents a reflection of the true underlying kidney function once hyperfiltration is corrected. In this regard, many patients with PA may be found to have some degree of chronic kidney disease postadrenalectomy that was previously not recognized. The clinical changes induced by curative adrenalectomy may include a cure of hypertension, reductions in cardiometabolic risk, and reduction in the risk of death. Cure of hypertension is observed in the minority of cases and more commonly among younger patients with a shorter duration of hypertension. More commonly, hypertension is not cured, but blood pressure control is improved and/or the number of antihypertensive medications required is decreased. Early observational data from a small sample size of 54 patients comparing long-­term cardiovascular outcomes between PA patients treated with adrenalectomy, PA patients treated with MR antagonist therapy, and a comparator group of patients with essential hypertension suggested no difference in long-­term cardiovascular risk among these groups.151 However, more recent observational data from large studies152 have shown that, despite similar blood pressure control between the groups, patients with PA treated with MR antagonists had a 3-­fold higher risk for incident cardiovascular events compared with patients with PA treated with curative adrenalectomy and a 2-­fold higher risk compared with patients with essential hypertension. In contrast, patients with PA treated with curative adrenalectomy had a 40% lower risk for incident cardiovascular events compared with patients with essential hypertension.152-­154 However, when MR antagonist therapy resulted in a substantial increase in renin activity, the risk for incident cardiovascular events was observed to be no different than comparable patients with essential hypertension and similar to those who underwent curative adrenalectomy. These observational findings underscore the efficacy of surgical adrenalectomy, but also suggest that, when MR antagonist therapy can be titrated to achieve a similar physiology to adrenalectomy, adverse outcome rates may approximate, but not quite capitulate, the effects of adrenalectomy.150 Curative adrenalectomy in PA has been shown to have a beneficial effect in terms of preventing incident atrial fibrillation compared with MR antagonist therapy and in preventing incident diabetes when compared to essential hypertension. Several studies have shown that curative adrenalectomy is associated with a preservation of kidney function when compared to MR antagonist therapy, where declines in glomerular filtration rate and the incidence of chronic kidney disease are greater. Both curative adrenalectomy and medical therapy have been shown to improve quality of life; however, the improvements in quality-­of-­life metrics following curative adrenalectomy have been more substantial.150 Although surgical adrenalectomy is most commonly recommended as a curative procedure for unilateral PA, there is also evidence to support the efficacy of noncurative unilateral adrenalectomy in bilateral PA. Importantly, there is no high-­grade clinical trial evidence comparing noncurative unilateral adrenalectomy with lifelong MR antagonist therapy in bilateral PA; rather, supportive evidence stems from observational studies and anecdotal patient series. In a case series of 12 patients who underwent noncurative unilateral adrenalectomy for bilateral PA, patients who had noncurative adrenalectomy had a substantial and durable reduction in blood pressure, a modest decrease in aldosterone levels, a substantial increase in renin that paralleled the rise in renin seen in those who underwent curative unilateral adrenalectomy,

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and near-­normalization of serum potassium.155 In a larger study of 51 patients who underwent noncurative adrenalectomy for bilateral PA, the operation cured hypertension in 15% of patients, improved blood pressure control in 40%, decreased aldosterone levels, substantially increased renin levels (and consequently decreased the ARR), normalized serum potassium, and increased serum creatinine.156 Notably, the adrenalectomy in this series was performed to target the morphologically larger adrenal and/or where adrenal venous sampling suggested greater lateralization. These findings, in combination with anecdotal clinical experiences, suggest that unilateral noncurative adrenalectomy for bilateral PA could be considered as an adjunct method to induce improvements in pathophysiology and clinical parameters in selected patients, particularly when guided by some degree of lateralization and/or greater morphologic abnormalities. In some centers, the use of super-­selective adrenal venous sampling to detect specific foci of aldosterone production, as well as thermal and arterial ablations, have also been shown to be effective targeted therapies. Bilateral adrenalectomy for bilateral PA is rarely employed for sporadic cases of PA, and is not recommended or supported by any reliable studies. Because bilateral adrenalectomy can cure the aldosteronism, it may be used as an option in extreme instances where unilateral adrenalectomy and/or medical therapy are unable to improve clinical and biochemical parameters, and when the risk for incident cardiometabolic disease is considered to be more severe than the risk for lifelong primary adrenal insufficiency. Detailed discussions regarding the risks, benefits, and ethics of such a decision should occur in conjunction with an experienced endocrinologist with knowledge in managing adrenal insufficiency and PA. In summary, surgical adrenalectomy is effective when utilized to unilateral PA, but sometimes also when used to attenuate the severity of the excess aldosterone production in bilateral PA. The hallmark characteristics suggestive of efficacy include improvements (or cure) of hypertension, reductions in aldosterone and increases in renin (such that the ARR is markedly reduced), normalization of serum potassium, and increases in serum creatinine indicative of a resolution in glomerular hyperfiltration. These features are associated with a reduction in the incident risk for cardiometabolic disease and death.

Medical Therapy. Medical therapy, here defined as any nonsurgical therapy, is the approach of choice when PA is deemed to be bilateral in origin by adrenal venous sampling, or when patients are unable or unwilling to undergo surgical adrenalectomy. Prior to initiating targeted MR antagonists, the critical importance of dietary sodium restriction should be underscored for patients, as it is a foundational intervention for all patients with hypertension. The efficacy of dietary sodium restriction for PA is high, and this is particularly true when combined with MR antagonists. Sodium is the substrate that fuels the pathophysiology of PA. Dietary sodium restriction results in reductions in effective arterial circulating volume, decreases in glomerular filtration, reductions in distal nephron sodium delivery, and consequently reductions in MR-­mediated sodium reabsorption and potassium and hydrogen ion excretion. Most patients in industrialized societies consume more than 150 to 200 mmol of sodium per day. In contrast, the American Heart Association recommends consuming less than 65 mmol of sodium per day for patients with hypertension. In one study, restricting dietary sodium intake to less than 50 mmol per day in patients with PA dramatically lowered blood pressure, substantially increased renin, and normalized the ARR in over 50% of participants.157 While the efficacy of dietary sodium restriction is established, the ability for most patients to adhere to these degrees of sodium restriction is a major challenge. Therefore, involving professional

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dieticians and nutrition support can be a valuable adjunct to pharmacologic therapy. In addition to dietary sodium restriction, MR antagonists are the mainstay of pharmacologic medical therapy. By blocking the MR, MR antagonists reduce sodium reabsorption and volume expansion, and consequently potassium and hydrogen ion excretion decline. In this manner, MR antagonists are capable of improving blood pressure, hypokalemia, and alkalosis. The main risk of MR antagonist therapy is hyperkalemia, which is more likely to occur when patients have concomitant reductions in glomerular filtration rate or use renin-­ angiotensin-­ aldosterone system inhibitors. Spironolactone and eplerenone are the most well-­known MR antagonists; both have a steroidal structure that permits competitive inhibition of aldosterone. Spironolactone is generally regarded as the first choice, whereas eplerenone should be considered for patients who cannot tolerate spironolactone. Spironolactone is approximately twice as potent as eplerenone and also inhibits the androgen receptor and activates the progesterone receptor; therefore, the most common adverse reaction leading to intolerance is gynecomastia in adult men, with menstrual irregularities in women being less common. The starting dose of spironolactone is usually 12.5 to 25 mg daily, but it can be titrated up to 200 mg daily. Eplerenone is usually initiated at 25 to 50 mg daily and can be titrated up to 200 to 400 mg daily. Beyond these conventional MR antagonists, new agents in development include finerenone and esaxerenone. Both finerenone and esaxerenone are nonsteroidal MR antagonists that been shown to reduce the progression of kidney disease. Esaxerenone has shown efficacy in lowering blood pressure, and finerenone is undergoing further study in heart failure. It should be noted that epithelial sodium channel antagonists, such as amiloride and triamterene, are also effective for blood pressure and potassium control in PA; however, these agents also carry the risk for hyperkalemia when kidney function is impaired, and further, they do not block the MR. Therefore, whether long-­term use of amiloride can mitigate MR-­mediated cardiovascular disease beyond blood pressure control remains unknown. The primary goals of medical therapy (dietary sodium restriction in combination with MR antagonists and/or epithelial sodium channel inhibitors) are normalization of blood pressure and serum potassium. The secondary goals are to optimize biomarkers associated with a decreased risk for incident cardiometabolic adverse outcomes. MR antagonists should be titrated to adequately block the renal and extrarenal MR. Optimized medical therapy is ideally defined as normal blood pressure, normal or high-­normal serum potassium, and an increase in renin activity or concentration.150 The achievement of these clinical and biochemical milestones is associated with similar cardiovascular risk reduction achieved by surgical adrenalectomy. When MR antagonist therapy is unable to optimize these clinical and biochemical parameters, amiloride can be added, or noncurative unilateral adrenalectomy considered. KEY POINTS  • There are multiple modalities to effectively treat primary aldosteronism. When possible, curative surgical adrenalectomy is the preferred method to improve blood pressure and risk for future cardiovascular events. Alternatively, medical therapy that combines dietary sodium restriction and mineralocorticoid receptor antagonists can also impart similar reductions in blood pressure and cardiovascular risk. In some select cases, the use of epithelial sodium channel inhibitors or noncurative unilateral adrenalectomy may be beneficial.

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CHAPTER 96  Primary Aldosteronism 22. Funder JW, Carey RM, Mantero F, et al. The management of primary aldosteronism: case detection, diagnosis, and treatment: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2016;101:1889– 1916. 23. Pallauf A, Schirpenbach C, Zwermann O, et al. The prevalence of familial hyperaldosteronism in apparently sporadic primary aldosteronism in Germany: a single center experience. Horm Metab Res. 2012;44:215–220. 24. Aglony M, Martinez-­Aguayo A, Carvajal CA, et al. Frequency of familial hyperaldosteronism type 1 in a hypertensive pediatric population: clinical and biochemical presentation. Hypertension. 2011;57:1117–1121. 25. Litchfield WR, Anderson BF, Weiss RJ, et al. Intracranial aneurysm and hemorrhagic stroke in glucocorticoid-­remediable aldosteronism. Hypertension. 1998;31:445–450. 26. Gordon RD, Stowasser M, Tunny TJ, et al. Clinical and pathological diversity of primary aldosteronism, including a new familial variety. Clin Exp Pharmacol Physiol. 1991;18:283–286. 27. Stowasser M, Gordon RD. Familial hyperaldosteronism. J Steroid Biochem Mol Biol. 2001;78:215–229. 28. Stowasser M, Gordon RD, Tunny TJ, et al. Familial hyperaldosteronism type II: five families with a new variety of primary aldosteronism. Clin Exp Pharmacol Physiol. 1992;19:319–322. 29. Scholl UI, Stolting G, Schewe J, et al. CLCN2 chloride channel mutations in familial hyperaldosteronism type II. Nat Genet. 2018;50:349–354. 30. Fernandes-­Rosa FL, Daniil G, Orozco IJ, et al. A gain-­of-­function mutation in the CLCN2 chloride channel gene causes primary aldosteronism. Nat Genet. 2018;50:355–361. 31. Scholl UI, Nelson-­Williams C, Yue P, et al. Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5. Proc Natl Acad Sci USA. 2012;109:2533–2538. 32. Mulatero P, Tauber P, Zennaro MC, et al. KCNJ5 mutations in European families with nonglucocorticoid remediable familial hyperaldosteronism. Hypertension. 2012;59:235–240. 33. Daniil G, Fernandes-­Rosa FL, Chemin J, et al. CACNA1H mutations are associated with different forms of primary aldosteronism. EBioMedicine. 2016;13:225–236. 34. Geller DS, Zhang J, Wisgerhof MV, et al. A novel form of human mendelian hypertension featuring nonglucocorticoid-­remediable aldosteronism. J Clin Endocrinol Metab. 2008;93:3117–3123. 35. Choi M, Scholl UI, Yue P, et al. K+ channel mutations in adrenal aldosterone-­producing adenomas and hereditary hypertension. Science. 2011;331:768–772. 36. Oki K, Plonczynski MW, Luis Lam M, et al. Potassium channel mutant KCNJ5 T158A expression in HAC-­15 cells increases aldosterone synthesis. Endocrinology. 2012;153:1774–1782. 37. Gomez-­Sanchez CE, Qi X, Gomez-­Sanchez EP, et al. Disordered zonal and cellular CYP11B2 enzyme expression in familial hyperaldosteronism type 3. Mol Cell Endocrinol. 2017;439:74–80. 38. Tong A, Liu G, Wang F, et al. A novel phenotype of familial hyperaldosteronism type III: concurrence of aldosteronism and Cushing’s syndrome. J Clin Endocrinol Metab. 2016;101:4290–4297. 39. Scholl UI, Stolting G, Nelson-­Williams C, et al. Recurrent gain of function mutation in calcium channel CACNA1H causes early-­onset hypertension with primary aldosteronism. Elife. 2015;4:e06315. 40. Scholl UI, Goh G, Stolting G, et al. Somatic and germline CACNA1D calcium channel mutations in aldosterone-­producing adenomas and primary aldosteronism. Nat Genet. 2013;45:1050–1054. 41. Semenova NA, Ryzhkova OR, Strokova TV, et al. [The third case report a patient with primary aldosteronism, seizures, and neurologic abnormalities (PASNA) syndrome de novo variant mutations in the CACNA1D gene]. Zh Nevrol Psikhiatr Im S S Korsakova. 2018;118:49–52. 42. De Mingo Alemany MC, Mifsud Grau L, Moreno Macian F, et al. A de novo CACNA1D missense mutation in a patient with congenital hyperinsulinism, primary hyperaldosteronism and hypotonia. Channels. 2020;14:175–180. 43. Nanba K, Omata K, Else T, et al. Targeted molecular characterization of aldosterone-­producing adenomas in White Americans. J Clin Endocrinol Metab. 2018;103:3869–3876.

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44. Nanba K, Omata K, Gomez-­Sanchez CE, et al. Genetic characteristics of aldosterone-­producing adenomas in Blacks. Hypertension. 2019;73:885– 892. 45. De Sousa K, Boulkroun S, Baron S, et al. Genetic, cellular, and molecular heterogeneity in adrenals with aldosterone-­producing adenoma. Hypertension. 2020;75:1034–1044. 46. Nanba K, Yamazaki Y, Bick N, et al. Prevalence of somatic mutations in aldosterone-­producing adenomas in Japanese patients. J Clin Endocrinol Metab. 2020;105:e4066–e4073. 47. Teo AE, Garg S, Shaikh LH, et al. Pregnancy, primary aldosteronism, and adrenal CTNNB1 mutations. N Engl J Med. 2015;373:1429–1436. 48. Scholl UI, Healy JM, Thiel A, et al. Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype. Clin Endocrinol. 2015;83:779–789. 49. Akerstrom T, Maharjan R, Sven Willenberg H, et al. Activating mutations in CTNNB1 in aldosterone producing adenomas. Sci Rep. 2016;6:19546. 50. Rhayem Y, Perez-­Rivas LG, Dietz A, et al. PRKACA somatic mutations are rare findings in aldosterone-­producing adenomas. J Clin Endocrinol Metab. 2016;101:3010–3017. 51. Stindl J, Tauber P, Sterner C, et al. Pathogenesis of adrenal aldosterone-­ producing adenomas carrying mutations of the Na(+)/K(+)-­ATPase. Endocrinology. 2015;156:4582–4591. 52. Rege J, Nanba K, Blinder AR, et al. Identification of somatic mutations in CLCN2 in aldosterone-­producing adenomas. J Endocr Soc. 2020;4:bvaa123. 53. Tauber P, Aichinger B, Christ C, et al. Cellular pathophysiology of an adrenal adenoma-­associated mutant of the plasma membrane Ca(2+)-­ ATPase ATP2B3. Endocrinology. 2016;157:2489–2499. 54. Azizan EA, Poulsen H, Tuluc P, et al. Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension. Nat Genet. 2013;45:1055–1060. 55. Fernandes-­Rosa FL, Williams TA, Riester A, et al. Genetic spectrum and clinical correlates of somatic mutations in aldosterone-­producing adenoma. Hypertension. 2014;64:354–361. 56. Lenzini L, Rossitto G, Maiolino G, et al. A meta-­analysis of somatic KCNJ5 K(+) channel mutations in 1636 patients with an aldosterone-­ producing adenoma. J Clin Endocrinol Metab. 2015;100:E1089–E1095. 57. Dutta RK, Arnesen T, Heie A, et al. Case report: a somatic mutation in CLCN2 identified in a sporadic aldosterone producing adenoma. Eur J Endocrinol. 2019;181:K37–K41. 58. Omata K, Satoh F, Morimoto R, et al. Cellular and genetic causes of idiopathic hyperaldosteronism. Hypertension. 2018;72:874–880. 59. Boulkroun S, Samson-­Couterie B, Dzib JF, et al. Adrenal cortex remodeling and functional zona glomerulosa hyperplasia in primary aldosteronism. Hypertension. 2010;56:885–892. 60. Nishimoto K, Nakagawa K, Li D, et al. Adrenocortical zonation in humans under normal and pathological conditions. J Clin Endocrinol Metab. 2010;95:2296–2305. 61. Nanba K, Vaidya A, Williams GH, et al. Age-­related autonomous aldosteronism. Circulation. 2017;136:347–355. 62. Williams TA, Peitzsch M, Dietz AS, et al. Genotype-­specific steroid profiles associated with aldosterone-­producing adenomas. Hypertension. 2016;67:139–145. 63. Guo Z, Nanba K, Udager A, et al. Biochemical, histopathological, and genetic characterization of posture-responsive and unresponsive APAs. J Clin Endocrinol Metab. 2020;105:e3224–e3235. 64. Maiolino G, Ceolotto G, Battistel M, et al. Macrolides for KCNJ5-­ Mutated Aldosterone-­Producing Adenoma (MAPA): Design of a Study for Personalized Diagnosis of Primary Aldosteronism. Vol. 27. Blood Press; 2018:200–205. 65. Newton-­Cheh C, Guo CY, Gona P, et al. Clinical and genetic correlates of aldosterone-­to-­renin ratio and relations to blood pressure in a community sample. Hypertension. 2007;49:846–856. 66. Hannemann A, Bidlingmaier M, Friedrich N, et al. Screening for primary aldosteronism in hypertensive subjects: results from two German epidemiological studies. Eur J Endocrinol. 2012;167:7–15.

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PART 7  Adrenal

67. Inoue K, Goldwater D, Allison M, et al. Serum aldosterone concentration, blood pressure, and coronary artery calcium: the multi-­ethnic study of atherosclerosis. Hypertension. 2020;76:113–120. 68. Monticone S, Burrello J, Tizzani D, et al. Prevalence and clinical manifestations of primary aldosteronism encountered in primary care practice. J Am Coll Cardiol. 2017;69:1811–1820. 69. Mosso L, Carvajal C, Gonzalez A, et al. Primary aldosteronism and hypertensive disease. Hypertension. 2003;42:161–165. 70. Williams JS, Williams GH, Raji A, et al. Prevalence of primary hyperaldosteronism in mild to moderate hypertension without hypokalaemia. J Hum Hypertens. 2006;20:129–136. 71. Omura M, Saito J, Yamaguchi K, et al. Prospective study on the prevalence of secondary hypertension among hypertensive patients visiting a general outpatient clinic in Japan. Hypertens Res. 2004;27:193–202. 72. Rossi GP, Bernini G, Caliumi C, et al. A prospective study of the prevalence of primary aldosteronism in 1,125 hypertensive patients. J Am Coll Cardiol. 2006;48:2293–2300. 73. Rossi E, Regolisti G, Negro A, et al. High prevalence of primary aldosteronism using postcaptopril plasma aldosterone to renin ratio as a screening test among Italian hypertensives. Am J Hypertens. 2002;15:896–902. 74. Strauch B, Zelinka T, Hampf M, et al. Prevalence of primary hyperaldosteronism in moderate to severe hypertension in the Central Europe region. J Hum Hypertens. 2003;17:349–352. 75. Xu Z, Yang J, Hu J, et al. Primary aldosteronism in patients in China with recently detected hypertension. J Am Coll Cardiol. 2020;75:1913– 1922. 76. Hu Y, Zhang J, Liu W, et al. Determining the prevalence of primary aldosteronism in patients with new-­onset type 2 diabetes and hypertension. J Clin Endocrinol Metab. 2020:105;(4):1079-1085. 77. Yozamp N, Hundemer GL, Moussa M, et al. Intraindividual variability of aldosterone concentrations in primary aldosteronism: implications for case detection. Hypertension. 2020;77(3):891-899. 78. Tsiavos V, Markou A, Papanastasiou L, et al. A new highly sensitive and specific overnight combined screening and diagnostic test for primary aldosteronism. Eur J Endocrinol. 2016;175:21–28. 79. Parasiliti-­Caprino M, Lopez C, Prencipe N, et al. Prevalence of primary aldosteronism and association with cardiovascular complications in patients with resistant and refractory hypertension. J Hypertens. 2020;38(9):1841–1848. 80. Brown JM, Siddiqui M, Carey R, et al. The unrecognized prevalence of primary aldosteronism. Ann Int Med. 2020;173(1):10–20. 81. Funder JW. Primary aldosteronism: at the tipping point. Ann Intern Med. 2020. 82. Calhoun DA, Nishizaka MK, Zaman MA, et al. Hyperaldosteronism among black and white subjects with resistant hypertension. Hypertension. 2002;40:892–896. 83. Williams B, Macdonald TM, Morant S, et al. Spironolactone versus placebo, bisoprolol, and doxazosin to determine the optimal treatment for drug-­resistant hypertension (PATHWAY-­2): a randomised, double-­ blind, crossover trial. Lancet. 2015;386:2059–2068. 84. Williams B, MacDonald TM, Morant SV, et al. Endocrine and haemodynamic changes in resistant hypertension, and blood pressure responses to spironolactone or amiloride: the PATHWAY-­2 mechanisms substudies. Lancet Diabet Endocrinol. 2018;(6):464–475. 85. Adlin EV. Subclinical primary aldosteronism. Ann Int Med. 2017;167(9):673–674. 86. Markou A, Pappa T, Kaltsas G, et al. Evidence of primary aldosteronism in a predominantly female cohort of normotensive individuals: a very high odds ratio for progression into arterial hypertension. J Clin Endocrinol Metab. 2013;98:1409–1416. 87. Piaditis G, Markou A, Papanastasiou L, et al. Progress in aldosteronism: a review of the prevalence of primary aldosteronism in pre-­hypertension and hypertension. Eur J Endocrinol. 2015;172:R191–R203. 88. Baudrand R, Guarda FJ, Fardella CE, et al. Continuum of Renin-­ independent Aldosteronism in Normotension. Hypertension. 2017;69(5):950–956. 89. Carey RM, Ayers CR, Vaughan Jr ED, et al. Activity of [des-­aspartyl1]-­ angiotensin II in primary aldosteronism. J Clin Invest. 1979;63:718–726.

90. Wisgerhof M, Brown RD. Increased adrenal sensitivity to angiotensin II in low-­renin essential hypertension. J Clin Invest. 1978;61:1456–1462. 91. Vasan RS, Evans JC, Larson MG, et al. Serum aldosterone and the incidence of hypertension in nonhypertensive persons. N Engl J Med. 2004;351:33–41. 92. Perschel FH, Schemer R, Seiler L, et al. Rapid screening test for primary hyperaldosteronism: ratio of plasma aldosterone to renin concentration determined by fully automated chemiluminescence immunoassays. Clin Chem. 2004;50:1650–1655. 93. Brown JM, Robinson-­Cohen C, Luque-­Fernandez MA, et al. The spectrum of subclinical primary aldosteronism and incident hypertension: a cohort study. Ann Int Med. 2017;167(9):630–641. 94. Rich GM, Ulick S, Cook S, et al. Glucocorticoid-­remediable aldosteronism in a large kindred: clinical spectrum and diagnosis using a characteristic biochemical phenotype. Ann Intern Med. 1992;116:813–820. 95. Stowasser M, Huggard PR, Rossetti TR, et al. Biochemical evidence of aldosterone overproduction and abnormal regulation in normotensive individuals with familial hyperaldosteronism type I. J Clin Endocrinol Metab. 1999;84:4031–4036. 96. Mulatero P, Stowasser M, Loh KC, et al. Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents. J Clin Endocrinol Metab. 2004;89:1045–1050. 97. Stowasser M, Gordon RD, Gunasekera TG, et al. High rate of detection of primary aldosteronism, including surgically treatable forms, after ‘non-­ selective’ screening of hypertensive patients. J Hypertens. 2003;21:2149–2157. 98. Sonino N, Tomba E, Genesia ML, et al. Psychological assessment of primary aldosteronism: a controlled study. J Clin Endocrinol Metab. 2011;96:E878–E883. 99. Ahmed AH, Gordon RD, Sukor N, et al. Quality of life in patients with bilateral primary aldosteronism before and during treatment with spironolactone and/or amiloride, including a comparison with our previously published results in those with unilateral disease treated surgically. J Clin Endocrinol Metab. 2011;96:2904–2911. 100. Sonino N, Fallo F, Fava GA. Psychological aspects of primary aldosteronism. Psychother Psychosom. 2006;75:327–330. 101. Sukor N, Kogovsek C, Gordon RD, et al. Improved quality of life, blood pressure, and biochemical status following laparoscopic adrenalectomy for unilateral primary aldosteronism. J Clin Endocrinol Metab. 2010;95:1360–1364. 102. Gordon RD, Tunny TJ. Aldosterone-­producing-­adenoma (A-­P-­A): effect of pregnancy. Clin Exp Hypertens. 1982;4:1685–1693. 103. Ronconi V, Turchi F, Zennaro MC, et al. Progesterone increase counteracts aldosterone action in a pregnant woman with primary aldosteronism. Clin Endocrinol. 2011;74:278–279. 104. Murphy BF, Whitworth JA, Kincaid-­Smith P. Malignant hypertension due to an aldosterone producing adrenal adenoma. Clin Exp Hypertens. 1985;7:939–950. 105. Zarifis J, Lip GY, Leatherdale B, et al. Malignant Hypertension in Association with Primary Aldosteronism. Vol. 5. Blood Press; 1996:250–254. 106. Taylor PJ, Cooper DP, Gordon RD, et al. Measurement of aldosterone in human plasma by semiautomated HPLC-­tandem mass spectrometry. Clin Chem. 2009;55:1155–1162. 107. Peitzsch M, Dekkers T, Haase M, et al. An LC-­MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism. J Steroid Biochem Mol Biol. 2015;145:75–84. 108. Guo Z, Poglitsch M, McWhinney BC, et al. Aldosterone LC-­MS/MS assay-­specific threshold values in screening and confirmatory testing for primary aldosteronism. J Clin Endocrinol Metab. 2018;103:3965–3973. 109. Thuzar M, Young K, Ahmed AH, et al. Diagnosis of primary aldosteronism by seated saline suppression test-­variability between immunoassay and HPLC-­MS/MS. J Clin Endocrinol Metab. 2020:105:E447–E483. 110. Sealey JE, Laragh JH. Radioimmunoassay of plasma renin activity. Semin Nucl Med. 1975;5:189–202. 111. Ferrari P, Shaw SG, Nicod J, et al. Active renin versus plasma renin activity to define aldosterone-­to-­renin ratio for primary aldosteronism. J Hypertens. 2004;22:377–381. 112. Montori VM, Schwartz GL, Chapman AB, et al. Validity of the aldosterone-­renin ratio used to screen for primary aldosteronism. Mayo Clin Proc. 2001;76:877–882.

CHAPTER 96  Primary Aldosteronism 113. Campbell DJ, Nussberger J, Stowasser M, et al. Activity assays and immunoassays for plasma renin and prorenin: information provided and precautions necessary for accurate measurement. Clin Chem. 2009;55:867–877. 114. Oelkers WK. Effects of estrogens and progestogens on the renin-­ aldosterone system and blood pressure. Steroids. 1996;61:166–171. 115. Morganti A. European study group for the validation of DiaSorin LDRA. A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin. J Hypertens. 2010;28:1307–1312. 116. Guo Z, Poglitsch M, McWhinney BC, et al. Measurement of equilibrium angiotensin II in the diagnosis of primary aldosteronism. Clin Chem. 2020;66:483–492. 117. Gordon RD, Stowasser M. Primary aldosteronism: the case for screening. Nat Clin Pract Nephrol. 2007;3:582–583. 118. Zarnegar R, Young Jr WF, Lee J, et al. The aldosteronoma resolution score: predicting complete resolution of hypertension after adrenalectomy for aldosteronoma. Ann Surg. 2008;247:511–518. 119. Stowasser M, Gordon RD. Primary aldosteronism-­careful investigation is essential and rewarding. Mol Cell Endocrinol. 2004;217:33–39. 120. Mulatero P, Rabbia F, Milan A, et al. Drug effects on aldosterone/plasma renin activity ratio in primary aldosteronism. Hypertension. 2002;40:897– 902. 121. Stowasser M, Ahmed AH, Pimenta E, et al. Factors affecting the aldosterone/renin ratio. Horm Metab Res. 2012;44:170–176. 122. Rossi GP, Seccia TM, Palumbo G, et al. Within-­patient reproducibility of the aldosterone: renin ratio in primary aldosteronism. Hypertension. 2010;55:83–89. 123. Young WFJ. Primary aldosteronism: update on diagnosis and treatment. Endocrinololgist. 1997;7:213–221. 124. Gordon RD. Primary aldosteronism. J Endocrinol Invest. 1995;18:495– 511. 125. Kem DC, Weinberger MH, Mayes DC, et al. Saline suppression of plasma aldosterone in hypertension. Arch Intern Med. 1971;128:380–386. 126. Holland OB, Brown H, Kuhnert L, et al. Further evaluation of saline infusion for the diagnosis of primary aldosteronism. Hypertension. 1984;6:717–723. 127. Ahmed AH, Cowley D, Wolley M, et al. Seated saline suppression testing for the diagnosis of primary aldosteronism: a preliminary study. J Clin Endocrinol Metab. 2014;99:2745–2753. 128. Stowasser M, Ahmed AH, Cowley D, et al. Comparison of seated with recumbent saline suppression testing for the diagnosis of primary aldosteronism. J Clin Endocrinol Metab. 2018;103:4113–4124. 129. Iwaoka T, Umeda T, Naomi S, et al. The usefulness of the captopril test as a simultaneous screening for primary aldosteronism and renovascular hypertension. Am J Hypertens. 1993;6:899–906. 130. Westerdahl C, Bergenfelz A, Isaksson A, et al. Captopril suppression: limitations for confirmation of primary aldosteronism. J Renin Angiotensin Aldosterone Syst. 2011;12:326–332. 131. Mulatero P, Bertello C, Garrone C, et al. Captopril test can give misleading results in patients with suspect primary aldosteronism. Hypertension. 2007;50:e26–e27. 132. Lifton RP, Dluhy RG, Powers M, et al. A chimaeric 11 beta-­hydroxylase/ aldosterone synthase gene causes glucocorticoid-­remediable aldosteronism and human hypertension. Nature. 1992;355:262–265. 133. Jonsson JR, Klemm SA, Tunny TJ, et al. A new genetic test for familial hyperaldosteronism type I aids in the detection of curable hypertension. Biochem Biophys Res Commun. 1995;207:565–571. 134. Hundemer GL, Curhan GC, Yozamp N, et al. Incidence of atrial fibrillation and mineralocorticoid receptor activity in patients with medically and surgically treated primary aldosteronism. JAMA Cardiol. 2018;3:768– 774. 135. Rossi GP, Maiolino G, Flego A, et al. Adrenalectomy lowers incident atrial fibrillation in primary aldosteronism patients at long term. Hypertension. 2018;71:585–591.

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136. Kupers EM, Amar L, Raynaud A, et al. A clinical prediction score to diagnose unilateral primary aldosteronism. J Clin Endocrinol Metab. 2012;97:3530–3537. 137. Young WF, Stanson AW, Thompson GB, et al. Role for adrenal venous sampling in primary aldosteronism. Surgery. 2004;136:1227–1235. 138. Gordon RD, Gomez-­Sanchez CE, Hamlet SM, et al. Angiotensin-­ responsive aldosterone-­producing adenoma masquerades as idiopathic hyperaldosteronism (IHA: adrenal hyperplasia) or low-­renin essential hypertension. J Hypertens Suppl. 1987;5:S103–S106. 139. Guo Z, Nanba K, Udager A, et al. Biochemical, Histopathological, and genetic characterization of posture-­responsive and unresponsive APAs. J Clin Endocrinol Metab. 2020;105;e:3224–3335. 140. O’Shea PM, O’Donoghue D, Bashari W, et al. 11) C-­Metomidate PET/ CT is a useful adjunct for lateralization of primary aldosteronism in routine clinical practice. Clin Endocrinol. 2019;90:670–679. 141. Powlson AS, Gurnell M, Brown MJ. Nuclear imaging in the diagnosis of primary aldosteronism. Curr Opin Endocrinol Diabetes Obes. 2015;22:150–156. 142. Daunt N. Adrenal vein sampling: how to make it quick, easy, and successful. Radiographics. 2005;25:S143–S158. 143. Doppman JL, Gill Jr JR. Hyperaldosteronism: sampling the adrenal veins. Radiology. 1996;198:309–312. 144. Rossi GP, Auchus RJ, Brown M, et al. An expert consensus statement on use of adrenal vein sampling for the subtyping of primary aldosteronism. Hypertension. 2014;63:151–160. 145. Young WF, Stanson AW. What are the keys to successful adrenal venous sampling (AVS) in patients with primary aldosteronism? Clin Endocrinol. 2009;70:14–17. 146. Satoh F, Morimoto R, Seiji K, et al. Is there a role for segmental adrenal venous sampling and adrenal sparing surgery in patients with primary aldosteronism? Eur J Endocrinol. 2015;173:465–477. 147. Yoneda T, Karashima S, Kometani M, et al. Impact of new quick gold nanoparticle-­based cortisol assay during adrenal vein sampling for primary aldosteronism. J Clin Endocrinol Metab. 2016;101:2554–2561. 148. Goupil R, Wolley M, Ungerer J, et al. Use of plasma metanephrine to aid adrenal venous sampling in combined aldosterone and cortisol over-­ secretion. Endocrinol Diabetes Metab Case Rep. 2015;2015:150075. 149. Williams TA, Lenders JWM, Mulatero P, et al. Outcomes after adrenalectomy for unilateral primary aldosteronism: an international consensus on outcome measures and analysis of remission rates in an international cohort. Lancet Diabet Endocrinol. 2017;5:689–699. 150. Hundemer GL, Vaidya A. Management of endocrine disease: the role of surgical adrenalectomy in primary aldosteronism. Eur J Endocrinol. 2020;183:R185–R196. 151. Catena C, Colussi G, Nadalini E, et al. Cardiovascular outcomes in patients with primary aldosteronism after treatment. Arch Intern Med. 2008;168:80–85. 152. Hundemer GL, Curhan GC, Yozamp N, et al. Cardiometabolic outcomes and mortality in medically treated primary aldosteronism: a retrospective cohort study. Lancet Diabet Endocrinol. 2018;6:51–59. 153. Hundemer G, Curhan G, Yozamp N, et al. Incidence of atrial fibrillation and mineralocorticoid receptor activity in patients with medically and surgically treated primary aldosteronism. JAMA Cardiol. 2018;3:768–774. 154. Hundemer G, Curhan G, Yozamp N, et al. Renal outcomes in medically treated primary aldosteronism. Hypertension. 2018;72:658–666. 155. Groth H, Vetter W, Stimpel M, et al. Adrenalectomy in primary aldosteronism: a long-­term follow-­up study. Cardiology. 1985;72:107–116. 156. Sukor N, Gordon RD, Ku YK, et al. Role of unilateral adrenalectomy in bilateral primary aldosteronism: a 22-­year single center experience. J Clin Endocrinol Metab. 2009;94:2437–2445. 157. Baudrand R, Guarda FJ, Torrey J, et al. Dietary sodium restriction increases the risk of misinterpreting mild cases of primary aldosteronism. J Clin Endocrinol Metab. 2016. jc20161963.

97 Adrenocorticotropic Hormone– Independent Cushing Syndrome Maria Candida Barisson Villares Fragoso, Annabel Berthon, and Jérôme Bertherat OUTLINE Introduction, 1614 Epidemiology, 1614 Pathophysiology––Molecular Genetics and Animal Models, 1615 Molecular and Genetic Causes of Cortisol-­Producing Adrenocortical Lesions, 1615 Animal Models, 1618 Diagnosis, 1619 Clinical Manifestations, 1619 Hormonal Evaluation, 1619 Etiology, 1622 Unilateral Tumors, 1622

Adrenocortical Adenoma, 1622 Adrenocortical Cancer, 1622 Bilateral Adrenocortical Tumors, 1624 Micronodular Adrenal Hyperplasia and Primary Pigmented Nodular Adrenal Disease, 1624 Primary Macronodular Adrenal Hyperplasia, 1625 Management of Adrenal Cushing Syndrome, 1626 Medical Treatment with Steroidogenesis Inhibitors, 1626 Surgery, 1627 Perspectives, 1628

 

INTRODUCTION

EPIDEMIOLOGY

KEY POINTS  • Cushing syndrome is a rare disease, characterized by chronic and excessive exposure to glucocorticoids. • Cushing syndrome is a prototype of metabolic syndrome and results in high morbidity and mortality if not treated. • During the last few years there have been improvements in our knowledge of the molecular mechanisms involved in the pathogenesis of adrenal Cushing syndrome.

KEY POINTS  • Adrenal Cushing syndrome accounts for approximately 20%–25% of all cases of endogenous Cushing syndrome, second only to benign and malignant adrenal disorders. • Adrenocortical tumors are frequent in the general population (3%–10% of adults), but the subset responsible for overt Cushing are rare, with the incidence being estimated at approximately 1.5/million/year, with a higher prevalence in females.

Cushing syndrome (CS) is a rare endocrine condition, firstly described in 1912 by Harvey Cushing,1 and is characterized by chronic and excessive exposure to glucocorticoids. CS can include significant morbidity and even mortality (if untreated), and its diagnosis and treatment can be challenging.2 In humans, cortisol is the primary active glucocorticoid produced by the adrenal cortex under the control of pituitary adrenocorticotropic hormone (ACTH), and its actions include mobilization of fats, proteins, and carbohydrates. CS can be secondary to chronic stimulation of the adrenal cortex by ACTH secreted by a pituitary or ectopic ACTH-­secreting tumor, or by a primary adrenal dysregulation. In the latter case, the causative adrenal lesions are independent of physiological regulation by pituitary ACTH, as the activity of the pituitary corticotroph cells is typically suppressed by glucocorticoid-­mediated negative feedback; this can be classified as “adrenal Cushing.” The adrenal lesions responsible for adrenal Cushing can be unilateral or bilateral, and can represent benign or malignant disease. This chapter will discuss clinical aspects of the diagnosis and management of the various causes of adrenal Cushing and recent advances in our understanding of their molecular genetics.

CS is a rare disease, with an estimated incidence ranging between 1 to 5 per million each year,3 although the estimations, as is the case for many rare diseases, are imprecise. Approximately 20% to 25% of endogenous CS cases are secondary to adrenal gland disorders including adenomas, carcinomas, and nodular hyperplasias (which may be macronodular and micronodular, based on nodules size [>1 cm or 20–30 mg/dL) can be associated with more adverse effects, whereas a response is less likely at a concentration less than 14 mg/L. Thus, mitotane has a narrow therapeutic window. However, some patients may respond to lower mitotane concentrations. Due to the long half-­life of mitotane, target levels are often achieved only after weeks to months of therapy.62 Treatment is often initiated with 1.5 g/day and rapidly increased to 4.5 to 6 g/ day until target concentrations have been reached. While most adverse effects are related to mitotane plasma concentrations, adverse gastrointestinal effects such as diarrhea seem to be more related to the daily dose. During long-­term treatment, the dose of mitotane can usually be reduced, and many patients need only 2 to 3 g/day during long-­ term therapy. Ingestion of mitotane together with a lipid-­rich drink or meal may enhance mitotane absorption and decreases gastrointestinal side effects. Due to its adrenolytic activity, mitotane treatment induces adrenal insufficiency. Furthermore, mitotane also increases the metabolic clearance of glucocorticoids by a very strong induction of cytochrome P450 3A4 (CYP3A4) and increases the concentration of cortisol-­binding globulin.63-­65 Patients on mitotane often require a higher dose of glucocorticoid replacement (averaging 40–60 mg per day) compared with patients with other forms of adrenal insufficiency (average requirements approximately 15–30 mg per day). Insufficient glucocorticoid replacement enhances mitotane intolerance. Aldosterone secretion is less often affected, as mitotane primarily acts on the zona fasciculata and the zona reticularis. However, aldosterone secretion should be monitored, and replacement with fludrocortisone may become necessary with long-­term use of mitotane. In addition, mitotane increases sex hormone–binding globulin63 and often leads to low free testosterone concentrations in males. With long-­term therapy, free thyroid hormone concentrations decline in the presence of low to low-­normal thyroid-­stimulating hormone, suggesting secondary hypothyroidism. Due to its estrogenic activity, mitotane may facilitate the development of gynecomastia.

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TABLE 98.3  Recommended Drug Regimens

for Advanced Adrenocortical Carcinoma*

Mitotane Monotherapy In mitotane-­naïve patients in good clinical condition and with presumably less aggressive disease: -­ Start with 1.5 g/day and increase daily dose within 4–6 days to 6 g/day. -­ Measure mitotane blood level every 3–4 weeks. -­ Adapt dosage according to tolerability and blood level (maximum dose 12 g/day; most patients do not tolerate >8 g/day). -­ In case of relevant adverse effects, lowering the dosage or even interrupting treatment may be required to allow compliance. For patients in poor clinical condition or with concomitant cytotoxic drugs, a lower dosage (e.g., 3 g/day) is recommended. Etoposide, Doxorubicin and Cisplatin (EDP) Plus Mitotane (EDP/M) Every 28 days: -­ day 1 40mg/m2 D -­ day 2 100mg/m2 E -­ day 3+4 100mg/m2 E + 40mg/m2 P Plus oral mitotane aiming at a blood level between 14 and 20 mg/L. However, in most patients we do not administer more than 4–5 g/day of mitotane. In patients unfit for the EDP-­M regimen, P-­M may constitute a reasonable alternative. Second-­Line/Salvage Therapies Gemcitabine plus capecitabine -­ 800 mg/m2 gemcitabine on day 1 and 8 (repeated every 3 weeks) -­ 1500 mg capecitabine orally per day in a continuous fashion -­ additional administration of mitotane should be consideredStreptozotocin plus Mitotane (Sz/M) -­ induction: day 1 to 5: 1g Sz/d -­ afterwards 2 g/day Sz every 21 days -­ plus oral mitotane aiming at a blood level between 14 and 20 mg/L *Consider enrollment of patients in clinical trials (www.clinicaltrial.gov). From Fassnacht M, Kroiss M, Allolio B. Update in adrenocortical carcinoma. J Clin Endocrinol Metab. 2013;98:4551–4564; Berruti A, Baudin E, Gelderblom H, et al. Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2012; 23(Suppl 7):vii131–vii38; and Sperone P, Ferrero A, Daffara F, et al. Gemcitabine plus metronomic 5-fluorouracil or capecitabine as a second-/third-line chemotherapy in advanced adrenocortical carcinoma: a multicenter phase II study. Endocr Relate Cancer. 2010;17:445–453

Changes in hepatic gamma glutamyl transaminase levels are so frequent that their absence calls into question patient compliance. However, in some cases, serious hepatotoxicity and even liver failure have been observed. Mitotane prolongs bleeding time, and it is advisable to stop mitotane for a minimum of 1 week prior to major surgery. High low-­density lipoprotein cholesterol and/or triglyceride concentrations are regularly observed.66 Despite the plethora of adverse effects of mitotane, most patients can be managed with acceptable toxicity in the long term.67 This is particularly important in an adjuvant setting, as treatment must be given for a minimum of 2 years. The abovementioned effect of mitotane as one of the strongest available inducers of hepatic CYP3A464 has a major impact on the care of ACC patients, because mitotane lowers the blood levels of many frequently coadministered drugs (including but not limited to steroids, antihypertensives, and antibiotics)65 (Table 98.3).

Cytotoxic Chemotherapy Only two cytotoxic regimens have been investigated in a randomized controlled trial.68 This trial, FIRM-­ACT, included a total of 304 patients and compared etoposide, doxorubicin, cisplatin, and mitotane (EDP-­M) with streptozotocin and mitotane (Sz-­M), both as first-­and second-­line cytotoxic treatment. Although overall survival was not significantly different (14.8 months [EDP-­M] vs. 12.0 months [Sz-­M]; HR 0.79; P = 0.07), objective response rate and progression-­ free survival clearly favored EDP-­M (23.2% vs. 9.2%, P < 0.001 and 5.0 months vs. 2.1 months; HR 0.55; P < 0.001). Most experts now advocate EDP-­M as first-­line therapy for patients requiring cytotoxic treatment.69 Because children were not included in the FIRM-­ACT trial, there is no proof that EDP-­M is also the first treatment of choice in children, and other regimens might be considered (see Fig. 98.3B).70 For patients who fail EDP-­M chemotherapy, an individualized approach is needed, with a strong preference for receiving therapy through a clinical trial.21 Over the past decade, multiple cytotoxic regimens were reported to have limited efficacy in the management of progressive ACC after failing first-­line therapy. The combination of gemcitabine and capecitabine (or metronomic 5-­fluorouracil) has been studied as a second-­/third-­line regimen based on earlier reports showing disease stabilization for at least 6 months in eight of 28 patients (29%) when using this combination.71 A more recent report of 145 patients assessed retrospectively after having gemcitabine-­based chemotherapy (132 in combination with capecitabine) found more limited impact on survival, with partial responses seen in 4.9% and stable disease in 25% of subjects.72 In another retrospective report of 28 patients with advanced ACC who were treated with temozolomide (200 mg/m2/day for 5 consecutive days every 28-­day cycle), median progression-­free survival was 3.5 months, with six patients ( 21%) showing evidence of short-­lived objective response, though one subject had complete response to therapy.73 Other potential salvage therapies might be trofosfamide or thalidomide. However, both have only modest antitumor activity (150 mg/ day).74,75

Targeted Therapies The current success with cytotoxic chemotherapy in ACC is not satisfactory, with less than 50% of tumors responding to treatment, and most of them for only a limited time. Small-­molecule multikinase inhibitors (MKIs) have transformed the care of multiple malignancies; however, so far, no breakthrough has been accomplished in ACC. While the experience with MKIs (e.g., sunitinib, sorafenib, dovitinib, axitinib) has been largely disappointing,76 it is important to consider multiple factors. First, the patients studied in these trials had virtually all received several different cytotoxic treatments prior to their targeted therapy. Thus, targeted therapies were used as salvage treatment in very high-­risk cohorts with poor prognoses. Furthermore, most of these patients had received previous or even concomitant mitotane leading to strong induction of CYP3A4, which frequently plays a major role in the degradation of MKIs and other targeted therapies, thereby profoundly reducing levels of these compounds. Accordingly, studies evaluating earlier treatment with targeted therapies in mitotane-­naive patients are urgently needed and may generate an entirely different picture. c-­Met signaling appears to be a worthy target, based on the finding of increased expression of c-­Met after exposing an ACC cell line in vitro to commonly used treatments in ACC (cisplatin, mitotane, radiation). Thus, it is believed that c-­Met signaling could be one of the resistance mechanisms in ACC.77 In a retrospective report of 16 ACC

CHAPTER 98  Adrenocortical Carcinoma patients who failed prior therapies, the use of cabozantinib resulted in partial response in three, stable disease in five, and progressive disease in eight patients, with a median progress-­free survival of approximately 4 months.78 Two ongoing phase II trials aim to ascertain the efficacy and safety of cabozantinib in ACC (ClinicalTrials.Gov identifiers: NCT03612232 and NCT03370718). As ACCs express high levels of insulin-like growth factor (IGF)-­2, which acts via the IGF-­1 receptor, blockade of the IGF-­1 receptor has been studied in ACC. A randomized, phase III trial (139 patients) using the small-­molecule IGF-­1 receptor inhibitor linsitinib (90 on linsitinib and 49 on placebo) was terminated early due to lack of significant improvement in progression-­free survival or overall survival, despite a few patients having an objective response to linsitinib.79 In a phase I study, the combination of an IGF-­1 receptor antibody (cixutumumab) with the mTOR inhibitor temsirolimus led to stable disease for at least 6 months in 11 of 26 patients,80 whereas other studies exploring monotherapy with the mTOR inhibitor everolimus did not show benefit.81 Using selective radiopharmaceuticals has been explored in ACC. 131I-­iodometomidate binds selectively to adrenal CYP11B enzymes as a targeted radionuclide therapy for advanced ACC. 131I-­iodometomidate was given on a compassionate use basis to 11 patients with advanced ACC. One patient experienced a partial response lasting for 26 months, and another five patients had stable disease.82

Immunotherapy In the past decade, the use of immune checkpoint inhibitors has transformed cancer care, and many drugs have been approved to treat different malignancies. The experience with immunotherapy in ACC is evolving, and the results of four small clinical trials were recently published. In a phase Ib clinical trial, avelumab (anti–programmed cell death ligand-­1 antibody) was studied in 50 ACC patients, out of whom three (6%) had partial response and 21 (42%) had stable disease. It is important to note that half of the study participants were also on mitotane therapy, including two of the patients who had partial response.83 In a phase II clinical trial, nivolumab (anti–programmed cell death-­1 [PD-­1] monoclonal antibody) was used in 10 ACC patients but had minimal efficacy without objective responses, and only two patients showed stable disease.84 Pembrolizumab (anti–PD-­1 antibody) was studied in two parallel phase II clinical trials and was associated with partial response rates of 14% to 23%, with reasonable tolerance and low levels of severe adverse events.85,86 The role of using immunotherapy with mitotane or MKIs is emerging, and so far there are limited retrospective data to suggest the potential for enhancing the activity of immunotherapy when combined with mitotane or anti–vascular endothelial growth factor therapy.87,88 These early findings are encouraging to propose future clinical trials combining MKIs or mitotane with immune checkpoint inhibitors.

FOLLOW-­UP Close follow-­up is particularly important after seemingly complete surgery for ACC stage I to stage III, as surgery for tumor recurrence is an important treatment option. Initially, staging is repeated every 3 months for a minimum of 2 years (CT of abdomen + chest). Even after 2 years without recurrence, there remains a high risk for relapse. Thus, further follow-­up is required, but imaging intervals may increase. Steroid metabolomics are currently being investigated for their potential to detect tumor recurrence early and may lead to less frequent imaging and lower radiation exposure. Regular surveillance of the patient should continue for a minimum of 5 years.

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CHAPTER 98  Adrenocortical Carcinoma 69. Berruti A, Baudin E, Gelderblom H, et al. Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-­up. Ann Oncol. 2012;23:131–138. vii. 70. Redlich A, Boxberger N, Strugala D, et al. Systemic treatment of adrenocortical carcinoma in children: data from the German GPOH-­MET 97 trial. Klin Pädiatr. 2012;224:366–371. 71. Sperone P, Ferrero A, Daffara F, et al. Gemcitabine plus metronomic 5-­fluorouracil or capecitabine as a second-­/third-­line chemotherapy in advanced adrenocortical carcinoma: a multicenter phase II study. Endocr Relat Cancer. 2010;17:445–453. 72. Henning JEK, Deutschbein T, Altieri B, et al. Gemcitabine-­based chemotherapy in adrenocortical carcinoma: a multicenter study of efficacy and predictive factors. J Clin Endocrinol Metab. 2017;102:4323–4332. 73. Cosentini D, Badalamenti G, Grisanti S, et al. Activity and safety of temozolomide in advanced adrenocortical carcinoma patients. Eur J Endocrinol. 2019;181:681–689. 74. Kroiss M, Deutschbein T, Schlotelburg W, et al. Salvage treatment of adrenocortical carcinoma with trofosfamide. Horm Cancer. 2016;7:211–218. 75. Kroiss M, Deutschbein T, Schlotelburg W, et al. Treatment of refractory adrenocortical carcinoma with thalidomide: analysis of 27 patients from the European Network for the Study of Adrenal Tumours Registry. Exp Clin Endocrinol Diabetes. 2019;127:578–584. 76. Altieri B, Ronchi CL, Kroiss M, et al. Next-­generation therapies for adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab. 2020;34:101434. 77. Phan LM, Fuentes-­Mattei E, Wu W, et al. Hepatocyte growth factor/ cMET pathway activation enhances cancer hallmarks in adrenocortical carcinoma. Cancer Res. 2015;75:4131–4142. 78. Kroiss M, Megerle F, Kurlbaum M, et al. Objective response and prolonged disease control of advanced adrenocortical carcinoma with cabozantinib. J Clin Endocrinol Metab. 2020;105(5):1461–1468.

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79. Fassnacht M, Berruti A, Baudin E, et al. Linsitinib (OSI-­906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-­blind, randomised, phase 3 study. Lancet Oncol. 2015;16:426–435. 80. Naing A, Lorusso P, Fu S, et al. Insulin growth factor receptor (IGF-­1R) antibody cixutumumab combined with the mTOR inhibitor temsirolimus in patients with metastatic adrenocortical carcinoma. Br J Cancer. 2013;108:826–830. 81. De Martino MC, Feelders RA, Pivonello C, et al. The role of mTOR pathway as target for treatment in adrenocortical cancer. Endocr Connect. 2019;8:R144–R156. 82. Hahner S, Kreissl MC, Fassnacht M, et al. [131I]iodometomidate for targeted radionuclide therapy of advanced adrenocortical carcinoma. J Clin Endocrinol Metab. 2012;97:914–922. 83. Le Tourneau C, Hoimes C, Zarwan C, et al. Avelumab in patients with previously treated metastatic adrenocortical carcinoma: phase 1b results from the JAVELIN solid tumor trial. J Immunother Cancer. 2018;6(1):111. 84. Carneiro BA, Konda B, Costa RB, et al. Nivolumab in metastatic adrenocortical carcinoma: results of a phase 2 trial. J Clin Endocrinol Metab. 2019;104:6193–6200. 85. Habra MA, Stephen B, Campbell M, et al. Phase II clinical trial of pembrolizumab efficacy and safety in advanced adrenocortical carcinoma. J Immunother Cancer. 2019;7:253. 86. Raj N, Zheng Y, Kelly V, et al. PD-­1 blockade in advanced adrenocortical carcinoma. J Clin Oncol. 2020;38(1):71–80. 87. Head L, Kiseljak-­Vassiliades K, Clark TJ, et al. Response to immunotherapy in combination with mitotane in patients with metastatic adrenocortical cancer. J Endocr Soc. 2019;3:2295–2304. 88. Bedrose S, Miller KC, Altameemi L, et al. Combined lenvatinib and pembrolizumab as salvage therapy in advanced adrenal cortical carcinoma. J Immunother Cancer. 2020;8(2):e001009.

99 Pheochromocytoma Karel Pacak, Henri J.L.M. Timmers, David Taieb, Jacques W.M. Lenders, and Graeme Eisenhofer

OUTLINE Introduction and History, 1642 Catecholamine Production, Secretion, and Metabolism, 1643 Clinical Presentation, 1643 Who to Test According to Presentation?, 1644 Signs and Symptoms, 1644 Differential Diagnosis, 1644 Pathology, 1645 Genetics of Pheochromocytoma, 1646 Multiple Endocrine Neoplasia Syndromes, 1646 Von Hippel–Lindau Syndrome, 1646 Neurofibromatosis Type 1, 1648 Succinate Dehydrogenase Gene Family, 1648 Less Common Hereditary or Other Causes, 1648 Biochemical Diagnosis of Pheochromocytoma, 1649 Preanalytics and Analytics, 1649 Interpretative Considerations, 1649

Follow-­Up Biochemical Testing, 1650 Localization of Pheochromocytoma, 1650 Anatomic Imaging, 1650 Differential Diagnosis, 1651 Functional Imaging, 1651 Metaiodobenzylguanidine Scintigraphy, 1651 Positron Emission Tomography, 1652 Management of Pheochromocytoma, 1652 Medical Therapy and Preparation for Surgery, 1653 Postoperative Management, 1654 Special Presentations and Therapeutic Problems, 1655 Multifocal and Metastatic Pheochromocytoma/ Paraganglioma, 1655 Pediatric Pheochromocytoma, 1656 Pheochromocytoma in Pregnancy, 1657 Future Directions, 1657

 

INTRODUCTION AND HISTORY KEY POINTS  • Pheochromocytomas and paragangliomas are dangerous neuroendocrine (chromaffin cell) tumors that can have lethal consequences if overlooked but are typically curable by surgery. • Pheochromocytomas, which arise from adrenal medullary chromaffin tissue, are more common (80%–85%) than paragangliomas (15%–20%), which arise from extraadrenal paraganglia.

The term pheochromocytoma derives from the Greek words phaios (“dusky”) and chroma (“color”); it refers to the staining that occurs when the tumors are treated with chromium salts. The first diagnosis of pheochromocytoma was made in 1886 by Fränkel, and the first large series of successful surgical removals with pharmacologic blockade was reported by Kvale and colleagues in 1956. Pheochromocytomas are rare but treacherous catecholamine-­producing tumors that, if missed or not properly treated, almost invariably prove fatal.1 Prompt initial diagnosis therefore is essential for effective treatment, usually by surgical resection. The clinical manifestations of the tumor are diverse and can mimic a variety of conditions, often resulting in erroneous diagnoses.1,2 Pheochromocytomas comprise 80% to 85% of all chromaffin cell tumors and typically arise from adrenal medullary chromaffin tissue. In approximately 15% to 20% of cases tumors arise from extraadrenal paraganglia and are commonly referred to as paragangliomas.

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Paragangliomas are divided into two groups: those that arise from parasympathetic paraganglia of the skull base and neck (e.g., glomus: caroticum, jugulare, tympanicum, and vagale) as well as anterior mediastinum and those that arise from sympathetic-­associated chromaffin tissue. Sympathetic paragangliomas arise from sympathetic ganglia in the abdomen (90%), less commonly from the pelvis, and rarely from the posterior mediastinum (2%). Many abdominal paragangliomas arise from large sympathetic ganglia located around the origin of the inferior mesenteric artery called the organ of Zuckerkandl.2 Pheochromocytoma occurs at any age, but most often in the fourth and fifth decades, and it occurs equally in men and women. In this chapter, the term “pheochromocytoma” is used to collectively refer to the tumors at both adrenal (pheochromocytoma) and extraadrenal locations (sympathetic paraganglioma), but excluding those at head and neck, as dealt with separately. Most pheochromocytomas arise sporadically, but up to 35% are hereditary.3,4 In contrast to sporadic pheochromocytomas that are usually solitary, hereditary pheochromocytomas are often multifocal and bilateral.5 An early autopsy series indicated a prevalence of pheochromocytoma of 1:1000; significant numbers of tumors remained undiagnosed until death, and up to 50% of these may have contributed to patient mortality.6 Since then there have been numerous advances in genetics, biochemical diagnosis, localization, and surgical approaches to pheochromocytoma. According to a Dutch nationwide study,7 there has been an increase in annual incidence of pheochromocytoma to eight cases per million, this presumably reflecting improved detection.

CHAPTER 99  Pheochromocytoma

Adrenomedullary chromaffin cells

PNMT NE

COMT NMN

NE

E COMT E

MN

Bloodstream 7% DHPG

25%

NE 93%

NMN

MAO

NE

90%

MN 10%

U2

COMT NE

E

75%

U2 NE NE

U1

95%

E MN COMT

NMN

MHPG MAO

Extraneuronal Sympathetic nerve cells varicosities Figure 99.1  Pathways of catecholamine uptake, vesicular storage and leakage, secretion, and metabolism in sympathetic varicosities compared with adrenal medullary chromaffin cells and other extraneuronal cells. NE, norepinephrine; E, epinephrine; NMN, normetanephrine; MN, metanephrine; DHPG, dihydroxyphenylglycol; MHPG, methoxyhydroxyphenylglycol; PNMT, phenylethanolamine-­N-­ methyltransferase; COMT, catechol-­O-­methyltransferase; MAO, monoamine oxidase; U1, uptake 1 (by neuronal transporters); U2, uptake 2 (by extraneuronal transporters). Percentages indicate proportions of circulating amines derived from intraadrenal vs. intraneuronal or extraneuronal metabolism. Note: most metabolism occurs initially intraneuronally by deamination of NE leaking from storage vesicles, whereas O-­methylation is a relatively minor pathway but important in adrenal chromaffin cells and their tumor derivatives.

CATECHOLAMINE PRODUCTION, SECRETION, AND METABOLISM KEY POINTS  • The major hormonal products of pheochromocytomas and paragangliomas are the catecholamines, norepinephrine, and epinephrine; these are responsible for most of the clinical signs and symptoms of these tumors. • Most catecholamines produced by the pheochromocytomas and paragangliomas are metabolized to metanephrines within tumor cells rather than after secretion.

Pheochromocytomas are often described as tumors that secrete catecholamines. The tumors, however, also metabolize catecholamines, and this is a more consistent process than catecholamine secretion (Fig. 99.1).8 Failure to recognize this key feature probably reflects several misconceptions about the storage and metabolism of catecholamines, perhaps the most pervasive being that metabolism occurs after catecholamine secretion. In fact, substantial amounts of

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catecholamines are metabolized in the same cells where the amines are synthesized (Fig. 99.1).9 Because the enzymes responsible for the metabolism of catecholamines have intracellular locations, the primary mechanism limiting the lifespan of catecholamines in the extracellular space is uptake by active transport, not metabolism by enzymes.10 Catecholamine metabolism occurs through an array of enzymes, resulting in a variety of metabolites, but in general the most important first step is metabolism within cells of synthesis, and primarily from catecholamines leaking from storage vesicles into the cytoplasm (Fig. 99.1).9 Most norepinephrine is synthesized within sympathetic neurons; because these neurons contain monoamine oxidase, but not catechol-­O-­methyltransferase, initial metabolism is by deamination, mainly from leakage of the catecholamine from vesicles and secondly from norepinephrine recaptured after secretion. In contrast to neurons, chromaffin cells contain catechol-­O-­ methyltransferase so that these cells, unlike neurons, produce methoxytyramine from dopamine, normetanephrine from norepinephrine, and metanephrine from epinephrine (Fig. 99.1).11 These O-­methylated metabolites are produced in small amounts and either only within chromaffin cells after leakage from storage vesicles or in other extraneuronal cells, after uptake following secretion of the catecholamines from sympathetic nerves or the adrenals. The single largest source of O-­methylated metabolites represents adrenal chromaffin cells, which account for more than 90% of circulating metanephrine and approximately 25% of circulating normetanephrine (Fig. 99.1).11 Because these metabolites are similarly produced in chromaffin cell tumors from catecholamines continuously leaking from storage vesicles, a process that is independent of catecholamine secretion, they are superior as diagnostic biomarkers compared with the parent catecholamines. The final main product of the deamination pathway is vanillylmandelic acid (VMA).9 Because rates of catecholamine metabolism through the deamination pathway are far larger than for pathways involving extraneuronal and chromaffin cell O-­methylation, VMA and other deaminated metabolites are poor biomarkers of catecholamine-­ producing tumors. In contrast to norepinephrine and epinephrine, most dopamine in the body is produced in peripheral tissues other than sympathetic nerves and chromaffin cells.12 Gastrointestinal tissues are a particularly major source of dopamine, large amounts of which are metabolized to sulfate-­conjugated derivatives by a specific sulfate-­conjugating enzyme confined to these tissues.13 That same enzyme is also responsible for sulfate conjugation of O-­methylated metabolites, which are commonly measured in urine as metanephrines after acid hydrolysiscatalyzed deconjugation; thus, these urinary deconjugated metabolites are largely distinct from the free metanephrines usually measured in plasma. Urinary free dopamine and methoxytyramine are derived from renal uptake and decarboxylation of circulating L-­ dopa,14,15 which explains why these metabolites in urine are poor biomarkers of dopamine-­producing tumors.

CLINICAL PRESENTATION KEY POINTS  • The clinical manifestations of a tumor are diverse, variable, and typically non-specific. This can make pheochromocytoma difficult to recognize. • Signs and symptoms are typically paroxysmal in nature (spells). Spells can occur spontaneously or be triggered by various factors, including certain medications. • Careful assessment of key signs and symptoms determine the indications for further testing and can assist with interpretation of test results.

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PART 7  Adrenal

TABLE 99.1  Signs and Symptoms of

Pheochromocytoma Symptoms

Signs

Headaches Palpitations

++ +++#

Sweating Anxiety/nervousness Tremulousness Nausea/emesis Pain in chest/abdomen

+++# ++ ++# ++# ++

Weakness/fatigue Dizziness Heat intolerance Paresthesias

+++# + + +

Constipation Dyspnea Visual disturbances Seizures, grand mal

++# + + very rare

Hypertension Tachycardia or reflex bradycardia Postural hypotension Hypertension, paroxysmal Weight loss Pallor Hypermetabolism Fasting hyperglycemia Tremor Increased respiratory rate Decreased gastrointestinal motility Psychosis (rare) Flushing, paroxysmal (rare)

++++ +++=# ++ ++ ++# ++# ++ ++ ++# ++ ++ very rare +

#indicates

a significant difference between patients with and without pheochromocytoma/paraganglioma. Incidence: ++++, 76%–100%; +++, 51%–75%; ++, 26%–50%; +, 1%–25%. Adapted from Lenders et al.1 and Geroula et al.16

Who to Test According to Presentation? The following patients should be evaluated for a pheochromocytoma: 1) anyone with paroxysmal profuse sweating, pallor, tremor, and tachycardia, especially in a subject with episodic hypertension; 2) as part of routine screening for anyone with a known mutation of one of the susceptibility genes and/or a family history of pheochromocytoma or past history of pheochromocytoma; 3) anyone with an incidentally discovered adrenal mass or mass at an extraadrenal location that may represent a paraganglioma; and 4) anyone who has had episodic hypertension, stroke, tachycardia, or an arrhythmia in response to anesthesia, surgery, medications, or food known to precipitate symptoms in patients with pheochromocytoma (Table 99.1). Among ptients evaluated as part of routine screening or because of an incidentaloma, signs and symptoms of catecholamine excess are not always present or, when present, may be overlooked in part due to their nonspecific nature.16 Nevertheless, for all presentations, there should be careful consideration of the nature of key signs and symptoms to ascertain or pretest likelihood of disease and thereby further interpret results of subsequent biochemical testing.

Signs and Symptoms The clinical signs and symptoms related to the presence of pheochromocytoma, as listed in the Table 99.1, result from hemodynamic and metabolic actions of excessive circulating catecholamines and rarely of other amines or cosecreted neuropeptides. Most signs and symptoms considered alone are nonspecific; nevertheless, the episodic presence of palpitations, tremor, and sweating in patients with hypertension with or without pallor should arouse immediate suspicion for a pheochromocytoma, because these symptoms and signs are significantly more prevalent among patients with than without pheochromocytoma.16 Paroxysmal hypertension is one of the most common clinical signs (40%–50%). The extent of the increase and types of catecholamine

secreted, as well as the nature of episodic versus continuous secretion, are important determinants of signs and symptoms. Moreover, adrenoceptor downregulation caused by consistently high circulating catecholamines as well as tumor necrosis or hemorrhage may significantly impact pheochromocytoma-­associated elevations in blood pressure. Pheochromocytoma also may present with hypotension, particularly in patients with epinephrine-­secreting tumors. Pheochromocytoma-­induced attacks (so-called spells) may usually last from a few seconds to 1 hour, with intervals between attacks varying widely; some occur only once every few months. Initially, the episodes may be mild, of short duration, and infrequent. A typical paroxysm is often characterized by a sudden major increase in blood pressure or heart rate associated with a constellation of one or more signs and symptoms: a severe, often pounding headache; profuse sweating over most of the body, especially the trunk; palpitations with tachycardia; anxiety or a sense of doom; skin pallor, especially on the face; nausea, with or without emesis; and pain in the abdomen, the chest, or both. After an episode, patients usually feel drained, exhausted, and weak, and some may urinate more frequently. Unusual symptoms related to paroxysmal blood pressure elevations during procedures such as endoscopy or administration of anesthesia (particularly during the induction phase of general anesthesia), or associated with ingestion of food or beverages containing tyramine, should arouse immediate suspicion of pheochromocytoma.17,18 The use of certain drugs such as metoclopramide, droperidol, glucocorticoids, monoamine oxidase inhibitors, tricyclics and other antidepressants, opiates (e.g., morphine, fentanyl), naloxone, glucagon, certain antibiotics (linezolid), drugs for obesity management (phentermine, sibutramine), and chemotherapy may precipitate a hypertensive episode.17,18 Causes of episodic catecholamine secretion often remain unclear, but in some situations a surge in catecholamine secretion may be caused by intentional or accidental tumor manipulation with or without an increase in intraabdominal pressure from palpation, defecation, severe stressful situations, or pregnancy. However, approximately 8% to 10% of patients may be completely asymptomatic; this is often the situation for small (24–48 hours), a shunt procedure may be performed. Typically, a distal shunt between the corpora and the glans is created using a percutaneous or open technique,126-­128 and tunneling procedures may also be indicated in refractory cases.127-­130 For patients with ischemia lasting longer than 2 days, early implantation of a penile prosthesis may be an option if there is zero response to injectable phenylephrine. Early prosthesis may result in fewer intraoperative and postoperative complications than implantation delayed for several months, with patients reporting higher satisfaction and less penile shortening with early implantation.131 Early implantation of a penile prosthesis is not obligatory. Nonischemic priapism is less common and is almost always the consequence or perineal or penile trauma.132 This condition is not a medical emergency. After trauma, vascular injury to the cavernosal artery or helicine arteries is exacerbated by cyclical penile erection.133,134 When a fistula forms between an artery and the sinusoidal spaces of the corpora, unregulated arterial inflow results in persistent, painless penile tumescence. Because venous drainage is preserved, the penile tissue does not become ischemic, and the condition is generally painless.135 Treatment progresses from least to most invasive and includes observation with penile compression, medical management with androgen ablation, selective arterial embolization, and surgical intervention as the last resort.121 Androgen ablation, which reduces sleep-­ related erection to facilitate closure of the vascular injury, for 1 to 3 months can provide an effective option if there is damage to the cavernous artery.133,134 If androgen ablation fails, more invasive therapies are then recommended.136 Recurrent ischemic, or stuttering, priapism is defined as periodic episodic prolonged erections that may evolve into ischemia priapism. Because this condition affects patients with sickle cell disease more

CHAPTER 109  Erectile Dysfunction commonly than others, microvascular occlusion secondary to hemolysis with stasis of sickled erythrocytes is thought to be a significant contributing factor.137 Animal models and clinical studies have revealed a role for improper PDE5 regulation and abnormal NO and cGMP signaling and regulation in the pathogenesis of stuttering priapism.138-­140 Therapeutic options for this condition are limited, and the primary goal of contemporary management of stuttering priapism is to prevent additional episodes to reduce the risk of progression to a major ischemic event.141 Androgen ablation therapy is an effective, proven therapy to eliminate the erection-­promoting effects of androgens.142 In men with sickle cell disease, treatment with continuous, low-­dose daily PDE5 inhibitors may normalize dysregulated PDE5 levels and decrease the frequency of priapism episodes.143,144

KEY POINTS • Patients may refer to all sexual complaints, including problems with penile sensation, premature/delayed ejaculation, emission, orgasm, or penile deformity, as ED. Recording a good sexual history is essential to defining the actual sexual concerns as each issue has specific treatment options. Peyronie’s disease is an acquired, progressive, connective tissue disease of the tunica albuginea that causes penile deformities, such as curvature, hourglass, indent, and shortening. Priapism is defined as a prolonged penile erection that is independent of sexual stimulation. There are two main categories of priapism: ischemic (emergency) and non-­ischemic (nonurgent). Ejaculatory disorders and orgasmic disorders may also present as ED.

FUTURE DIRECTIONS FOR THE TREATMENT OF ERECTILE DYSFUNCTION With the exceptions of lifestyle modifications and revascularization procedures, currently available interventions for ED merely treat the problem, offering symptomatic relief rather than a cure for the underlying disease process. The collective desire to develop a cure has stimulated interest in development of novel therapeutics for ED. Multiple preliminary trials have investigated the restorative potential of promising regenerative therapies, including stem cell therapy (SCT), low-­ intensity extracorporeal shock wave therapy (LiESWT), and platelet-­rich plasma (PRP) injection therapy. The underlying mechanisms and the short-­and long-­term biological effects of these therapies remain to be fully elucidated. Well-­designed, rigorous studies are needed to further define the mechanisms of action, safety profiles, efficacy, and protocols for treatment in humans. At this time, these treatments should be reserved for use only in the context of institutional review board–approved clinical trials until such time as a sufficient body of evidence has demonstrated their efficacy and safety.3

New Pharmacotherapeutics Ongoing basic science research is attempting to identify effective therapeutics that target alternative erectogenic molecular pathways for PDE5 inhibitor–refractory patients. There is a specific need for pharmacotherapeutics that induce smooth muscle relaxation in the absence of NO. ROCK inhibitors and soluble guanylyl cyclase activators are of particular interest for PDE5 inhibitor nonresponders, as they are independent of the NO pathway. The ROCK pathway plays an important role in maintaining the flaccid state of the penis, in that ROCK phosphorylates and inactivates myosin light chain phosphatase, allowing myosin light chain to stay bound to smooth muscle actin.145 Inhibition of ROCK produces a mechanism for smooth muscle relaxation. Soluble guanylyl cyclase is

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an enzyme in the NO pathway that can be directly activated to result in smooth muscle relaxation in the absence of NO production. Further research is warranted regarding these and other pathways and to identify additional promising treatments for ED patients.

Stem Cell Therapy Over the past decades, there has been considerable excitement about the potential use of stem cell (SC)-­based therapies in medicine in general and in the field of urology in specific.146 SCT has been in use in other fields, such as for graft-­versus-­host-­disease to enhance engraftment in patients receiving allogeneic hematopoietic SC transplantation, since the 1990s.147 In addition, the antiinflammatory, restorative, and immunomodulatory qualities of SCs are currently being exploited clinically to treat hematologic diseases,148-­150 CVD,151 neurologic diseases such as Parkinson disease152 and stroke,153 and autoimmune disease such as multiple sclerosis153,154 and lupus,155 as well as refractory wounds,156 spinal cord injury,157 and cartilage defects.158 It makes sense that the field of urology would also be interested in utilization of this type of approach for acute and chronic urologic illnesses. SCs have been investigated for ED based on the premise that SCs can differentiate into various cell types, including endothelial cells, smooth muscle cells, Schwann cells, and neurons.159 One hypothesis suggests that SC transplantation into the penis may replenish these cell concentrations in the corporal tissue in order to restore erectile function. An alternative hypothesis is that transplanted SCs may encourage regeneration of the host’s own endothelial and smooth muscle cells or may restore proper interactions between these cells via a paracrine effect.160 The exact mechanisms through which SCs may augment erectile function are poorly understood at this time. There have been many preclinical studies utilizing SCs in animal models of ED. For animal trials, most authors utilize an animal model that simulates either an acute iatrogenic trauma to the neurovascular bundle or a chronic disease state, such as aging, or hyperlipidemia. In acute ED models, the mechanism of action of SCs is presumed to be by paracrine action.161-­163 In contrast, in chronic ED the theoretical method of SC action is proposed to be both engraftment and cellular differentiation.164 However, the exact mechanism of action of SCs in the treatment of ED remains uncertain.165 There have been a limited number of clinical trials investigating SC therapy for male sexual dysfunction. Small studies have utilized SCs in human patients with no adverse outcomes, but little can be concluded from these studies regarding the potential restorative effects of SCT. Many questions remain unanswered. Future studies should focus on robust, placebo-­controlled, double-­blind, randomized design in order to assess the true efficacy of SCT for male sexual dysfunction.

Low-­Intensity Extracorporeal Shock Wave Therapy On the horizon for the treatment of ED is LiESWT, modified as low-­intensity pulsed ultrasound or low-­energy shock wave therapy (LESWT) in different studies. Provoking widespread enthusiasm from urologists and patients alike for novel noninvasive therapies, LiESWT has already been discussed in the first-­line therapy section of the European Association of Urology Sexual Dysfunction Guidelines.166 The FDA has not yet approved LiESWT for mainstream treatment of ED in the United States, as it is important first to establish the mechanisms of action of the treatment in addition to performing rigorous clinical trials with large numbers of patients, using validated and standardized protocols, and ensuring long-­term follow-­up to ensure the safety and efficacy of treatment. Many studies have investigated the mechanisms of action of LiESWT for ED, and it is believed that this therapy has the ability to activate SCs in situ to produce such downstream effects as endogenous

l

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PART 9  Male Reproduction

SC differentiation with subsequent angiogenesis and tissue regeneration. Animal studies reveal that LiESWT stimulates cell proliferation through activation of integrin receptors and the Rho/ROCK/Src/ERK signaling pathway167 and by promoting multilineage differentiation of mesenchymal SC lines through the ROCK-­Cot/Tpl2-­MEK-­ERK signaling pathway.168 Additionally, endogenous SC recruitment and Schwann cell activation coincided with angiogenesis, tissue regeneration, and nerve generation in a rat model of pelvic neurovascular injury after LiESWT treatment.169 The angiogenesis-­promoting effect of LiESWT is believed to involve upregulation of vascular endothelial growth factor. Additionally, LESWT has been shown to markedly improve erectile function170 through downregulation of receptors for advanced glycation end products171 and through recruitment of endogenous mesenchymal SCs.172 There have been many small, short-­term clinical trials and subsequent systematic reviews and metaanalyses to evaluate the efficacy of LiESWT for the treatment of ED. Though there is heterogeneity regarding study design, treatment protocol (dosing, frequency, number of shocks, linear versus focuses device), and follow-­up interval, the overall conclusions suggest that there is clinical improvement with LiESWT, with no significant adverse events reported. Proponents of LiESWT suggest that this regenerative treatment modality will be able to potentially mitigate or eliminate the safety and methodological concerns about SC acquisition, preparation, and delivery and also reduce or eliminate the need for expensive and invasive medical and surgical therapies for ED in many patients. As with all regenerative therapies, the underlying mechanisms of therapeutic ultrasound and the biological effects it has on the human body remain to be thoroughly investigated. Additional well-­designed, rigorous studies are needed to further define the mechanisms of action, safety profile, efficacy, and optimal treatment protocols for this promising therapy.

Platelet-­Rich Plasma In recent years, PRP injection therapies have been utilized in many fields of medicine in hopes of restoring structure and function to diseased tissues. This therapy is based on the hypothesis that biologically active molecules including growth factors can upregulate downstream cell regeneration. For the PRP procedure, whole blood is withdrawn from the patient and then centrifuged to produce autologous plasma containing four times the normal concentration of platelets and growth factors. This concentrated plasma is then injected into the structure of concern. Beyond promising initial studies, which were limited by a low number of animals and unconventional ICP monitoring techniques, there has been a lack of significant basic science exploring the mechanisms and efficacy of PRP for ED. Additional animal studies are necessary for elucidation of the mechanisms of RPR in erectile recovery. Across the globe, however, there are already human trials underway to investigate the use and safety of PRP in human subjects. Thus far, there have been no major adverse outcomes, and no patients have reported worsening of their ED. These studies, however, have had low patient numbers, have suffered from the lack of a control group, and have shown uncertain efficacy. Overall, current data suggest that PRP KEY POINTS • Multiple regenerative therapies, including stem cell therapy, low intensity extracorporeal shock wave therapy, and platelet rich plasma, are under investigation in hopes of providing either a durable treatment or curative therapy for ED. At the time of this writing, these potentially restorative and regenerative therapies remain under investigation and are not recommended by professional guidelines for the treatment of ED outside of clinical trials.

for ED is safe in humans, but no studies have determined whether there is any long-­term functional benefit in the context of sexual medicine.173 Good-­quality randomized, prospective, placebo-­controlled trials are needed before PRP can be recommended as a therapy for ED.

CONCLUSIONS Over the past three decades, the treatment algorithm for ED has been transformed by discovery. Whereas psychosexual therapy was once the only therapeutic option, patients can now choose from multiple efficacious pharmacologic agents and progress, if necessary, to highly effective and safe surgical interventions. New and improved therapies for ED will continue to emerge as our understanding of the basic mechanisms of penile erection continues to evolve. Penile erection is a complex physiological phenomenon that is reliant not only on neurovascular components, but also on psychological wellness. A thorough understanding of the mechanisms of penile erection and the medical, surgical, and psychosocial conditions that contribute to ED facilitates the evaluation and management of patients with this distressing condition.

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PART 9  Male Reproduction

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153. Bang OY, Lee JS, Lee PH, et al. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol. 2005;57:874. 154. Lee JS, Hong JM, Moon GJ, et al. A long-­term follow-­up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke. Stem Cell. 2010;28:1099. 155. Sun L, Akiyama K, Zhang H, et al. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cell. 2009;27:1421. 156. Dash NR, Dash SN, Routray P, et al. Targeting nonhealing ulcers of lower extremity in human through autologous bone marrow-­derived mesenchymal stem cells. Rejuvenation Res. 2009;12:359. 157. Pal R, Venkataramana NK, Bansal A, et al. Ex vivo-­expanded autologous bone marrow-­derived mesenchymal stromal cells in human spinal cord injury/paraplegia: a pilot clinical study. Cytotherapy. 2009;11:897. 158. Wakitani S, Okabe T, Horibe S, et al. Safety of autologous bone marrow-­ derived mesenchymal stem cell transplantation for cartilage repair in 41 patients with 45 joints followed for up to 11 years and 5 months. J Tissue Eng Regen Med. 2011;5:146. 159. Lin CS, Xin ZC, Deng CH, et al. Recent advances in andrology-­related stem cell research. Asian J Androl. 2008;10:171. 160. Lin CS. Advances in stem cell therapy for the lower urinary tract. World J Stem Cells. 2010;2:1. 161. Bochinski D, Lin GT, Nunes L, et al. The effect of neural embryonic stem cell therapy in a rat model of cavernosal nerve injury. BJU Int. 2004;94:904. 162. Kendirci M, Trost L, Bakondi B, et al. Transplantation of nonhematopoietic adult bone marrow stem/progenitor cells isolated by p75 nerve growth factor receptor into the penis rescues erectile function in a rat model of cavernous nerve injury. J Urol. 2010;184:1560. 163. Zhang H, Yang R, Wang Z, et al. Adipose tissue-­derived stem cells secrete CXCL5 cytokine with neurotrophic effects on cavernous nerve regeneration. J Sex Med. 2011;8:437. 164. Soebadi MA, Moris L, Castiglione F, et al. Advances in stem cell research for the treatment of male sexual dysfunctions. Curr Opin Urol. 2016;26:129. 165. Albersen M, Lin CS, Lue T. Stem-­cell therapy for erectile dysfunction. Arab J Urol. 2013;11:237. 166. Hatzimouratidis K, Eardley I, Giuliano F, et al. EAU guidelines on penile curvature. Eur Urol. 2012;62:543. 167. Zhou S, Schmelz A, Seufferlein T, et al. Molecular mechanisms of low intensity pulsed ultrasound in human skin fibroblasts. J Biol Chem. 2004;279:54463. 168. Kusuyama J, Bandow K, Shamoto M, et al. Low intensity pulsed ultrasound (LIPUS) influences the multilineage differentiation of mesenchymal stem and progenitor cell lines through ROCK-­Cot/Tpl2-­MEK-­ERK signaling pathway. J Biol Chem. 2014;289:10330. 169. Li H, Matheu MP, Sun F, et al. Low-­energy shock wave therapy ameliorates erectile dysfunction in a pelvic neurovascular injuries rat model. J Sex Med. 2016;13:22. 170. Vardi Y, Appel B, Jacob G, et al. Can low-­intensity extracorporeal shockwave therapy improve erectile function? A 6-­month follow-­up pilot study in patients with organic erectile dysfunction. Eur Urol. 2010;58:243. 171. Liu J, Zhou F, Li GY, et al. Evaluation of the effect of different doses of low energy shock wave therapy on the erectile function of streptozotocin (STZ)-­induced diabetic rats. Int J Mol Sci. 2013;14:10661. 172. Qiu X, Lin G, Xin Z, et al. Effects of low-­energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med. 2013;10:738. 173. Campbell JD, Milenkovic U, Usta MF, et al. The good, bad, and the ugly of regenerative therapies for erectile dysfunction. Transl Androl Urol. 2020;9:S252.

110 Misuse and Abuse of Anabolic Hormones Thiago Gagliano-­Jucá and Shehzad Basaria

OUTLINE AAS: Use, Misuse, and Abuse, 1846 Use, 1846 Misuse, 1846 Abuse, 1846 History and Epidemiology of Anabolic Steroid Abuse, 1847 History of Anabolic Steroid Abuse, 1847 Epidemiology of Anabolic Steroid Abuse, 1848 Ergogenic Potential of Anabolic Steroids, 1848 Types and Patterns of Androgen Doping, 1848 Direct Doping, 1849 Indirect Doping, 1850 Detection of Androgen Doping, 1850 Detection of Compounds Used in Direct Androgen Doping, 1850 Detection of Compounds Used in Indirect Androgen Doping, 1852 Evaluation Process of AAS Abuse in Elite Athletes, 1852 Assessment of Men Abusing AAS in the Clinics, 1852 Strategies Used by Athletes to Avoid Detection of Androgen Abuse, 1853

Administration of Epitestosterone, 1853 Masking Agents, 1853 Health Consequences of Anabolic Steroid Abuse, 1853 Cardiovascular, 1853 Reproductive, 1855 Gynecomastia, 1855 Hepatic, 1855 Musculoskeletal, 1855 Renal, 1855 Dermatologic, 1856 Neuropsychiatric and Behavioral, 1856 Dependence, 1856 Management Options of Anabolic Steroid Abuse, 1856 Elite Athletes, 1856 General Public, 1856 Conclusion, 1857

 

Anabolic androgenic steroids (AAS) are a group of steroid hormones, including testosterone and its synthetic derivatives, that have been in use for the past eight decades. In addition to their anabolic effects on the skeletal muscles and bone, androgens promote and maintain male secondary sexual characteristics, sexual function, and sexual behavior, and are used in the treatment of male androgen deficiency. However, there is an important distinction between treatment with testosterone at physiologic doses in organic male hypogonadism and abuse of AAS as pharmacotherapy for the purposes of achieving competitive advantage in sports or for “image enhancement” for cosmetic reasons. Indeed, few topics in medicine have generated more interest and controversy, both among the clinicians and lay public, than abuse of AAS for nonmedical reasons. This controversy is further compounded by the fact that the long-­term safety of these steroids remains unclear. Additionally, there are no effective strategies to treat men who have discontinued or plan to discontinue AAS and are either experiencing or are concerned about AAS-­withdrawal syndrome. The paucity of high-­ quality data in these areas foments the disregard toward medical advice related to adverse effects associated with AAS abuse,1,2 with users often assuming that the physicians are misinformed regarding the consequences of AAS abuse.3 This chapter focuses on the pharmacological use of AAS in men, including (1) the history and epidemiology of AAS abuse, (2) types of doping, (3) methods used in the detection of AAS, (4) adverse health

1846

consequences of AAS abuse, and (5) management options in men who are rendered hypogonadal as a result of prior AAS abuse.

AAS: USE, MISUSE, AND ABUSE At the beginning of this chapter, we highlight various terms associated with the use of AAS.

Use The use of AAS implies taking prescription androgens (mainly testosterone) for medical conditions. An example is the treatment of hypogonadal men with organic diseases of the hypothalamus, pituitary, or testes. Testosterone therapy, at physiologic dose, is indicated to induce or maintain secondary sexual characteristics.

Misuse The misuse constitutes systematic prescribing of AAS for unproven medical indications in men who do not have organic androgen deficiency. This includes prescribing androgens to treat obesity, diabetes, impaired cognition, or reverse aging.

Abuse The abuse of AAS constitutes their use for nonmedical purposes where they are often used in supraphysiologic doses for either the purposes of performance enhancement (competitive sports) or image

CHAPTER 110  Misuse and Abuse of Anabolic Hormones

1939

Ruzicka and Butenandt isolate testosterone

Russians use AAS in weightlifting competition in Vienna

1968

1991

Establishment of WADA by IOC

Enactment of the Anabolic Steroid Control Act of 2004

2007

1988

East German state-sponsored doping program exposed Anabolic Steroid Control Act of 1990 classifies AAS as schedule III controlled substances

1999

1977

American College of Sports Medicine revises statement saying that AAS might be ergogenic Federal Anti-Drug Abuse Act of 1988 makes AAS distribution a felony

1990

1954

International Olympic Committee bans AAS

American College of Sports Medicine states that AAS are not ergogenic

1987

1847

2004

George Mitchel publishes report on AAS in Major League Baseball Designer Steroid Control Act closes loopholes and gives full regulation to the Justice Department

2014

Figure 110.1  Timeline of important events in the discovery and abuse of anabolic androgenic steroids (AAS). IOC, International Olympic Committee; WADA, World Anti-­Doping Agency.

enhancement. In these situations, AAS are usually procured illicitly from compounding pharmacies, fitness centers, or veterinary clinics. This chapter will focus on this patient population.

HISTORY AND EPIDEMIOLOGY OF ANABOLIC STEROID ABUSE History of Anabolic Steroid Abuse The culture of using ergogenic (performance-­enhancing) compounds among athletes dates to ancient Greece, where herbal remedies and animal extracts were used by athletes to gain competitive edge. Thus, the history of “doping” began long before the discovery of testosterone by Butenandt in the 1930s. However, the attribution of athletic prowess to testicular secretion dates back to the 18th century when experiments by John Hunter showed that testicular transplantation in any anatomical location maintained cock combs.4 In the 1870s, Charles Edouard Brown-­Séquard, a 72-­year-­old physician-­scientist, hypothesized that injections of testicular extracts from young animals possessed rejuvenating effects and decided to use himself as the first human subject. He self-­administered 10 doses of this extract subcutaneously over 2 weeks and reported improvement in strength and endurance.5 The news of Brown-­Séquard’s “Elixir of Life” quickly spread throughout Europe

and North America, where it was commercially sold for decades. However, replication of Brown-­Séquard’s published methods demonstrated that his elixir had insufficient concentrations of testosterone to produce a biological response6; thus, the birth of Andrology began with demonstration of the placebo effect. Six decades later, Butenandt and Ruzicka won the Nobel Prize for the synthesis of testosterone. Subsequently, chemists and compounding pharmacies made numerous derivatives of testosterone in the 1950s. Although powerlifters were the first group of athletes to abuse AAS for competitions (Fig. 110.1),7 AAS abuse soon spread to other athletes, including those participating in the Olympic Games.8 The most well-­known phase of AAS abuse in the history of Olympics was the systematic administration of AAS to the Soviet weightlifting team in the 1952 and 1956 Olympic Games.8 In the United States, the credit for introducing AAS to American athletes goes to Dr. John Ziegler, a physician member of the US Weightlifting Team, who learned about the use of AAS by the Russian team during his trip to weightlifting championships in Vienna in 1954.9 Upon return, he administered testosterone to weightlifters in the York Babel Club in Pennsylvania. In the 1960s, the German Democratic Republic instituted a program of systematic doping of their Olympic athletes with AAS, which led to winning 153 Olympic gold medals from 1968 to 1988, despite having a population of only 18 million.7

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PART 9  Male Reproduction

While athletes were well aware of the ergogenic effects of AAS, the scientific community and the general public remained skeptical. Although the International Olympic Committee (IOC) prohibited the use of AAS in 1974, a position paper from the American College of Sports Medicine in 1977 still raised doubts regarding the ergogenic potential of AAS.10 It was not until 1996 that the effects of AAS on strength were demonstrated in a well-­controlled study.11 Currently, the scrutiny of doping in sports is conducted by the World Anti-­Doping Agency (WADA), a subsidiary of IOC. Every year WADA publishes an ever-­growing list of prohibited drugs; however, androgens remain the most commonly used ergogenic drugs in sports.7

Epidemiology of Anabolic Steroid Abuse The epidemiology of AAS abuse in athletes and in the general public has been difficult to ascertain.12,13 Questionnaire-­based studies of elite college athletes in the United States have demonstrated a lifetime prevalence of 20%. In one frequently cited study, Goldman et al. presented a Faustian bargain to world-­class athletes biannually between 1982 and 1995, asking if they would agree to take a drug that would guarantee an Olympic gold medal without being caught, but which would also result in their death within 5 years (The Goldman Dilemma).14 Interestingly, 52% of the athletes said they would accept the “death-­for-­gold” deal. In 2013, another study revisited the Goldman Dilemma in 212 elite track athletes and found that 12% would still take a hypothetical illegal performance-­enhancing drug if it was undetectable and guaranteed an Olympic gold medal, but only 2% would accept the death-­for-­gold deal.15 In the general American public, questionnaire-­based studies have suggested lifetime prevalence of up to 15%,13 with 98% of users being men.16 It is estimated that 3 to 4 million males between age 13 and 50 years in the United States have used AAS. The lifetime prevalence of AAS use is higher among gay and bisexual men (21%) as well as among ethnic minorities (25%).17 Over the decades, the abuse of AAS has permeated beyond competitive sports, as elite athletes account for only a small fraction of AAS users in the United States.16 Most of the users are noncompetitive weightlifters and men who are keen to acquire muscular appearance for image-­enhancing purposes. The desire to become muscular among some men is influenced by exposure to muscular male images in mass media while others may have muscle dysmorphia, a body image disorder that manifests as an obsessive preoccupation with a muscular appearance. It is estimated that approximately 45% of men with muscle dysmorphia may have used AAS.18 Worldwide, the lifetime prevalence of AAS abuse among men is estimated to be 1% to 5%. Although most Western cultures currently have an endemic problem with AAS abuse, East Asian cultures, such as Chinese and Japanese cultures, appear to be largely shielded from this public health issue; this is likely due to the fact that Confucian tradition associates masculinity with intellect and integrity rather than physical strength.19 In contrast, in the Nordic culture, where physical strength has been valued since the origins of Norse mythology, the lifetime prevalence of AAS abuse is high at 2.4% in Norwegians and 4.4% among the Swedes.20 KEY POINTS  • AAS abuse, initially limited to elite athletes, has now permeated the general population where it is mainly used for the purposes of image enhancement. • The lifetime prevalence of AAS abuse in the general population is estimated to be 5%, 98% of these being men.

ERGOGENIC POTENTIAL OF ANABOLIC STEROIDS While elite and amateur athletes had been aware of the anabolic effects of AAS on skeletal muscles for decades, the scientific community continued to attribute ergogenic effects of AAS solely to their influence on motivation, arguing that only men who trained harder and longer while taking AAS experienced improved performance. This skepticism started to abate in 1987 when the American College of Sports Medicine revised its original conclusion that AAS did not increase muscle strength.21 However, it was only in 1996 that the medical community fully accepted the anabolic effects of AAS after a well-­controlled study conclusively demonstrated that administration of supraphysiologic doses of testosterone (600 mg/week) increased muscle strength in young healthy men; this study also showed that the effects of testosterone and exercise were additive.11 Men who only received testosterone experienced an increase in fat-­free mass of 3.2 kg compared to the placebo plus exercise group who had an increase of 1.9 kg. On the other hand, the increase in fat-­free mass in the testosterone plus exercise group was 6.1 kg.11 The synergistic effects of testosterone and resistance training on muscle strength have since been verified in other studies.22 Similarly, studies have shown that suppression of endogenous testosterone in men reduces muscle mass and strength.23 The anabolic actions of AAS on skeletal muscle involve several mechanisms that include: (1) muscle fiber hyperplasia and hypertrophy, (2) differentiation of mesenchymal pluripotent stem cells toward myogenic rather than adipogenic lineage,24 (3) stimulation of skeletal muscle protein synthesis,25,26 and (4) reduction of muscle catabolism via inhibition of the ubiquitin proteasome and myostatin pathways.25,27 Testosterone administration also leads to the activation of muscle satellite cells.28,29 In addition to their direct effects via androgen receptor (AR), AAS also exert their anabolic effects via stimulation of growth hormone (GH) secretion. Indeed, testosterone replacement in hypogonadal men has been shown to increase serum concentrations of GH30 and an increase in intramuscular insulin-­like growth factor-­1 (IGF-­1) mRNA expression. Additionally, AAS may also improve physical performance via stimulation of erythropoiesis, which results in an increase in oxygen-­carrying capacity and endurance.

KEY POINTS  • The ergogenic effects of AAS are due to their direct anabolic action on skeletal muscles where they stimulate muscle fiber hyperplasia and hypertrophy, promote differentiation of mesenchymal pluripotent stem cells toward myogenic lineage, and increase protein incorporation. • The end result is an increase in muscle mass and strength. Additionally, stimulation of erythropoiesis by androgens increases endurance.

TYPES AND PATTERNS OF ANDROGEN DOPING Men who abuse AAS rely on two main doping strategies (Table 110.1). Indirect Doping: This involves use of drugs that increase endogenous testosterone production. Direct Doping: This includes direct use of both natural and synthetic steroids (including testosterone). In addition to traditional AAS, there has been a remarkable proliferation of new nonsteroidal anabolic agents that are increasingly being used in conjunction with traditional AAS, such as selective androgen receptor modulators (SARMs).31–33 The practice of abusing multiple anabolic agents simultaneously is known as “stacking,” which involves

CHAPTER 110  Misuse and Abuse of Anabolic Hormones

1849

TABLE 110.1  Drugs Used in Direct and

increasingly being used. The following are the classes of drugs used in direct doping.

Direct Doping

Indirect Doping

Naturally Occurring AAS. The naturally occurring steroids com-

Naturally Occurring Anabolic Steroids Testosterone Epitestosterone Dihydrotestosterone Nandrolone Boldenone

Testosterone Precursors DHEA Androstenedione

monly used in direct doping include testosterone, nandrolone, and boldenone. Testosterone esters are by far the most commonly abused AAS.34–36 The physiological dose of testosterone replacement in hypogonadal men is 100 mg every week intramuscularly; men who abuse testosterone may take as much as 6000 mg weekly. Testosterone is aromatized to estradiol by the aromatase enzyme complex via two successive hydroxylations on the C19 methyl group of testosterone; Nandrolone (19-­nortestosterone) is a penultimate step in the aromatization process,37 thus making it a naturally occurring AAS. Exogenous nandrolone is poorly aromatized, but it upregulates aromatase expression.38 Therefore, men “stacking” nandrolone with testosterone are more likely to develop gynecomastia. Nandrolone is also 5α-­reduced, a process that increases its androgenic effects. Nandrolone was previously used medically in the treatment of anemia of chronic kidney disease39 but is no longer manufactured in the United States. However, it still remains a popular AAS, both for the purposes of performance and image enhancement.34,36 Boldenone, also known as equipoise, is a naturally occurring androgen in animals that can rarely be detected in urine samples of nondoping athletes.40 Boldenone is currently marketed for veterinary purposes and is among the most abused AAS.

Indirect Doping

Synthetic Anabolic Steroids Clostebol Danazol Drostanolone Ethylestrenol Fluoxymesterone Gestrinone Methandienone Methenolone Methyltestosterone Norethandrolone Oxandrolone Oxymetholone Stanozolol Tibolone Trenbolone Designer Steroids Boldione (precursor to boldenone) Dimethazine Mentabolan Methasteron Methoxygonadiene Methylclostebol Methylepistiostanol Methylstenbone Methylstenbolone Prostanozolol (precursor to stanozolol)

Selective Estrogen ­Receptor Modulators Clomiphene Droloxifene Raloxifene Tamoxifen Toremifene Gonadotropins Human chorionic gonadotropin Recombinant human LH Aromatase inhibitors Anastrozole Letrozole Vorozole Atamestane Exemestane Testolactone Selective Estrogen ­Receptor Degraders Fulvestrant

Selective Androgen Receptor Modulators GTx-­024 LGD-­4033 RAD140 S-­4 S-­23 YK11

mixing a variety of oral and injectable drugs that are considered to have synergistic effects on muscle mass and strength. Some men follow the practice of “cycling,” with planned periods of AAS use (“on”) followed by abstinence (“off”). It is believed that the “off” period prevents desensitization of AR (an unproven concept). Some users apply the “blast and cruise” technique, in which continuous “blasts” of high-­ dose AAS use is interspaced with lower-­dose “cruise.”

Direct Doping This type of doping involves the direct use of testosterone and its steroid derivatives. Additionally, other compounds, such as SARMs (designed to have selective effects on some but not all tissues), are also

Synthetic Androgenic Steroids. Shortly after the isolation and synthesis of testosterone, several synthetic AAS were developed for clinical use (Table 110.1). Among the most commonly abused synthetic AAS are stanozolol, oxandrolone, trenbolone, and methandienone.34,36 Some of these steroids are still used for select medical conditions. For example, oxandrolone is often used as an anabolic agent in patients with severe burn injuries to counter catabolism41 and in patients with Turner syndrome receiving GH therapy. However, the remaining synthetic AAS developed are no longer used for medical purposes. Designer Steroids. A “designer steroid” is an AAS that is synthesized from a known parent compound and is chemically modified with the intent to circumvent substance control laws.42 These agents were initially sold legally as supplements until 2015 but were labeled as controlled substances after passing of the Designer Anabolic Steroid Control Act of 2014.43 The modifications in these compounds are also thought to alter the anabolic and androgenic effects of these steroids. Norbolethone has been credited as the first designer androgen that was originally discovered in the 1960s and its chemical signature was identified in 2002.44 A year later, tetrahydrogestrinone, a derivative of gestrinone (a progestin), was identified.45 In 2005, a third designer androgen, desoxymethyltestosterone, was identified. These compounds are banned by the World Anti-­Doping Agency (WADA), though their ergogenic potential remains unclear. SARMs. For decades, scientists have been on a quest to dissect out the anabolic properties of androgens from their androgenic potential. With the discovery of SARMs, this endeavor came to fruition, as these compounds achieved tissue selectivity by exerting anabolic effects on the skeletal muscle and bone while sparing the prostate. This tissue selectivity is considered important in the elderly, who are at a higher risk of prostate abnormalities.46 The first SARM was synthesized in 1998 and derived from AR antagonists, flutamide and bicalutamide. Over the years, four groups of SARMs have been synthesized: (1) arylpropionamides, (2) bicyclic hydantoins, (3) quinolines, and (4) tetrahydroquinolines.

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Indirect Doping

Androgen Precursors. This class of agents include drugs like

Indirect doping denotes use of drugs that increase endogenous testosterone secretion. This can be achieved with compounds that (1) stimulate the hypothalamic-­pituitary-­gonadal (HPG) axis such as estrogen receptor (ER) antagonists and aromatase inhibitors; (2) directly stimulate the testes, including luteinizing hormone (LH) and its analogs like human chorionic gonadotropin (hCG); and (3) testosterone precursors such as dehydroepiandrosterone (DHEA) and androstenedione (Table 110.1).

androstenedione and DHEA. Androstenedione is a prohormone that is produced in both the gonads and the adrenals in both sexes. It is synthesized from DHEA and is converted to testosterone by 17β-­ hydroxysteroid dehydrogenase. On the other hand, DHEA is predominantly secreted by the adrenal glands. Although the IOC had banned these compounds in the late 1990s, they were sold in an uncontrolled fashion as dietary supplements for the subsequent two decades under the Dietary Supplement Health and Education Act until 2004, when the US Congress added androstenedione to the list of banned drugs.55 Data suggest that androstenedione at a dose of 300 mg increases serum testosterone by 30%,56 whereas a dose of 1500 mg improves lean body mass and muscle strength. Similarly, DHEA at high doses (100 mg) improves muscle strength.57 Although banning of these prohormones by the IOC is justified because of their ergogenic potential, there is still no conclusive evidence that testosterone precursors significantly impact the performance of young athletes.

Estrogen Blockers. The rationale for using ER antagonists and aromatase inhibitors stems from the fact that estradiol is a more potent negative regulator of the HPG axis than testosterone.47 This is evident in men with congenital aromatase deficiency in whom serum concentrations of estradiol are undetectable but both serum testosterone and gonadotropins are elevated; estrogen administration suppresses the HPG axis in these patients.48 Aromatase is a product of the CYP19 gene and converts testosterone to estradiol. Aromatase inhibitors inhibit aromatase activity, thereby decreasing estradiol synthesis; as a result, serum testosterone concentrations increase by ∼50% to 65%,49 which can still induce myotrophic effects. Among steroidal aromatase inhibitors, aminoglutethimide was the first drug discovered in this class, followed by testolactone, atamestane, and exemestane. Nonsteroidal aromatase inhibitors include anastrozole, letrozole, and vorozole. The ER antagonists increase gonadotropins by blocking the negative feedback of estradiol on the hypothalamus and the pituitary, which in turn increases serum testosterone concentrations. The original antiestrogens were nonsteroidal drugs such as clomiphene and tamoxifen that bind to both ERα and ERβ. Newer nonsteroidal agents have since become available such as raloxifene, toremifene, and droloxifene. In a trial of clomiphene administered for 30 days to healthy eugonadal young men, there was a progressive increase in serum testosterone concentrations by ∼146% by day 30, well into the supraphysiological range.50 Notably, testosterone concentrations were still increasing at the end of the planned intervention period of 30 days.50 Thus, it remains unclear if a longer treatment duration could result in even higher testosterone levels. Both ER antagonists and aromatase inhibitors are on the WADA’s list of banned drugs. Men who abuse aromatizable AAS commonly “stack” ER antagonists or aromatase inhibitors to avoid development of gynecomastia.34,35,51 Some men use these drugs after completing a cycle of AAS in an attempt to “hasten” the recovery of the HPG axis, known as “post-­ cycle therapy.”35 Gonadotropins. Another form of indirect doping is the use of gonadotropins, such as recombinant LH or hCG, to stimulate the testes directly, bypassing the hypothalamus and the pituitary. Both drugs are currently on the WADA’s list of prohibited drugs for male athletes. hCG is a dimeric glycoprotein containing an α and a β subunit that is normally produced by the human placenta52; its α-­subunit is similar to other glycoprotein hormones, namely, follicle-­stimulating hormone (FSH), LH, and thyroid-­stimulating hormone (TSH). It undergoes glycosylation with sialic acid residues that prolongs its half-­life and makes it a long-­acting analog of LH. Clinically, recombinant LH or hCG are used to stimulate spermatogenesis in men who have central hypogonadism and desire fertility.53 These therapies are expensive and require injections several times per week, but the evidence regarding their ergogenic potential is sparse. Small trials in older men have shown that hCG therapy increases serum testosterone levels and lean body mass but does not improve muscle strength.54 Men abusing AAS also use hCG to prevent testicular atrophy.

KEY POINTS  • Direct doping involves the use of testosterone, its synthetic derivatives, and designer androgens (such as SARMs). • Indirect doping involves the use of drugs that increase endogenous testosterone secretion by stimulating the HPG axis (ER antagonists and aromatase inhibitors) or via direct stimulation of the testes (LH and hCG).

DETECTION OF ANDROGEN DOPING Detection of Compounds Used in Direct Androgen Doping Although some athletes use both natural and synthetic AAS to circumvent the detection of synthetic androgens, athletes have resorted to doping mainly with testosterone. Hence, the detection of illegal use depends upon distinguishing between endogenous and exogenous testosterone. Detection of exogenous testosterone abuse can be accomplished by two methods: measurement of testosterone-­to-­epitestosterone (T/E) ratio and isotope ratio mass spectrometry (IRMS), the latter is only performed by specialized laboratories working with antidoping agencies to test elite athletes (not available to clinicians).

Testosterone-­to-­Epitestosterone Ratio. Epitestosterone (17α-­ hydroxy-­ 4-­ androsten-­ 3-­ one), a 17-­ epimer of testosterone, is also secreted by the Leydig cells. Epitestosterone is biologically inactive, and there is no interconversion between testosterone and epitestosterone. It is mainly excreted in the urine as a glucuronide.58 Its production rate is 4. Athletes doping with testosterone may concurrently administer epitestosterone, which decreases T/E ratio and results in a false-­negative result.62 As urinary excretion of epitestosterone is about one-­third that of testosterone, coadministration of testosterone and epitestosterone in 30:1 ratio will generally result in a normal T/E ratio.73 Deviations in the T/E ratio from an athlete’s biological passport trigger further investigation but in itself do not constitute a doping violation. These urine samples can be submitted for IRMS confirmation.

Masking Agents

Adverse effects of AAS Hepatic

Renal

Neuropsychiatric and behavioral

The WADA’s list of prohibited substances also includes drugs that are used to mask detection of prohibited agents. Masking agents belong to different classes of medications and have different mechanisms of action. In this section, we will discuss such agents and how they mask detection of AAS.

Reproductive

Cardiovascular

Probenecid. Probenecid is a uricosuric agent that is used in the treatment of gout. Probenecid is known to interfere with renal excretion of steroid glucuronide conjugates and has been shown to reduce urinary excretion of endogenous (testosterone, epitestosterone, androsterone) and exogenous (norandrosterone, noretiocholanolone) steroid conjugates.73 Probenecid is detected by GC-­MS, and its identification alone is considered sufficient evidence to establish a doping violation.

Steroid 5α-­Reductase Inhibitors. These drugs inhibit 5α-­reductase, the enzyme that inhibits conversion of testosterone to DHT, and are used in the treatment of benign prostatic hyperplasia and male-­ pattern baldness. Endogenously produced 5α-­reduced steroids (DHT and androsterone) are part of the biological passport in athletes. Concomitant use of finasteride with testosterone alters the urinary steroid profile by reducing the excretion of 5α-­reduced steroids without changing the excretion of corresponding 5β-­reduced steroids, resulting in a decreased ratio between epimeric 5α-­and 5β-­reduced steroids. If a 5α-­reduced metabolite is targeted for testing, the use of finasteride can mask its detection. Similarly, finasteride inhibits the formation of 19-­norandrosterone in nandrolone abuse. Finasteride can be measured by GC-­MS and high-­performance liquid chromatography (HPLC).

Diuretics. There are several classes of diuretics used in clinical practice to treat hypertension and hypervolemic states. Athletes use diuretics to achieve rapid weight loss in sports with weight restrictions such as boxing and wrestling. Diuretics are also effective masking agents as they increase urine volume and dilute urinary concentrations of AAS and their metabolites to either below the limits of detection or below allowed limits.74 KEY POINTS  • Athletes use several methods to avoid detection of AAS, including administration of epitestosterone and use of masking agents such as probenecid, 5α-­reductase inhibitors, and diuretics.

Atherosclerosis

Arrhythmia

Hypertension Figure 110.3  Adverse effects of anabolic androgenic steroids (AAS) by physiological systems.

HEALTH CONSEQUENCES OF ANABOLIC STEROID ABUSE High-­quality data on adverse health effects of AAS are scarce as there are no systematic prospective studies in individuals abusing AAS. Additionally, the current secrecy surrounding AAS abuse further confounds information from retrospective studies. Thus, the majority of the data on the adverse effects of AAS are derived from case reports, case series and cross-­sectional case-­control studies. Furthermore, there is evidence that many men who abuse AAS also indulge in other illicit drugs,77 which could potentially contribute to the observed adverse effects. Additionally, these men often take high doses of dietary supplements and nutraceuticals that have not been carefully studied for safety or content.77 As the widespread use of AAS in the general population only began 30 years ago, most abusers are still relatively young and may not have yet experienced adverse effects from AAS abuse.78 Thus, most healthcare providers have not yet seen a large number of patients with complications of AAS abuse but will likely see them in their clinics as this cohort gets older. Below is the summary of the potential adverse effects of AAS across various organ systems (Fig. 110.3).

Cardiovascular The AR is expressed in the human myocardium, vascular smooth muscle, and endothelium79; therefore, the abuse of AAS can have an

PART 9  Male Reproduction 80 70 Left ventricular ejection fraction (%)

impact on several aspects of human cardiovascular physiology. Indeed, a growing body of evidence supports that AAS abuse directly or indirectly impacts cardiovascular morbidity. Blood Pressure: Supraphysiological doses of AAS have been associated with the incident or exacerbation of hypertension via multiple mechanisms. These include (1) inhibition of extraneuronal uptake of catecholamines,80 (2) salt and water retention by inhibition of 11β-­ hydroxylase activity (which results in cortisol-­induced activation of the mineralocorticoid receptor),81 and (3) reduced aortic elasticity. Studies have shown that normalization of blood pressure after discontinuation of AAS may take up to a year.82,83 To the contrary, some studies have not found such an association.84 This lack of consensus is likely a result of the observational design and small sample size of these studies. Dyslipidemia: AAS abuse is known to result in dyslipidemia. The most common observation is a 20% to 70% reduction in HDL cholesterol85; this reduction is more profound with the use of oral nonaromatizable AAS. In addition, HDL-­ mediated cholesterol efflux (a measure of HDL functionality) is also reduced in men who abuse AAS. This is important as HDL cholesterol efflux capacity is inversely associated with subclinical cardiovascular risk in young adults. Reductions in apolipoprotein A186 and increases in low-­density lipoprotein (LDL) cholesterol and triglycerides have also been observed.85 This atherogenic lipid profile may develop within a few weeks of AAS abuse and may take several months to resolve after their discontinuation. Erythrocytosis: Erythrocytosis is the most common adverse effect of androgen use87 and can result in increased blood viscosity and blood-­flow resistance.88 In vitro studies have shown a direct correlation between hematocrit and platelet aggregation. Mechanisms by which androgens stimulate erythrocytosis include stimulation of erythropoietin secretion, increasing iron availability by suppression of hepcidin, and direct stimulation of erythroid elements in the bone marrow.89 Elevation of hematocrit above 52% may result in thromboembolic events.85,90 Myocardial Function: Long-­term AAS abuse influences cardiac remodeling. Autopsy studies in young men who have abused AAS have shown left ventricle hypertrophy and increased overall cardiac mass.91 These post mortem findings are corroborated by echocardiographic studies that show impairment of cardiac function in individuals with long-­term abuse of AAS.92,93 In a recent report, young men with at least 2 years of exposure to supraphysiologic doses of AAS had impaired systolic and diastolic function on echocardiography compared with experienced weightlifters reporting no exposure to AAS.93 Cardiac dysfunction was worse in men who were still taking AAS at the time of evaluation compared with previous users, suggesting that discontinuation of AAS at least partially restores cardiac function (Fig. 110.4).93 Systolic dysfunction with AAS abuse has also been observed by other authors.92 However, as left ventricular hypertrophy is considered a physiologic adaptation to endurance and resistance training,94–96 it is difficult to differentiate between the impact of exercise training versus the direct effect of AAS on this remodeling. Long-­term exposure to high doses of AAS results in a decrease in myocardial contractile reserve to β-­adrenoceptor stimulation,97 which likely contributes to cardiac dysfunction. Furthermore, subtle histological changes in the myocardium as a result of AAS abuse are seen even before the onset of overt ventricular hypertrophy.98 Arrhythmias: Long-­term abuse of AAS may lead to myocardial scarring and fibrosis even in individuals without coronary artery disease,99 possibly by inducing cardiomyocyte apoptosis. Myocardial fibrosis alters cardiac electrophysiology predisposing to lethal arrhythmias.100 Indeed, animal studies have suggested that nandrolone can potentiate the arrhythmogenic effects of cardiac ischemia by altering cardiomyocyte calcium handling. Arrhythmias in AAS abusers often

60

Lower lomit of normal

50 40 30 20 10 Current use (n = 58)

Previous use (n = 28)

Ever users (n = 86)

Never users (n = 54)

16 14 Early diastolic left ventricular tissue velocity (cm/s)

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12 10 8

Lower lomit of normal

6 4 2

Current Previous Ever Never use use users users (n = 58) (n = 28) (n = 86) (n = 54) Figure 110.4  Left ventricular systolic and diastolic function in anabolic-­ androgenic steroid users and nonusers. (Adapted from Baggish AL, Weiner RB, Kanayama G, et al. Cardiovascular toxicity of illicit anabolic-­ androgenic steroid use. Circulation 2017;135:1991–2002.)

occur during or shortly after physical activity101 and include atrial fibrillation, supraventricular or ventricular tachycardia, and ventricular fibrillation.102 Arrhythmias appear to be a common cause of sudden death in young AAS abusers, as some autopsy studies did not find evidence of either atherosclerosis or thrombosis in coronary arteries of these patients.103,104 Atherosclerosis: Abuse of AAS has been associated with accelerated atherosclerosis. Men who abuse AAS have greater coronary artery plaque volume on computed tomography coronary angiography compared with nonusers, with greater plaque burden associated with cumulative AAS exposure.93,105 These findings are corroborated by the numerous case reports of myocardial infarction in young men abusing AAS, which is likely a result of plaque rupture and thrombus formation.91,106 Many cases of stroke in young AAS abusers have also been reported; however, most are thought to be due to embolic events.107,108 Indeed, testosterone treatment has been shown to augment ex vivo platelet aggregability by increasing thromboxane A2 receptor density on human platelets.109 Androgens also decrease production of prostacyclin, an inhibitor of platelet aggregation. Additionally, current AAS abusers have reduced fibrinolysis compared to nonusers and former users.110

CHAPTER 110  Misuse and Abuse of Anabolic Hormones

many men take ER antagonists or aromatase inhibitors. Gynecomastia is also seen in men who have persistent androgen deficiency after discontinuation of both aromatizable and nonaromatizable AAS.117

300 AAS > 14 weeks ago Sperm concentration (millions/mL)

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Hepatic

200 AAS 3–14 weeks ago Current AAS users 100 50 0 50 100 Normal volunteers 200

Figure 110.5  Sperm concentrations in 41 bodybuilders currently using anabolic androgenic steroids (AAS), those who discontinued AAS 3 to 14 weeks ago or discontinued >14 weeks ago (red bars) and in 41 drug-­free volunteers (blue bars). (Adapted from Nieschlag E, Vorona E. Mechanisms in endocrinology: medical consequences of doping with anabolic androgenic steroids: effects on reproductive functions. Eur J Endocrinol 2015;173:R47–R58.)

Reproductive Considering that exogenous androgens suppress the HPG axis, it is not surprising that individuals involved in direct doping have suppression of testicular testosterone secretion and spermatogenesis. Although systematic evaluation of reproductive function in men who abuse AAS is lacking, data from studies of male hormonal contraception that used supraphysiologic doses of testosterone, usually in combination with a progestin, show profound oligozoospermia during the intervention phase.111 Reassuringly, sperm concentrations returned to normal in 90% of participants within 12 months of cessation of intervention and in 100% within 24 months. The results of semen analysis in men abusing AAS show a spectrum that ranges from normal sperm count to azoospermia; the recovery of spermatogenesis is associated with time since last AAS (Fig. 110.5).112,113 As the seminiferous tubules account for ∼95% of the testicular volume, abuse of AAS often results in testicular atrophy. This explains why some AAS users choose to co-­ administer hCG to maintain testicular volume and spermatogenesis. Sexual dysfunction is also a common adverse effect of AAS abuse.78 As estrogens play an important role in male libido,114 men who abuse nonaromatizable AAS experience low libido and erectile dysfunction as endogenous testosterone and estradiol are both suppressed. Additionally, even aromatizable synthetic AAS may not generate metabolites with similar estrogenic activity as estradiol, resulting in a detrimental effect on libido similar to nonaromatizable AAS. For these reasons, testosterone remains the most abused AAS, both among the athletes and general public. However, hypogonadism and sexual dysfunction are an integral part of the “AAS-­withdrawal syndrome” that occurs upon discontinuation of all AAS,115 and long-­term abuse may result in prolonged androgen deficiency that may persist for months to years after discontinuation of AAS.116

Gynecomastia Abuse of AAS may result in gynecomastia in some men, particularly those using aromatizable AAS in supraphysiologic doses. To prevent this,

Hepatic adverse effects of AAS are generally limited to oral 17α-­ alkylated agents, such as methyltestosterone, methandrostenolone, oxandrolone, oxymetholone, and stanozolol. However, case reports of hepatic injury due to abuse of SARMs are emerging. The clinical assessment of AAS-­ induced hepatotoxicity poses some challenges. Rhabdomyolysis from high-­intensity exercise can increase transaminases that may mislead clinicians into considering these elevations as a sign of hepatic injury.78 To the contrary, AAS-­induced hepatic injury sometimes does not result in an increase in transaminase levels.118 Long-­term use of 17α-­alkylated agents reduces the activities of the microsomal drug-­metabolizing enzyme system and the hepatic mitochondrial respiratory chain complex, and increases hepatic lysosomal hydrolases, without any increase in liver function tests. Macroscopic signs of AAS-­induced hepatotoxicity are related to the cumulative dose and duration of abuse of 17α-­alkylated steroids, and include cholestatic jaundice and hepatic peliosis (proliferation of sinusoidal capillaries that result in blood-­filled cysts).118 Data suggest that peliosis and hepatic adenomas are rare and not clinically apparent, with most cases found incidentally or on autopsy, although there have been rare reports of spontaneous hepatic rupture in men abusing AAS.119

Musculoskeletal Compared with bodybuilders who do not abuse AAS, men abusing androgens have a much higher risk of tendon ruptures, mainly of the upper body.120 This might be due to a rapid increase in skeletal muscle mass that outpaces adaptation by the connective tissues. This discrepancy in the speed of adaptation between the muscles and the tendons may also contribute to the higher patellar tendon stiffness observed in men with long-­term AAS abuse. Some data also suggest altered metabolism of type-­1 and type-­3 collagen fibers in these men.121. Another important musculoskeletal adverse effect of AAS abuse is premature closure of the epiphyses in teenagers, which mainly occurs with the use of aromatizable AAS, resulting in reduced final height. Other musculoskeletal complications include myositis ossificans, a form of heterotopic ossification, that occurs at the site of intramuscular injections,122 and generalized necrotizing myopathy, which rarely leads to severe rhabdomyolysis resulting in renal injury or multiorgan failure.

Renal In addition to renal injury related to rhabdomyolysis, some cases of acute bile nephropathy secondary to AAS have been reported.123 As previously discussed, oral AAS can cause cholestasis, increasing circulating concentrations of bile acids that lead to formation of bile casts in the renal tubules resulting in acute tubular injury; chronic exposure leads to progressive interstitial injury and tubulointerstitial fibrosis. Additionally, there have been reports of AAS-­induced focal segmental glomerulosclerosis.124 Though these serious forms of renal injury have been reported, the use of AAS generally presents as progressive subclinical deterioration of renal function. Indeed, a recent report that assessed creatinine and cystatin-­C–derived estimates of glomerular filtration rate (eGFR) in weightlifters (57 current AAS users, 28 past users, and 52 never users) showed that eGFR was lowest in the current users, intermediate among the past users, and highest in nonusers.125 Animal studies suggest that increases in oxidative stress and proinflammatory cytokines lead to renal cell apoptosis. Similarly, an increase in glomerular expression of CD34, a marker of endothelial dysfunction, has also been reported. Clinicians should be aware that higher muscle mass in these men is reflected by an increase

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in serum creatinine, which does not necessarily suggest impaired renal function. Thus, they should consider measurement of cystatin-­C and its clearance to assess renal function.

Dermatologic AR is expressed in the skin, and testosterone has been shown to dose-­ dependently increase sebum production.126 Use of AAS affects the pilosebaceous unit and can lead to or exacerbate acne vulgaris and folliculitis; these conditions generally resolve upon discontinuation of AAS. Men abusing AAS may also develop striae distensae (stretch marks) due to the rapid increase in muscle size that outpaces expansion of the skin, likely due to reduced cutaneous elasticity as a result of AAS-­induced reduced collagen production.127 Keloid formation is also seen in some men.

Neuropsychiatric and Behavioral Although there is a widespread notion that AAS abuse increases aggressive behavior, this is observed in a small number of individuals. In addition to experiencing neuropsychiatric symptoms during AAS abuse, androgen withdrawal is also associated with major depression, including suicidal ideation.116 These mood changes might be related to a decrease in functional connectivity between the amygdala and the default mode network, as well as with other cerebral centers involved in control of emotions.128 Moreover, reduced volumes of total gray matter, cerebral cortex, and putamen have been demonstrated in weightlifters with cumulative exposure to AAS of at least 1 year compared with weightlifters who never used androgens.129 These data are also supported by preclinical studies showing that AAS induce neuronal apoptosis and reduce expression of nerve growth factor.

Dependence Since the late 1980s, reports of dependence on AAS have been emerging, but only in recent years, these dependence syndromes have been widely recognized. Pooled data from 10 international studies suggest that 30% of AAS abusers may develop dependence—a prevalence much greater than seen for other recreational drugs.16 Multiple factors likely contribute to the development of AAS dependence.130 First, men with muscle dysmorphia may become “addicted” to AAS to maintain their physique as they fear losing muscle mass upon discontinuation of androgens. Second, AAS suppresses the HPG axis115,116; thus, upon discontinuation of AAS (especially after prolonged use), many men experience androgen deficiency. Recent data suggest that this androgen deficiency may persist for months or even years following cessation of AAS116,131 and may result in sexual dysfunction, fatigue, and mood changes. Thus, many men resume AAS to avoid these adverse effects. Third, AAS may cause dependence via their hedonic properties. Although AAS use is not associated with intoxication that is associated with alcohol, opiates, or cocaine, their use is associated with feelings of self-­confidence and “invincibility.”78 Animal data show that when male hamsters are given the opportunity to inject themselves with AAS via a nose poke device, they will self-­administer AAS to the point of death.132 This phenomenon has been demonstrated even in those animal studies where AAS were administered directly into the central nervous system, suggesting that the effects of AAS are central. All these factors might be responsible in predisposing men to dependence on AAS. KEY POINTS  • Although high-­quality data on adverse health consequences of AAS abuse are lacking, evidence from case reports, case series, and cohort studies suggest that AAS abuse may result in cardiovascular dysfunction, erythrocytosis, infertility, suppression of gonadal axis, gynecomastia, acne, and neuropsychological disturbances.

MANAGEMENT OPTIONS OF ANABOLIC STEROID ABUSE Although the general principles of the medical management of AAS abuse are the same for both the elite athletes and the general public, there are some important differences that are highlighted in the section below.

Elite Athletes Therapeutic Use Exemption: As in the general public, athletes may also have hormonal deficiencies for which they might be receiving appropriate hormone replacement. These include athletes with androgen deficiency due to organic diseases of the hypothalamus, pituitary, or the testes, such as Klinefelter’s syndrome, chemotherapy-­induced testicular injury, or central hypogonadism after hypophysectomy. For elite athletes participating in competitive sports in which AAS are prohibited, the process for obtaining permission to continue treatment from the relevant antidoping agency is known as Therapeutic Use Exemption (TUE). Clinicians, particularly endocrinologists, may be asked to verify that testosterone therapy is truly indicated for a particular athlete. The granting of a TUE for testosterone follows rigid guidelines, including documentation of specific signs and symptoms of androgen deficiency and unequivocally low morning testosterone concentrations on more than one occasion. For documented organic male androgen deficiency, the TUE after vigorous review can be granted for testosterone therapy and in some circumstances for gonadotropins. If an elite athlete is found to be abusing AAS to purely achieve competitive advantage (i.e., not under TUE), the athlete is instructed to discontinue the use of the banned drug and may be blocked from competition for a proscribed period of time. These athletes are not given testosterone to mitigate the risk of AAS-­withdrawal syndrome if they are still actively competing. On the other hand, a clinician may encounter retired elite male athletes who previously abused AAS while competing and now present with persistently low serum testosterone concentrations. Many of these men have low or inappropriately normal serum gonadotropins suggesting continued suppression of their HPG axis. The medical management of these retired elite athletes with hypogonadal symptoms is similar to members of the general public who have previously abused AAS (see below).

General Public As there are no clinical trials on the management of long-­term AAS abuse in the general public, care is based on the likelihood of severe withdrawal symptoms, the goals of the patient, and a risk-­benefit analysis of the available treatment options. Men Not Willing to Discontinue AAS: Some men who abuse AAS, either for competing in amateur sports or for image enhancement, are not willing to discontinue AAS. In this case, the main role of the endocrinologist is to ensure that the patient is fully informed about the known adverse effects of all types of AAS such as erythrocytosis, reduced fertility, dyslipidemia, and potential cardiovascular risks. Additionally, clinicians should counsel on the formulation-­specific adverse effects of AAS (e.g., hepatotoxicity associated with 17α-­alkylated steroids). In men who are already using supraphysiologic doses of testosterone, a prudent approach would be to gradually reduce the dose over the course of weeks to months to a physiologic replacement dose. This approach may help avoid severe symptoms of AAS withdrawal. On the other hand, men who are taking AAS other than testosterone could be switched to prescription testosterone. Based on the current dose of their AAS, replacement with testosterone can be initiated up to twice the usual replacement dose with a gradual taper to physiological dose over subsequent weeks. Anecdotal experience suggests that this

CHAPTER 110  Misuse and Abuse of Anabolic Hormones approach (1) foments trust with the patient, (2) discourages purchase of AAS from unregulated sources, and (3) allows continuity of care with the physician. The clinician should continue to inform the patient that continued use of testosterone will result in continued suppression of the HPG axis. Men Willing to Discontinue AAS: There are no randomized controlled trials comparing medical therapy versus no treatment on the degree of withdrawal symptoms related to discontinuation of AAS. Furthermore, available medical therapies are not Food and Drug Administration approved and are used in the clinics in an off-­label fashion. For men who are willing to discontinue AAS, the management options include (1) discontinuation without additional medical therapy, (2) initiation of physiological testosterone replacement to avoid withdrawal symptoms (as mentioned earlier), and (3) short-­term treatment with clomiphene or hCG. The selection of these options is dictated by the predominant reason that has led these men to discontinue AAS; in most cases, it is infertility. Clinicians should perform the usual workup indicated in the evaluation of infertility including measurement of serum testosterone and gonadotropins and perform semen analyses. If the working diagnosis of infertility is prior AAS abuse, then the choice of therapy is based on the couples’ timeline for starting a family. If this is not an immediate goal, then the clinician could consider discontinuation of AAS with monitoring of serum testosterone and gonadotropins periodically. Once testosterone has normalized, serial semen analyses can be performed to assess resumption of spermatogenesis. This approach is based on the observation of spontaneous recovery of the HPG axis in trials of male contraception, where recovery of endogenous testosterone and spermatogenesis occurs within 3 and 12 months, respectively, after discontinuation of exogenous testosterone. Similarly, in men who have abused AAS for ≤1 year, recovery of the HPG axis occurs within a few months. To the contrary, among men who have abused high doses of AAS for >1 year, the recovery time is longer; such men might benefit from a short course of clomiphene or gonadotropin therapy after discontinuation of AAS. Clomiphene Therapy: Clomiphene is a selective estrogen receptor modulator (SERM) that increases serum gonadotropins and testosterone concentrations within 2 to 4 weeks in men with an intact HPG axis.133,134 Thus, the integrity of the HPG axis is a prerequisite for its efficacy. Limited data suggest that some men with a history of long-­term AAS abuse experienced recovery of their HPG axis with clomiphene therapy.135 The benefits of hastening the recovery of the HPG axis are of interest to men who are eager to have a child soon. Initiation of clomiphene at 25 to 50 mg every other day is a reasonable approach as it has been efficacious in normalizing serum testosterone concentrations. However, this benefit should be weighed against the potential side effects such as venous thrombosis and other cardiovascular events. The clinician should inform the patient that the safety and efficacy of this approach has not been shown in clinical trials. hCG Therapy: Treatment with hCG is another option in patients with similar presentation. Subcutaneous hCG therapy stimulates testosterone production and spermatogenesis in almost all men with postpubertal hypogonadotropic hypogonadism. The increase in serum testosterone that results from administration of hCG suppresses gonadotropins; therefore, the HPG axis will remain suppressed after discontinuation of hCG. The usual dose of hCG is 1000 to 2000 IU subcutaneously 2 to 3 times weekly. The dose is adjusted until serum total testosterone is in the normal range and continued at least until conception. Data on the comparative effectiveness of clomiphene and hCG are lacking.

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KEY POINTS  • Elite athletes abusing AAS should be advised to discontinue them, except in whom TUE has been granted. • In the general population, men who abuse AAS and are unwilling to discontinue could be transitioned to physiologic testosterone replacement. • Men who are willing to discontinue AAS could either be offered a drug holiday from testosterone, treatment with hCG, or a short-­term trial with clomiphene (in an attempt to hasten the recovery of the gonadal axis).

CONCLUSION The abuse of AAS continues to increase. Although legislative efforts have attempted to curtail access to these agents,136 they continue to be easily procured through the Internet and other unregulated sources.137,138 Although antidoping agencies tightly regulate doping among elite athletes, most men abusing AAS are not elite athletes but young men who want to enhance their image by improving their muscularity. Furthermore, the burden of adverse effects of these compounds will only become apparent when these men enter their middle age. Thus, tremendous amount of work needs to be done, including (1) organization of an education campaign to raise public awareness regarding the risks of AAS use, (2) collaboration between doping control laboratories and commercial laboratories to make doping tests available to clinicians, (3) creation of a steroid hormone user registry so that men abusing AAS can be followed systematically to assess doping practices and potential risks, and (4) clinical trials with ER antagonists and aromatase inhibitors to determine their efficacy in hastening the recovery of the HPG axis. This can be accomplished when patients, clinicians, researchers, and policy makers join hands and make a concerted effort toward achieving these objectives.

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13. Sagoe D, Molde H, Andreassen CS, et al. The global epidemiology of anabolic-­androgenic steroid use: a meta-­analysis and meta-­regression analysis. Ann Epidemiol. 2014;24:383–398. 14. Goldman B, Bush PJ, Klatz R. Death in the Locker Room. London: Century; 1984. 15. Connor J, Woolf J, Mazanov J. Would they dope? Revisiting the Goldman dilemma. Br J Sports Med. 2013;47:697–700. 16. Pope Jr HG, Kanayama G, Athey A, et al. The lifetime prevalence of anabolic-­androgenic steroid use and dependence in Americans: current best estimates. Am J Addict. 2014;23:371–377. 17. Blashill AJ, Safren SA. Sexual orientation and anabolic-­androgenic steroids in U.S. adolescent boys. Pediatrics. 2014;133:469–475. 18. Pope Jr HG, Khalsa JH, Bhasin S. Body image disorders and abuse of anabolic-­androgenic steroids among men. JAMA. 2017;317:23–24. 19. Yang CF, Gray P, Pope Jr HG. Male body image in Taiwan versus the west: Yanggang Zhiqi meets the Adonis complex. Am J Psychiatry. 2005;162:263–269. 20. Sagoe D, Tr T, Molde H, et al. Anabolic-­androgenic steroid use in the Nordic countries: a meta-­analysis and meta-­regression analysis. Nord Stud Alcohol Dr. 2017;32:7–20. 21. American College of Sports Medicine. Position stand on the use of anabolic-­androgenic steroids in sports. Med Sci Sports Exerc. 1987;19:534–539. 22. Storer TW, Magliano L, Woodhouse L, et al. Testosterone dose-­ dependently increases maximal voluntary strength and leg power, but does not affect fatigability or specific tension. J Clin Endocrinol Metab. 2003;88:1478–1485. 23. Basaria S, Lieb 2nd J, Tang AM, et al. Long-­term effects of androgen deprivation therapy in prostate cancer patients. Clin Endocrinol. 2002;56:779–786. 24. Singh R, Artaza JN, Taylor WE, et al. Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-­mediated pathway. Endocrinology. 2003;144:5081–5088. 25. Rossetti ML, Steiner JL, Gordon BS. Androgen-­mediated regulation of skeletal muscle protein balance. Mol Cell Endocrinol. 2017;447:35–44. 26. Ferrando AA, Sheffield-­Moore M, Paddon-­Jones D, et al. Differential anabolic effects of testosterone and amino acid feeding in older men. J Clin Endocrinol Metab. 2003;88:358–362. 27. Mendler L, Baka Z, Kovacs-­Simon A, et al. Androgens negatively regulate myostatin expression in an androgen-­dependent skeletal muscle. Biochem Biophys Res Commun. 2007;361:237–242. 28. Kadi F, Eriksson A, Holmner S, et al. Effects of anabolic steroids on the muscle cells of strength-­trained athletes. Med Sci Sports Exerc. 1999;31:1528–1534. 29. Sinha-­Hikim I, Roth SM, Lee MI, et al. Testosterone-­induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men. Am J Physiol Endocrinol Metab. 2003;285:E197– E205. 30. Illig R, Prader A. Effect of testosterone on growth hormone secretion in patients with anorchia and delayed puberty. J Clin Endocrinol Metab. 1970;30:615–618. 31. Krug O, Thomas A, Walpurgis K, et al. Identification of black market products and potential doping agents in Germany 2010-­2013. Eur J Clin Pharmacol. 2014;70:1303–1311. 32. Thevis M, Lagojda A, Kuehne D, et al. Characterization of a non-­ approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-­I metabolites for human sports drug testing. Rapid Commun Mass Spectrom. 2015;29:991–999. 33. Van Wagoner RM, Eichner A, Bhasin S, et al. Chemical composition and labeling of substances marketed as selective androgen receptor modulators and sold via the Internet. JAMA. 2017;318:2004–2010. 34. Ip EJ, Barnett MJ, Tenerowicz MJ, et al. The Anabolic 500 survey: characteristics of male users versus nonusers of anabolic-­androgenic steroids for strength training. Pharmacotherapy. 2011;31:757–766. 35. de Ronde W, Smit DL. Anabolic androgenic steroid abuse in young males. Endocr Connect. 2020;9(4):R102–R111.

36. Mullen C, Whalley BJ, Schifano F, et al. Anabolic androgenic steroid abuse in the United Kingdom: an update. Br J Pharmacol. 2020;177:2180–2198. 37. Simpson ER, Mahendroo MS, Means GD, et al. Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev. 1994;15:342–355. 38. Sirianni R, Capparelli C, Chimento A, et al. Nandrolone and stanozolol upregulate aromatase expression and further increase IGF-­I-­dependent effects on MCF-­7 breast cancer cell proliferation. Mol Cell Endocrinol. 2012;363:100–110. 39. Navarro JF. In the erythropoietin era, can we forget alternative or adjunctive therapies for renal anaemia management? The androgen example. Nephrol Dial Transplant. 2003;18:2222–2226. 40. Schanzer W. Metabolism of anabolic androgenic steroids. Clin Chem. 1996;42:1001–1020. 41. Ring J, Heinelt M, Sharma S, et al. Oxandrolone in the treatment of burn injuries: a systematic review and meta-­analysis. J Burn Care Res. 2020;41:190–199. 42. Rahnema CD, Crosnoe LE, Kim ED. Designer steroids -­over-­the-­ counter supplements and their androgenic component: review of an increasing problem. Andrology. 2015;3:150–155. 43. Congress US. Designer Anabolic Steroid Control Act of 2014; 2014. 44. Catlin DH, Ahrens BD, Kucherova Y. Detection of norbolethone, an anabolic steroid never marketed, in athletes’ urine. Rapid Commun Mass Spectrom. 2002;16:1273–1275. 45. Jasuja R, Catlin DH, Miller A, et al. Tetrahydrogestrinone is an androgenic steroid that stimulates androgen receptor-­mediated, myogenic differentiation in C3H10T1/2 multipotent mesenchymal cells and promotes muscle accretion in orchidectomized male rats. Endocrinology. 2005;146:4472–4478. 46. Basaria S, Dobs AS. Risks versus benefits of testosterone therapy in elderly men. Drugs Aging. 1999;15:131–142. 47. Winters SJ, Janick JJ, Loriaux DL, et al. Studies on the role of sex steroids in the feedback control of gonadotropin concentrations in men. II. Use of the estrogen antagonist, clomiphene citrate. J Clin Endocrinol Metab. 1979;48:222–227. 48. Bilezikian JP, Morishima A, Bell J, et al. Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. N Engl J Med. 1998;339:599–603. 49. Leder BZ, Rohrer JL, Rubin SD, et al. Effects of aromatase inhibition in elderly men with low or borderline-­low serum testosterone levels. J Clin Endocrinol Metab. 2004;89:1174–1180. 50. Miller GD, Moore C, Nair V, et al. Hypothalamic-­pituitary-­testicular axis effects and urinary detection following clomiphene administration in males. J Clin Endocrinol Metab. 2019;104:906–914. 51. Parkinson AB, Evans NA. Anabolic androgenic steroids: a survey of 500 users. Med Sci Sports Exerc. 2006;38:644–651. 52. Casarini L, Santi D, Brigante G, et al. Two hormones for one receptor: evolution, biochemistry, actions, and pathophysiology of LH and hCG. Endocr Rev. 2018;39:549–592. 53. Finkel DM, Phillips JL, Snyder PJ. Stimulation of spermatogenesis by gonadotropins in men with hypogonadotropic hypogonadism. N Engl J Med. 1985;313:651–655. 54. Liu PY, Wishart SM, Handelsman DJ. A double-­blind, placebo-­ controlled, randomized clinical trial of recombinant human chorionic gonadotropin on muscle strength and physical function and activity in older men with partial age-­related androgen deficiency. J Clin Endocrinol Metab. 2002;87:3125–3135. 55. Congress US. Anabolic Steroid Control Act of 2004; 2004. 56. Leder BZ, Longcope C, Catlin DH, et al. Oral androstenedione administration and serum testosterone concentrations in young men. JAMA. 2000;283:779–782. 57. Morales AJ, Haubrich RH, Hwang JY, et al. The effect of six months treatment with a 100 mg daily dose of dehydroepiandrosterone (DHEA) on circulating sex steroids, body composition and muscle strength in age-­advanced men and women. Clin Endocrinol. 1998;49:421–432. 58. Starka L. Epitestosterone. J Steroid Biochem Mol Biol. 2003;87:27–34.

CHAPTER 110  Misuse and Abuse of Anabolic Hormones 59. Wilson H, Lipsett MB. Metabolism of epitestosterone in man. J Clin Endocrinol Metab. 1966;26:902–914. 60. Basaria S. Androgen abuse in athletes: detection and consequences. J Clin Endocrinol Metab. 2010;95:1533–1543. 61. Schulze JJ, Lundmark J, Garle M, et al. Doping test results dependent on genotype of uridine diphospho-­glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab. 2008;93:2500–2506. 62. Dehennin L. Detection of simultaneous self-­administration of testosterone and epitestosterone in healthy men. Clin Chem. 1994;40:106–109. 63. Shackleton CH, Phillips A, Chang T, et al. Confirming testosterone administration by isotope ratio mass spectrometric analysis of urinary androstanediols. Steroids. 1997;62:379–387. 64. Michelini E, Cevenini L, Mezzanotte L, et al. A sensitive recombinant cell-­based bioluminescent assay for detection of androgen-­like compounds. Nat Protoc. 2008;3:1895–1902. 65. Kicman AT, Parkin MC, Iles RK. An introduction to mass spectrometry based proteomics-­detection and characterization of gonadotropins and related molecules. Mol Cell Endocrinol. 2007;260–262:212–227. 66. Mareck U, Sigmund G, Opfermann G, et al. Identification of the aromatase inhibitor letrozole in urine by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2005;19:3689–3693. 67. Mareck U, Geyer H, Guddat S, et al. Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:1954–1962. 68. Dehennin L, Ferry M, Lafarge P, et al. Oral administration of dehydroepiandrosterone to healthy men: alteration of the urinary androgen profile and consequences for the detection of abuse in sport by gas chromatography-­mass spectrometry. Steroids. 1998;63:80–87. 69. Bowers LD. Oral dehydroepiandrosterone supplementation can increase the testosterone/epitestosterone ratio. Clin Chem. 1999;45:295–297. 70. Cawley AT, Trout GJ, Kazlauskas R, et al. The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4-­hydroxyandrostenedione metabolite. Rapid Commun Mass Spectrom. 2008;22:4147–4157. 71. Catlin DH, Hatton CK, Starcevic SH. Issues in detecting abuse of xenobiotic anabolic steroids and testosterone by analysis of athletes’ urine. Clin Chem. 1997;43:1280–1288. 72. Evans NA. Gym and tonic: a profile of 100 male steroid users. Br J Sports Med. 1997;31:54–58. 73. van de Kerkhof DH, de Boer D, Thijssen JH, et al. Evaluation of testosterone/epitestosterone ratio influential factors as determined in doping analysis. J Anal Toxicol. 2000;24:102–115. 74. Cadwallader AB, de la Torre X, Tieri A, et al. The abuse of diuretics as performance-­enhancing drugs and masking agents in sport doping: pharmacology, toxicology and analysis. Br J Pharmacol. 2010;161:1–16. 75. Sonino N. The use of ketoconazole as an inhibitor of steroid production. N Engl J Med. 1987;317:812–818. 76. Oftebro H, Jensen J, Mowinckel P, et al. Establishing a ketoconazole suppression test for verifying testosterone administration in the doping control of athletes. J Clin Endocrinol Metab. 1994;78:973–977. 77. Hakansson A, Mickelsson K, Wallin C, et al. Anabolic androgenic steroids in the general population: user characteristics and associations with substance use. Eur Addict Res. 2012;18:83–90. 78. Pope Jr HG, Wood RI, Rogol A, et al. Adverse health consequences of performance-­enhancing drugs: an Endocrine Society scientific statement. Endocr Rev. 2014;35:341–375. 79. Cai J, Hong Y, Weng C, et al. Androgen stimulates endothelial cell proliferation via an androgen receptor/VEGF/cyclin A-­mediated mechanism. Am J Physiol Heart Circ Physiol. 2011;300:H1210–H1221. 80. Baker PJ, Ramey ER, Ramwell PW. Androgen-­mediated sex differences of cardiovascular responses in rats. Am J Physiol. 1978;235:H242–H246. 81. Johannsson G, Gibney J, Wolthers T, et al. Independent and combined effects of testosterone and growth hormone on extracellular water in hypopituitary men. J Clin Endocrinol Metab. 2005;90:3989–3994. 82. Urhausen A, Albers T, Kindermann W. Are the cardiac effects of anabolic steroid abuse in strength athletes reversible? Heart. 2004;90:496–501.

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83. Kuipers H, Wijnen JA, Hartgens F, et al. Influence of anabolic steroids on body composition, blood pressure, lipid profile and liver functions in body builders. Int J Sports Med. 1991;12:413–418. 84. Sader MA, Griffiths KA, McCredie RJ, et al. Androgenic anabolic steroids and arterial structure and function in male bodybuilders. J Am Coll Cardiol. 2001;37:224–230. 85. Achar S, Rostamian A, Narayan SM. Cardiac and metabolic effects of anabolic-­androgenic steroid abuse on lipids, blood pressure, left ventricular dimensions, and rhythm. Am J Cardiol. 2010;106:893–901. 86. Singh AB, Hsia S, Alaupovic P, et al. The effects of varying doses of T on insulin sensitivity, plasma lipids, apolipoproteins, and C-­reactive protein in healthy young men. J Clin Endocrinol Metab. 2002;87:136–143. 87. Bhasin S, Brito JP, Cunningham GR, et al. Testosterone therapy in men with hypogonadism: an endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2018;103:1715–1744. 88. Guo W, Bachman E, Vogel J, et al. The effects of short-­term and long-­ term testosterone supplementation on blood viscosity and erythrocyte deformability in healthy adult mice. Endocrinology. 2015;156:1623–1629. 89. Gagliano-­Juca T, Pencina KM, Ganz T, et al. Mechanisms responsible for reduced erythropoiesis during androgen deprivation therapy in men with prostate cancer. Am J Physiol Endocrinol Metab. 2018;315:E1185–E1193. 90. Lippi G, Banfi G. Doping and thrombosis in sports. Semin Thromb Hemost. 2011;37:918–928. 91. Frati P, Busardo FP, Cipolloni L, et al. Anabolic Androgenic Steroid (AAS) related deaths: autoptic, histopathological and toxicological findings. Curr Neuropharmacol. 2015;13:146–159. 92. Rasmussen JJ, Schou M, Madsen PL, et al. Cardiac systolic dysfunction in past illicit users of anabolic androgenic steroids. Am Heart J. 2018;203:49–56. 93. Baggish AL, Weiner RB, Kanayama G, et al. Cardiovascular toxicity of illicit anabolic-­androgenic steroid use. Circulation. 2017;135:1991–2002. 94. Scharf M, Oezdemir D, Schmid A, et al. Myocardial adaption to HI(R) T in previously untrained men with a randomized, longitudinal cardiac MR imaging study (Physical adaptions in Untrained on Strength and Heart trial, PUSH-­trial). PLoS One. 2017;12:e0189204. 95. Pluim BM, Zwinderman AH, van der Laarse A, et al. The athlete’s heart. A meta-­analysis of cardiac structure and function. Circulation. 2000;101:336–344. 96. Dickerman RD, Schaller F, McConathy WJ. Left ventricular wall thickening does occur in elite power athletes with or without anabolic steroid Use. Cardiology. 1998;90:145–148. 97. Norton GR, Trifunovic B, Woodiwiss AJ. Attenuated beta-­adrenoceptor-­ mediated cardiac contractile responses following androgenic steroid administration to sedentary rats. Eur J Appl Physiol. 2000;81:310–316. 98. Hassan NA, Salem MF, Sayed MA. Doping and effects of anabolic androgenic steroids on the heart: histological, ultrastructural, and echocardiographic assessment in strength athletes. Hum Exp Toxicol. 2009;28:273– 283. 99. Baumann S, Jabbour C, Huseynov A, et al. Myocardial scar detected by cardiovascular magnetic resonance in a competitive bodybuilder with longstanding abuse of anabolic steroids. Asian J Sports Med. 2014;5:e24058. 100. Lusetti M, Licata M, Silingardi E, et al. Pathological changes in anabolic androgenic steroid users. J Forensic Leg Med. 2015;33:101–104. 101. Sculthorpe N, Grace F, Jones P, et al. Evidence of altered cardiac electrophysiology following prolonged androgenic anabolic steroid use. Cardiovasc Toxicol. 2010;10:239–243. 102. Nieminen MS, Ramo MP, Viitasalo M, et al. Serious cardiovascular side effects of large doses of anabolic steroids in weight lifters. Eur Heart J. 1996;17:1576–1583. 103. Kennedy MC, Lawrence C. Anabolic steroid abuse and cardiac death. Med J Aust. 1993;158:346–348. 104. Fineschi V, Riezzo I, Centini F, et al. Sudden cardiac death during anabolic steroid abuse: morphologic and toxicologic findings in two fatal cases of bodybuilders. Int J Legal Med. 2007;121:48–53. 105. Souza FR, Dos Santos MR, Porello RA, et al. Diminished cholesterol efflux mediated by HDL and coronary artery disease in young male anabolic androgenic steroid users. Atherosclerosis. 2019;283:100–105.

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106. Ferenchick GS, Adelman S. Myocardial infarction associated with anabolic steroid use in a previously healthy 37-­year-­old weight lifter. Am Heart J. 1992;124:507–508. 107. Shimada Y, Yoritaka A, Tanaka Y, et al. Cerebral infarction in a young man using high-­dose anabolic steroids. J Stroke Cerebrovasc Dis. 2012;21:906–911. e909. 108. Nieschlag E, Vorona E. Doping with anabolic androgenic steroids (AAS): adverse effects on non-­reproductive organs and functions. Rev Endocr Metab Disord. 2015;16:199–211. 109. Ajayi AA, Mathur R, Halushka PV. Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses. Circulation. 1995;91:2742–2747. 110. Sidelmann JJ, Gram JB, Rasmussen JJ, et al. Anabolic-­androgenic steroid abuse impairs fibrin clot lysis. Semin Thromb Hemost. 2021;47(1):11–17. 111. Nieschlag E. Clinical trials in male hormonal contraception. Contraception. 2010;82:457–470. 112. Basaria S, Collins L, Dillon EL, et al. The safety, pharmacokinetics, and effects of LGD-­4033, a novel nonsteroidal oral, selective androgen receptor modulator, in healthy young men. J Gerontol A Biol Sci Med Sci. 2013;68:87–95. 113. Christou MA, Christou PA, Markozannes G, et al. Effects of anabolic androgenic steroids on the reproductive system of athletes and recreational users: a systematic review and meta-­analysis. Sports Med. 2017;47:1869– 1883. 114. Finkelstein JS, Lee H, Burnett-­Bowie SA, et al. Gonadal steroids and body composition, strength, and sexual function in men. N Engl J Med. 2013;369:1011–1022. 115. Coward RM, Rajanahally S, Kovac JR, et al. Anabolic steroid induced hypogonadism in young men. J Urol. 2013;190:2200–2205. 116. Kanayama G, Hudson JI, DeLuca J, et al. Prolonged hypogonadism in males following withdrawal from anabolic-­androgenic steroids: an under-­recognized problem. Addiction. 2015;110:823–831. 117. Narula HS, Carlson HE. Gynaecomastia-­-­pathophysiology, diagnosis and treatment. Nat Rev Endocrinol. 2014;10:684–698. 118. Neri M, Bello S, Bonsignore A, et al. Anabolic androgenic steroids abuse and liver toxicity. Mini Rev Med Chem. 2011;11:430–437. 119. Patil JJ, O’Donohoe B, Loyden CF, et al. Near-­fatal spontaneous hepatic rupture associated with anabolic androgenic steroid use: a case report. Br J Sports Med. 2007;41:462–463. 120. Kanayama G, DeLuca J, Meehan 3rd WP, et al. Ruptured tendons in anabolic-­androgenic steroid users: a cross-­sectional cohort study. Am J Sports Med. 2015;43:2638–2644. 121. Parssinen M, Karila T, Kovanen V, et al. The effect of supraphysiological doses of anabolic androgenic steroids on collagen metabolism. Int J Sports Med. 2000;21:406–411. 122. Schultzel MM, Johnson MH, Rosenthal HG. Bilateral deltoid myositis ossificans in a weightlifter using anabolic steroids. Orthopedics. 2014;37:e844–e847. 123. Luciano RL, Castano E, Moeckel G, et al. Bile acid nephropathy in a bodybuilder abusing an anabolic androgenic steroid. Am J Kidney Dis. 2014;64:473–476.

124. Herlitz LC, Markowitz GS, Farris AB, et al. Development of focal segmental glomerulosclerosis after anabolic steroid abuse. J Am Soc Nephrol. 2010;21:163–172. 125. Hudson JI, Kanayama G, Pope Jr HG, et al. Glomerular filtration rate and supraphysiologic-­dose anabolic-­androgenic steroid use: a cross-­ sectional cohort study. Am J Kidney Dis. 2020;76:152–155. 126. Bhasin S, Travison TG, Storer TW, et al. Effect of testosterone supplementation with and without a dual 5alpha-­reductase inhibitor on fat-­free mass in men with suppressed testosterone production: a randomized controlled trial. J Am Med Assoc. 2012;307:931–939. 127. Walker J, Adams B. Cutaneous manifestations of anabolic-­ androgenic steroid use in athletes. Int J Dermatol. 2009;48:1044– 1048. quiz 1048. 128. Westlye LT, Kaufmann T, Alnaes D, et al. Brain connectivity aberrations in anabolic-­androgenic steroid users. Neuroimage Clin. 2017;13:62–69. 129. Bjornebekk A, Walhovd KB, Jorstad ML, et al. Structural brain imaging of long-­term anabolic-­androgenic steroid users and nonusing weightlifters. Biol Psychiatry. 2017;82:294–302. 130. Kanayama G, Brower KJ, Wood RI, et al. Treatment of anabolic-­ androgenic steroid dependence: emerging evidence and its implications. Drug Alcohol Depend. 2010;109:6–13. 131. Rasmussen JJ, Selmer C, Ostergren PB, et al. Former abusers of anabolic androgenic steroids exhibit decreased testosterone levels and hypogonadal symptoms years after cessation: a case-­control study. PLoS One. 2016;11:e0161208. 132. Wood RI. Anabolic-­androgenic steroid dependence? Insights from animals and humans. Front Neuroendocrinol. 2008;29:490–506. 133. Handelsman DJ. Clinical review: the rationale for banning human chorionic gonadotropin and estrogen blockers in sport. J Clin Endocrinol Metab. 2006;91:1646–1653. 134. Habous M, Giona S, Tealab A, et al. Clomiphene citrate and human chorionic gonadotropin are both effective in restoring testosterone in hypogonadism: a short-­course randomized study. BJU Int. 2018;122:889–897. 135. Guay AT, Jacobson J, Perez JB, et al. Clomiphene increases free testosterone levels in men with both secondary hypogonadism and erectile dysfunction: who does and does not benefit? Int J Impot Res. 2003;15:156– 165. 136. Goldman AL, Pope HG, Bhasin S. The health threat posed by the hidden epidemic of anabolic steroid use and body image disorders among young men. J Clin Endocrinol Metab. 2019;104:1069–1074. 137. McBride JA, Carson 3rd CC, Coward RM. The availability and acquisition of illicit anabolic androgenic steroids and testosterone preparations on the Internet. Am J Men’s Health. 2018;12:1352–1357. 138. Westerman ME, Charchenko CM, Ziegelmann MJ, et al. Heavy testosterone use among bodybuilders: an uncommon cohort of illicit substance users. Mayo Clin Proc. 2016;91:175–182. 139. Nieschlag E, Vorona E. Mechanisms in endocrinology: medical consequences of doping with anabolic androgenic steroids: effects on reproductive functions. Eur J Endocrinol. 2015;173:R47–R58.

111 Regulation of Spermatogenesis Kate L. Loveland, Liza O’Donnell, David de Kretser, Robin M. Hobbs, and Mark P. Hedger

OUTLINE Macroscopic Organization of the Adult Human Testis, 1861 The Seminiferous Tubules, 1861 The Intertubular Tissue, 1861 Postnatal Growth, 1862 Cellular Development and Organization for Functional Control, 1862 The Spermatogenic Epithelium, 1862 Sertoli Cells, 1862 Spermatogonia and Spermatogonial Stem Cells, 1864 The Spermatogonial Stem Cell Niche, 1865 Morphological Features of Spermatogonia in Primate and Human, 1866 Spermatogonial Differentiation and Its Control, 1866 Meiosis, 1866 Spermiogenesis: Emergence of Spermatozoa, 1867

Germ Cell Associations and the Spermatogenic Cycle, 1869 Spermatogenic Efficiency and Germ Cell Loss, 1869 Genetic Defects and Spermatogenesis, 1870 Epigenetics in the Male Germline, 1870 The Intertubular Compartment, 1870 Leydig Cells, 1871 Other Interstitial Cells, 1871 The Immunologic Environment of the Testis, 1871 Endocrine and Local Control of Spermatogenesis, 1871 Interactions Between the Testis, Hypothalamus, and Pituitary Gland, 1871 Control of Sertoli Cell Function, 1871 Sertoli Cells During Fetal and Postnatal Development, 1872 Control of Spermatogenesis: Challenges Ahead, 1874

 

MACROSCOPIC ORGANIZATION OF THE ADULT HUMAN TESTIS The adult human testis is 4-­to 5-­cm long, 15 to 35 mL in weight, enclosed on anterolateral and medial surfaces by serous membranes of the tunica vaginalis. Posteriorly, the associated epididymis and spermatic cord include the vas deferens and testicular neurovascular pedicle. Vessels and autonomic nerves from renal and abdominal aortic plexuses traverse the tunica albuginea, a 1-­mm-­thick, collagen-­rich capsule deep within the tunica vaginalis. The testicular artery enters posteriorly, descending to the inferior pole under the tunica albuginea to form the tunica vasculosa. In human testes, the main artery stem ascends beneath the anterior surface before smaller branches penetrate the parenchyma. Posteriorly, the thickened tunica albuginea projects into the testis parenchyma, forming the honeycomb-­like mediastinum through which the rete testis links the testis seminiferous tubules via efferent ductules to the epididymis. Numerous imperfect, thin connective tissue septa extend from the tunica albuginea toward the mediastinum, creating several hundred incomplete pyramidal lobules containing the seminiferous tubules surrounded by loose connective tissue.

The Seminiferous Tubules Spermatogenesis takes place within the seminiferous tubules; these are ∼200 μm in diameter, ∼600-­m long, and occupy ∼50% of the human testis volume. The numerous individual seminiferous tubules are highly convoluted linear segments. Each tubule ends in the mediastinum and empties via straight extensions, termed tubuli recti.1 The seminiferous epithelium is formed by contiguous Sertoli cells extending from the tubule base to its lumen. Fine and elaborate Sertoli cell cytoplasmic extensions envelope spermatogenic cells at all

development phases. The most immature germ cells are mitotic spermatogonia that contact the tubule basement membrane. Spermatogonia undergo both self-­renewing and multiplicative divisions, the latter with incomplete cytokinesis; clonal siblings are connected by cytoplasmic bridges through all subsequent differentiation steps. Spermatogonia differentiate and move away from the tubule base into the epithelium, forming the primary spermatocytes that commence meiosis. After the first meiotic division, these become secondary spermatocytes that undergo a second nuclear division to complete meiosis and become haploid round spermatids. Spermatids do not divide but undergo the complex metamorphosis called spermiogenesis. They are released into the tubule lumen as individual spermatozoa by the processes of spermiation. Direct movement of substances between adjacent Sertoli cells is prevented by specialized tight cell junctions that also create basal and adluminal compartments in the seminiferous tubules. These junctions form the blood-­testis barrier, which transiently opens as spermatogonia lose contact with the basement membrane to enter the adluminal compartment when they mature. Peritubular myoid cells completely cover the external tubule surfaces; together with the Sertoli cells, they synthesize the basement membrane that surrounds the tubules and upon which spermatogonia reside. These myoid cells contract irregularly to move sperm and Sertoli cell-­secreted fluid in the tubule lumen through the mediastinum into the rete testis, which drains into the epididymis. Three successive rete testis zones are (1) straight tubules of the septal rete, (2) anastomosing mediastinal rete channels, and (3) wider extratesticular rete spaces that connect to the 6 to 12 fine efferent ductules that lead into the epididymal head.

The Intertubular Tissue Adult human seminiferous tubules are surrounded by multiple layers of myofibroblasts, macrophages, and fibroblasts interspersed with collagen and extracellular matrix1; The peristaltic contraction and

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relaxation of peritubular myoid cells is governed by local vasoconstrictive and vasorelaxant products such as endothelin, vasopressin, oxytocin, and nitric oxide. Peritubular cells cosynthesize the tubule basement membrane with Sertoli cells, secreting proteins that modify the intratubular environment and directly affect spermatogenesis.2 The loose connective tissue of the interstitial area contains a species-­ specific cell composition. For example, the frequency and proportion of the androgen-­secreting Leydig cells varies between species, typically occupying 10% to 20% of this area. Blood vessels occur randomly throughout the interstitium, and capillaries are nonfenestrated. Although lymphatic vessels and large lymphatic sinusoids exist in testes of several rodent species, in humans these reside only in the main connective tissue septa extending inward from the tunica albuginea. Because blood vessels do not enter the seminiferous tubules, germ cells embedded within the epithelium are dependent on Sertoli cells for direct support.1 Interstitial areas also contain fibroblasts and macrophages, both opposed to the tubules (peritubular macrophages) and also within larger spaces, usually adjacent to Leydig cells and blood vessels (interstitial macrophages). Lymphocytes and mast cells are less frequent and are typically more abundant near the tunica vasculosa. The macrophage and lymphocyte frequency and function relate to their roles in maintaining the testis as an immune-­privileged organ.3

Postnatal Growth Testis volume increases significantly from 1.1 mL (median paired testes volume) at 0 to 1 year of age, to 3 mL at 5 to 10 years, to 23 mL at 14 to 18 years, and reaching 40 mL in an adult group of 18 to 50 years.4 Seminiferous tubule length per testis increases in prepubertal and postpubertal age ranges in a biphasic pattern, reaching a median length of 600 m in adults, with a wide variation that exceeds 1000 m in some individuals. The tubule diameter does not appreciably increase from birth to 14 years (50 to 60 μm) but expands to ∼130 μm at 14 to 18 years and is ∼200 μm in adults. Growth in seminiferous tubule length, but not diameter, in the prepubertal period reflects the significant increase in total germ cell numbers, from 13 to 83 million from 1 to 10 years of age, as spermatogonia populate the growing seminiferous tubules by migrating along the tubule length and forming more expansive colonies. This is followed by an exponential numerical expansion in the germ cell population.5–7

CELLULAR DEVELOPMENT AND ORGANIZATION FOR FUNCTIONAL CONTROL The Spermatogenic Epithelium Adult male fertility requires sustained proliferation and maturation of self-­renewing germline stem cells and spermatogonial stem cells (SSCs) capable of differentiating into fully formed spermatozoa. The duration of one adult spermatogenic cycle (from stem cell to sperm) varies between species, taking 35 days in mice and hamsters, 50 days in rats, 45 to 65 days in several nonhuman primates, and 70 days in humans. The first release of spermatozoa (spermarche) in human puberty is at ∼13.5 years of age, or earlier. Adult men produce ∼1000 sperm per second, with 1.5 to 2.5 × 108 sperm typically present in an ejaculate. KEY POINTS  • Spermatogenesis occurs with all germ cells embedded in the seminiferous epithelium created by Sertoli cells.

Germ cells occupy up to 75% of the seminiferous epithelium, arranged in multiple layers with least mature germ cells adjacent to the base and more mature cells closer to the tubule lumen. Maturing cells

move apically through the epithelium connected as clonal siblings by cytoplasmic bridges until the most mature testicular germ cells, spermatozoa, are released into the tubule lumen (Figs. 111.1 and 111.2). This basal to luminal arrangement of maturing germ cells within the seminiferous epithelium is fundamentally similar in all mammals, although the associations between differentiating germ cell types along its length, termed stages, differ between species. Broadly speaking, regulation of adult spermatogenesis involves (1) endocrine stimulation of the testis by gonadotropins and steroid hormones (notably androgens), (2) local paracrine and autocrine factors, (3) tightly regulated gene expression within each germ and somatic cell type, (4) dynamic interactions between individual Sertoli cells and the changing variety of developing germ cells they support, and (5) Sertoli cell maintenance of the unique seminiferous tubule environment that restricts entry of cells, macromolecules, and immunogenic agents originating from the intertubular tissues and vasculature. Disturbance or impairment of any one of these parameters may disrupt or suppress spermatogenesis, contribute to defects in spermatozoa, and/or decrease sperm count.

Sertoli Cells In the adult testis, Sertoli cells are the only nondividing cell type within the seminiferous tubules, and their function is central to sperm production. A single Sertoli cell partially or completely surrounds and supports several dozen or more germ cells.8 The columnar Sertoli cell base rests on the basement membrane and overarches spermatogonia, extending apically to the lumen with elaborate lateral branches that interdigitate and surround all other germ cell types (Fig. 111.1). Each Sertoli cell contacts the base of five or six other Sertoli cells, simultaneously supporting up to 50 germ cells at different developmental stages.9 Sertoli cells change shape as germ cells multiply, move, and undergo their own shape and size transformations. Sertoli cells divide only during fetal and postnatal life, stimulated by follicle-­stimulating hormone (FSH), activin, and other factors; their number in an adult testis determines its spermatogenic capacity.10 Proliferation during puberty occurs in species with a lengthy interval between birth and puberty, ending by approximately 15 years of age in humans.11

Blood-­Testis Barrier. The blood-­testis barrier exists in all animals. It is a structural and physiological compartment created by inter–Sertoli cell tight junctions on the luminal side of spermatogonia and their immediate descendants, the preleptotene/leptotene primary spermatocytes (Figs. 111.1 and 111.2). It includes several junction types, including tight junctions between adjacent Sertoli cell membranes that overlay a region of smooth endoplasmic reticulum with microfilaments projecting into the cytoplasm. The blood-­testis barrier is stabilized by many intracellular components just beneath the cell membrane, including actin filaments and actin-­binding proteins such as espin.9,12 The junction positioning at the seminiferous epithelium base subdivides the anatomically and functionally distinct basal and adluminal compartments; spermatogonia develop within the basal compartment, while the meiotic and postmeiotic germ cells mature within the restricted adluminal compartment. Barrier integrity is essential for (1) excluding meiotic and postmeiotic germ cells from immunologic challenge arising from outside the seminiferous epithelium, (2) maintaining the stem and spermatogonial cell niche, (3) preventing intercellular fluid and macromolecular flow into the adluminal compartment and thus allowing its milieu to be determined by the Sertoli cells, and (4) enabling selective exposure of basal germ cells to regulatory factors originating from interstitial cells, such as Leydig and peritubular cells and macrophages.

.

l

CHAPTER 111  Regulation of Spermatogenesis The blood-­testis barrier forms first in the peripubertal period when spermatogenesis is initiated by gonadotropic stimulation and coincides with the appearance of zygotene-­pachytene primary spermatocytes.13 Developing spermatocytes move through this tight junction barrier, which opens during their transit into the adluminal compartment and then reforms basally.14 In humans, junctional specializations between Sertoli cells are observed after 8 years of age and assembled in early puberty (11 to

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13 years)15 accompanied by increasing spermatocyte numbers. Coordinated signals by hormones (testosterone and FSH), retinoic acid (RA), and other factors differentially regulate tight junction dynamics to maintain blood-­testis barrier integrity, yet allow germ cells to cross the barrier at the appropriate stage for meiotic maturation (see “Adult Sertoli Cell Function”). KEY POINTS  • The haploid spermatids undergo complex differentiation, termed spermiogenesis, involving sperm head nuclear compaction and acrosome development, sperm tail formation, cell organelle reorganization, and shedding of excess cytoplasm.

Plasticity and Links With Germ Cells. Concordant with elon-

Figure 111.1  Diagram of the human seminiferous epithelium showing the general relationship between Sertoli cells and germ cells. Type A pale (Ap), A dark (Ad), and B (B) spermatogonia are shown in the basal compartment, together with primary spermatocytes (P), early round spermatids (RS), and elongating spermatids (ES) in the adluminal compartment. Inter–Sertoli cell tight junctions are indicated by arrows.

gated spermatid release at the end of spermiogenesis, Sertoli cells form specialized junctions with a new cohort of elongating spermatids and restructure inter–Sertoli cell junctions to permit spermatocyte translocation into the adluminal compartment (Fig. 111.3). The elaborate and dynamic cytoskeleton of adult Sertoli cells maintains their columnar shape, determines the distribution of its organelles, and mediates adhesion with germ cells. The cytoplasm around its nucleus contains microtubules and abundant motor proteins, dynein and kinesin, that are especially abundant in crypts surrounding elongating spermatid heads.16 These cytoskeletal elements, together with intermediate filaments, are positioned to influence the dynamic shape and position of maturing germ cells as proceed through the spermatogenic cycle. Mouse models with defects in microtubule dynamics often exhibit phenotypes of premature germ cell release and sterility. The actin-­containing ectoplasmic specialization (ES) is another Sertoli cell cytoskeleton component that connects Sertoli and germ cells and facilitates spermatid orientation within the seminiferous epithelium. This structure in the Sertoli cells is positioned opposite the nucleus of newly elongating spermatids and facilitates their translocation in the epithelium as they elongate and develop an extensive flagellum.17 The abundant Sertoli cell microtubular system moves spermatids by coupling its associated motor proteins to the ES. The microtubule-­associated protein kinesin directs the

eS

eS eS

rS

rS PSc

S

Ap

S pSc

S Ad

Sg

A

Bm

PT

B

PT

Figure 111.2  Histological sections showing seminiferous epithelium cross sections of (A) rat and (B) human testes. Ad, spermatogonia types A dark; Ap, A pale; Bm, basement membrane; eS, step 19 elongating spermatids; P, pachytene spermatocytes; pS and eS, round and elongated spermatids; pSc, primary spermatocytes; PT, peritubular tissue; rS, step 8 round spermatids; S, Sertoli cell nucleus; Sg, spermatogonia.

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Spermatogonia and Spermatogonial Stem Cells

A

B

C Figure 111.3  Schema showing tight occluding junctions between adjacent Sertoli cells that constitute the blood-­testis barrier. (A) Tight occluding junctions are formed above the Type A and Type B spermatogonia positioned on the basement membrane, and (B) above and below meiotic spermatocytes (labeled), as developing germ cells move (C) from the basal to the adluminal epithelial compartment. Positions where tracers, representing macromolecules required for germline support, can penetrate are indicated with arrows.

initial basal movement of elongating spermatids into deep Sertoli cell crypts, then the microtubule motor protein dynein reverses spermatid translocation, toward the lumen.17 This transient penetration of elongating spermatids deep within the epithelium likely facilitates spermatid-­ Sertoli cell communication during spermiogenesis.

Phagocytosis, Endocytosis, and Secretion. Many germ cells are eliminated via apoptosis during normal spermatogenesis, and the spermatid residual cytoplasm remains within the epithelium as a residual body after spermiation. Sertoli cells process these apoptotic germ cells and residual bodies via phagocytic and endocytic pathways. Residual bodies move basally, while excess plasma membrane removal and luminal fluid absorption occur at the Sertoli cell apical cytoplasm.18 Sertoli cells must also transport or generate metabolites required for germ cells in the adluminal compartment, as the blood-­testis barrier prevents their direct delivery. Receptor-­mediated endocytosis of macromolecules occurs at both apex and base of the epithelium, including to deliver iron from serum to germ cells.19

Spermatogonial proliferation and differentiation are regulated by hormones, paracrine/autocrine factors, and systemic nutrients.20 The proliferative, sexually indifferent primordial germ cells formed in early embryogenesis adopt the male fate in response to somatic cell cues following SRY expression in Sertoli cells. Termed gonocytes, or prospermatogonia, in the fetal testis, they enter a period of mitotic quiescence then transform immediately after birth into mitotic spermatogonia in rodents or asynchronously in humans from fetal life to about 2 years. Fibroblast growth factor (FGF) signaling mediated by FGF8 helps initiate this transition by germ cells in late fetal life in mice21; transcripts encoding FGF receptors are present in human spermatogonia but their functionality is undetermined.22 All spermatogonia undergo mitosis. Continuous production and release of spermatozoa requires a stable population of SSCs that either self-­renew to generate more stem cells or produce progeny, which commence spermatogenesis and transform into sperm. KEY POINTS  • The entire spermatogenic process involves highly regulated developmental steps, mediated by local and endocrine somatic cell products and requiring ongoing spermatogonial stem cell replication for continuous sperm production over the life span.

All spermatogonia are partly surrounded by Sertoli cells and also directly contact the basal lamina, so secretory products of peritubular tissues, Leydig cells, immune cells, and circulating factors including hormones can influence their maturation (Fig. 111.4). In adult mammalian testes, spermatogonia span a range of differentiation states that include the least differentiated SSCs, progenitor spermatogonia primed to differentiate but that can still form a stem cell pool, and the differentiated spermatogonia that cannot function as stem cells. Spermatogonia in mammals were originally classified into types A and B according to their nuclear chromatin patterns in histologic preparations.23 Type A spermatogonia were classified into undifferentiated and differentiated subtypes, based on morphological criteria and their potential to function as stem cells,24 and type B spermatogonia are those that divide to produce preleptotene spermatocytes. These categories and species-­specific differences are being revealed by single cell transcriptome data (discussed later). Transplantation analyses functionally identify SSCs as those germ cells that can reconstitute full spermatogenesis when transferred into a recipient testis. This can assess how specific signaling pathways contribute to SSC maintenance and differentiation, with one goal being the preservation or restoration of fertility for individuals undergoing stem cell–damaging chemotherapy.25 Both organ and cell cryopreservation and culture are used for SSC preservation and recovery, and genetic modifications prior to transplantation can correct infertility-­ causing mutations in animals.25 Transplantation of heterologous or xenologous germ cells has also revealed fundamental or conserved features of testis development. For example, rat or hamster germ cells undergo spermatogenic development in recipient mouse testes26,27 with the rate of germ cell development intrinsic to the transplanted donor germ cells, rather than determined by the host niche. Mouse and other host testes will maintain human SSCs for months, but they do not differentiate25; however, allogeneic transplantation of Rhesus macaque spermatogonia into either juvenile or adult recipient testes can undergo complete spermatogenesis.28 Thus autologous transplantation of preserved self-­germline stem cells may enable men requiring chemotherapy to produce healthy sperm. The prevailing model of the rodent spermatogonial hierarchy is based on markers for undifferentiated cell subsets and development of

CHAPTER 111  Regulation of Spermatogenesis

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Basement membrane

Macrophages Peritubular myoid cells

Leydig cells Lymphatic endothelial cells

Spermatocytes

Sertoli Spermatids

Undifferentiated spermatogonia

Differentiating spermatogonia Meiosis

SSCs

GFR α1

c-KIT

NGN3 + RARγ PLZF Figure 111.4  Illustration of rodent spermatogonial hierarchy and components of the stem cell niche. Spermatogonia are attached to the seminiferous tubule basement membrane and surrounded by multiple supporting somatic cell types (top panel). A segment of the seminiferous tubule is shown. The spermatogonial population is divided into undifferentiated and differentiating fractions (bottom panel). The undifferentiated population contains spermatogonial stem cells (SSCs) and differentiation-­destined transit-­amplifying cells. Differentiating spermatogonia undergo a series of coordinated mitotic divisions before generating meiotic spermatocytes that produce haploid spermatids. Molecular markers of the distinct spermatogonial fractions are shown.

functional SSC assays (lineage-­tracing and transplantation).20 It proposes that SSCs of the homeostatic testis are spermatogonia expressing the coreceptor for growth factor GDNF (GFRα1), which includes most single (As) and a substantial fraction of paired (Apr) cells (Fig. 111.4). Transcriptional regulators ID4, EOMES, and PDX1 are linked to SSCs and are expressed within the GFRα1+ population.29 GFRα1+ cells continually interconvert between syncytial states (Apr plus short chains of aligned cells [Aal] and As through chain fragmentation).30 In contrast, undifferentiated cells positive for Neurog3 (Ngn3), primarily longer chains (Aal), are destined to differentiate under the influence of RA acting via RA receptor γ (RARγ).31 Spermatogonial differentiation is marked by KIT receptor induction and epigenetic changes, accompanied by loss of stem cell potential.32 Importantly, Ngn3+ Aal can fragment to As and Apr and contribute to the GFRα1+ stem cell pool, particularly under regenerative conditions, for example, following germline depletion or transplantation.24 The role of transit-­ amplifying populations in germline regeneration was confirmed by studies of cells expressing Piwil4 (Miwi2), a subset of the Ngn3+ population. Further, differentiation-­primed progenitors can revert to an SSC state under the influence of niche factors such as GDNF, as demonstrated through culture and transplant-­based studies.29 Markers such as GFRα1, RARγ, KIT, and others are routinely used to functionally categorize spermatogonia (Fig. 111.4).29 Rodents have a distinct first round of spermatogenesis in which the first sperm arise soon after birth directly from gonocytes that transition directly into differentiating spermatogonia; all subsequent rounds

of spermatogenesis derive from gonocytes that transition first into an undifferentiated spermatogonial state. Recent single cell-­RNA sequencing (scRNAseq) analyses indicate that this is different in humans.22,33 Human spermatogonia are maintained in one of two undifferentiated states in low numbers (States 0 and 1; 3% to 4% of all testis cells) from infancy through adulthood. This “reserve” population is amplified by multiplication at the onset of puberty, from 11 years; differentiating spermatogonia emerge (stages 2 to 4) with transcription of meiotic genes evident from 13 years.22 Thus the mechanisms that control the initiation of spermatogenesis in juveniles are likely to be both shared and distinct between rodents and humans, and they may also be distinct from those active in adults.

The Spermatogonial Stem Cell Niche The specialized stem cell niche microenvironment conveys extrinsic signals to modulate the intrinsic stem cell regulators that determine fate choice: between self-­renewal, proliferation, differentiation, or cell death. Adult fertility requires an uninterrupted germ cell supply along the complete length of each tubule, and hence stem cells must be appropriately distributed. In the postnatal prepubertal rodent testis, spermatogenesis is initiated nonuniformly throughout the testis, with RA a fundamental driver for this in both neonatal and adult rodents.34,35 Over time, complete cycles of the seminiferous epithelium are established with fairly precise spatial distribution along the tubules. Histological and real-­time video evidence from rodent testes indicates that SSCs are nonrandomly positioned close to the vascular-­rich intertubular regions rather than in

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regions where neighboring tubules are closely opposed; this distribution was reestablished if tagged tubule segments were freed of vessels and interstitial tissues subsequently transplanted into a recipient testis (Fig. 111.4). SSC density and localization are regulated by competition for limiting amounts of FGFs produced by lymphatic endothelial cells and other interstitial cells surrounding the tubules.36 The Sertoli cell– produced chemokine, CXCL12, provides a critical signal via the CXCR4 receptor on SSCs, maintaining them in an undifferentiated state and controlling their homing to the niche.37 CXCL12 may also influence the behavior of seminoma cells that arise in young men from fetal germ cells that fail to differentiate normally into spermatogonia.37,38 Glial cell–derived neurotrophic factor (GDNF) synthesis by Sertoli cells is essential for the maintenance and proliferation of mouse, rat, hamster, bull, and human SSCs. Its synthesis is stimulated by pituitary-­ derived hormone FSH and locally produced factors such RA, basic fibroblast growth factor (bFGF), tumor necrosis factor-­α (TNFα), and interleukin 1b (IL1β).39,40 Other cytokines including FGFs and colony-­ stimulating factor-­1 (CSF-­1) complement the role of GDNF in SSC renewal and proliferation20 but how circulating factors affect their synthesis is unknown. Balanced signaling by WNT ligands and inhibitors (via both canonical β-­catenin–mediated and noncanonical pathways) also contributes to SSC maintenance and differentiation.20

Morphological Features of Spermatogonia in Primate and Human Type A spermatogonia are precursors of all further differentiated germ cells. Primate and human testes exhibit dark and pale type A spermatogonia. The A dark (Ad) spermatogonia stain deeply with hematoxylin and have homogeneous, fine chromatin with an obvious central pale-­stained vacuole-­type area. Pale type A (Ap) spermatogonia have uniformly pale-­ staining nuclei and one or two nucleoli near the nuclear membrane. Type B spermatogonia nuclei feature coarse, deeply stained heterochromatin clumps next to the nuclear membrane, with a central nucleolus. Ad and Ap spermatogonia are in pairs and groups along the seminiferous tubule (see Fig. 111.2). Type Ad spermatogonia rarely divide in the human testis and are considered as reserve stem cells that only divide and transform into Ap cells after irradiation or cytotoxic treatment.41 Type Ap spermatogonia undergo both self-­renewing mitotic divisions and mitotic amplification as they transition into type B spermatogonia.42 Type B spermatogonia divide by mitosis then become primary and secondary spermatocytes while undergoing the two nuclear cell divisions of meiosis to yield the round haploid spermatids that then transform into spermatozoa. In humans, the ratio of Ad spermatogonia:Ap spermatogonia:type B spermatogonia:spermatocytes is 1:1:2:4. The relationship of these morphologically defined populations to those identified by scRNAseq is yet to be revealed.

Spermatogonial Differentiation and Its Control Application of scRNAseq to testes of different ages and in different species has identified the progressive expression of transcripts through their maturation, including those that encode signaling receptors and thereby reveal regulatory components that act successively. Common to both mouse and human spermatogonia is the delineation of sequential maturation states, transitioning generally from a more metabolically quiescent to active, replicative state, and acquiring machinery for meiosis.43,44 The first two spermatogonial states in adult human testes (referred to as States 0 and 1) express stem cell–associated factors including ID4 and PLZF, suggesting that they represent SSCs, while proliferation and differentiation-­ associated markers are upregulated in subsequent states (e.g., KI67 and KIT, respectively).33 State 0 cells most closely resemble germ cells of the immature infant testis, indicating they are more primitive than State 1 cells. Surprisingly, expression of GFRA1, an SSC marker commonly used

in rodents, is predominately observed in State 1 spermatogonia, while State 0 cells, a potential quiescent reserve SSC population, are uniquely marked by transcripts such as PIWIL4 plus TSPAN33 and lacking in rodents.33 However, in common with rodents, RNA-­velocity analyses indicate that human spermatogonia can dynamically interconvert between functional states, and their transplantation potential is not restricted to the most primitive state.33,45 This cellular plasticity is associated with limited changes in the epigenetic landscape between spermatogonial states.33 Notably, markers of State 0 and 1 cells are expressed by both Ad and Ap spermatogonia, indicating the disconnect between transcriptional and histological cell features historically used to assess human spermatogonial functions22,45 Multiple signaling molecules produced by testis somatic cells that are known, and new ones predicted from scRNAseq analysis, support germ cells transitioning through spermatogenesis.45 RA signaling is essential for spermatogonial differentiation in postnatal and adult testes. Induced RA deficiency in rats and mice through either dietary or pharmacological means reversibly arrests type A spermatogonial proliferation and differentiation.34 Produced by Sertoli cells, RA represses GDNF transcription40 and elevates transcripts associated with spermatogonial differentiation in both Sertoli (Kitl and Bmp4) and germ cells (Stra8 and Kit). Activation of the KIT tyrosine kinase receptor via KITL (termed KIT ligand or stem cell factor [SCF]) from Sertoli cells is essential for survival and proliferation of differentiating spermatogonia.46,47 This pathway is also central to human fertility; abnormal pathway component expression is documented in testes with defective spermatogenesis, and polymorphisms and mutations in KIT and KITL are frequently associated with infertility and testicular germ cell tumors.48 The transforming growth factor-­β (TGFβ) superfamily ligands BMP4 and activin A also influence spermatogonial differentiation, including by modulating levels of KIT, the hallmark of differentiating spermatogonia.49 Additional factors affecting spermatogonial differentiation and proliferation include nerve growth factor, epidermal growth factor, and platelet-­derived growth factor. Also key for postnatal spermatogenesis is the doublesex-­ related transcription factor (DMRT1) involved with both Sertoli and germ cell development; Dmrt1 deletion in mouse embryos resulted in azoospermic adults. Cell-­selective deletion of Dmrt1 in Sertoli cells prevented their postnatal differentiation and led to the loss of the male phenotype; deletion in germ cells impaired the initial establishment and subsequent maintenance of spermatogonia,50,51 premature entry into meiosis, and eventual stem cell depletion. This outcome was dependent on vitamin A actions, including through direct interaction of Dmrt1 with genes encoding proteins required at the onset of meiosis such as Stra8 and the mitotic inhibitor p21Cip1.52 Mutations and single-­nucleotide polymorphisms (SNPs) in DMRT1 are implicated in human infertility and increased testicular germ cell tumor risk, with the latter implicating a germ cell–intrinsic defect as critical in the establishment of this condition in some men. As mentioned in “Genetic Defects”, mutations in sex chromosomes may also affect spermatogonial proliferation and/or maturation.

Meiosis Type B spermatogonia enter prophase of the first meiotic division to form primary spermatocytes. Sibling spermatocytes remain connected via the cytoplasmic bridges through which mRNAs and other macromolecules can transit.53 Although these bridges first form as SSCs commence differentiation, their loss does not visibly impact progression until meiosis.54 Meiosis involves a long prophase whereby DNA is replicated and there is homologous recombination between paired chromosomes to facilitate genetic diversity of the games. Preleptotene spermatocytes commence DNA synthesis, replicating each chromosome to form twin copies called sister chromatids, marking the commencement of prophase. As a consequence, the preleptotene primary

CHAPTER 111  Regulation of Spermatogenesis spermatocyte chromosome number remains diploid, but the DNA content is 4C, twice that found in spermatogonia. Prophase is subdivided into five phases based on gross chromosomal activities and appearance: (1) leptotene, thread-­like chromosomes, (2) zygotene, homologous chromosome pairing, (3) pachytene, paired chromosomes thicken and undertake crossing over, (4) diplotene, paired chromosomes decondense and partially separate, and (5) diakinesis, chromosomes recondense. During prophase, homologous chromosomes pair and the synaptonemal complex facilitate the close apposition of each homologue pair, and the chromosomes form a synapse that permits crossover and exchange between them at chiasmata. Genetic exchange between the paired chromosomes requires error-­free double-­strand DNA breakage and repair; key to this is the strong upregulation in mid-­phase primary spermatocytes of highly conserved topoisomerases.55 Metaphase follows prophase, as the highly condensed chromosomes align on a metaphase spindle. During anaphase, the chromosome pairs move to opposite poles of the cell. During telophase, complete nuclear separation forms daughter cell nuclei, producing secondary spermatocytes. The second meiotic division proceeds rapidly (5 years Calcitonin postmenopause) Parathyroid hormone Treatment RANK ligand inhibitor Treatment Estrogens Prevention

Site of Action Vertebrae and hip Vertebrae Vertebrae Vertebrae and hip Vertebrae and hip

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action will only prevent further bone loss, as demonstrated by many RCTs. No HT product currently has government approval for the treatment of osteoporosis; however, most are approved for osteoporosis prevention. Observational studies and RCTs have demonstrated reductions in fractures of the vertebrae and possibly the hip in women on HT. A meta-­analysis of 22 trials of estrogen reported an overall 27% reduction in non-­vertebral fractures, while taking into account that the quality of individual studies varied.147 The Heart and Estrogen/ Progestin Replacement Study (HERS) and HERS II found no reduction in hip, wrist, vertebral, or total fractures with HT, but the follow-­up interval was relatively short.148 The WHI is the first RCT to demonstrate reduction of hip fracture risk with estrogen use149; risk reductions were also reported for vertebral and other osteoporotic fractures. The US Preventive Services Task Force concluded that good evidence suggests that HT prevents bone loss, and fair to good evidence indicates that it reduces fractures.150 Women experiencing an early menopause who require prevention of bone loss are probably best served by the administration of HT or oral contraceptives, rather than bone specific treatments, until they reach the median age of natural menopause (51.4 years), at which time treatment needs may be reassessed.106 For postmenopausal women with established osteoporosis without a symptom indication for HT, non-­hormonal modalities are available. Alendronate, a bisphosphonate that is a powerful antiresorptive agent and reduces fractures in menopausal women with osteoporosis, has been approved by the US FDA for the prevention (5 mg daily) and treatment (10 mg daily or 70 mg weekly) of osteoporosis. Risedronate is more potent and has fewer upper gastrointestinal side effects than alendronate; it reduces the incidence of fracture in osteoporotic women. Raloxifene is the only SERM approved for long-­term treatment in the prevention of osteoporotic fractures in postmenopausal women. It has been demonstrated to reduce vertebral fractures.151 Denosumab is a human monoclonal antibody to the receptor activator of nuclear factor-­ κB (NF-­κB) ligand, which binds to the receptor activator of NF-­κB on the surface of osteoclasts blocking proliferation and differentiation of the osteoclasts. Approved in 2010 for treatment of postmenopausal women with osteoporosis who are at high risk for fracture, it is administered subcutaneously every 6 months.152 Finally, recombinant parathyroid hormone, administered subcutaneously, daily, is approved for the treatment of osteoporosis in postmenopausal women who are at high risk for fractures, and can increase BMD substantially.153

Cardiovascular Disease. Observational studies have long suggested that combined HT is beneficial for the primary prevention of heart disease.154 The WHI, a primary prevention trial of 16,608 healthy postmenopausal women after 5.2 years of daily HT or placebo, initially reported a small but significantly increased risk for nonfatal heart attacks. Although CHD mortality was not increased, the risk for heart disease was 29% higher for women taking combined HT than for those on placebo.149 The WHI findings do not support the use of HT for primary prevention of heart disease in women. The HERS indicated that women with known CHD who were randomized to CEE and MPA had a 52% increased risk for myocardial infarction during the first year of use and experienced increased CHD deaths during the first 3 years of use.155 After 6.8 years of follow-­up in HERS II, no difference in primary or secondary CHD events was reported among estrogen users.156 Thus, for secondary prevention of heart disease, no clinical benefit is derived from HT. Furthermore, the American Heart Association has long recommended discontinuing treatment after an acute event in a woman who is currently using HT.157 Since the publication of the two major primary and secondary prevention trials, multiple observational cohorts and smaller RCTs have been performed to attempt to reconcile the discrepancy between the

apparent benefit of HT in cohort studies and a neutral to harmful outcome in RCTs. Unfortunately, the divide has only deepened with time, with further observational studies such as the Danish Osteoporosis Prevention Study supporting a benefit of HT,158 despite no clear benefit demonstrated from randomized clinical trials. The “timing hypothesis” seeks to explain this discrepancy by theorizing that HT has multiple long-­term beneficial effects when initiated before or during the early period after menopause, but not during the late postmenopausal period (i.e., more than 10 years after FMP). The ELITE study, a single-­site RCT of 643 healthy postmenopausal women, stratified the sample according to time since menopause (early postmenopause as < 6 years or late postmenopause as ≥ 10 years). Patients were randomly assigned to either oral 17β-­estradiol or placebo (with intermittent progesterone or placebo progesterone). ELITE found that oral estradiol therapy initiated within 6 years after menopause was associated with a decreased development of subclinical atherosclerosis (measured as CIMT) than placebo but not when it was initiated ≥ 10 years after menopause.84 At the same time, KEEPS, a multicenter RCT of 727 women, was performed to assess the effect of HT in women who started within 3 years of FMP. Treatment arms included (1) oral conjugated equine estrogens (O-­CEE) 0.45 mg/day, (2) transdermal estradiol (t-­E2) 50 mcg/day, and (3) placebo. Active arms received cyclical micronized progesterone 200 mg/day for 12 days/month, with a progesterone placebo for the placebo arm. After 48 months of follow-­up, O-­CEE and t-­E2 had no adverse effects on atherosclerosis progression or coronary calcium. O-­CEE and t-­E2 had neutral effects on blood pressure and mainly favorable or neutral effects on CHD biomarkers. These findings support a lack of harm, and imply the potential for benefit to early intervention with HT for symptomatic women, but they are a far cry from supporting widespread preventive use of HT. Aside from heart disease, HT may play a role in recurrent thrombotic events. The WHI reported an increased risk for stroke after 5 years of HT use149 and, in agreement with previous studies, a higher risk for thromboembolism in the first year of HT use, which diminishes thereafter to approximately a 2-­fold increased risk.149 Follow-­up analysis of the WHI that examined both coronary and venous thromboembolic events showed that the increased risk for both events in the combined HT group subsided over the 2.4-­ year postintervention follow-­ up period.159 Taking all of the data into account, guidelines on HT have evolved in their recommendations to include the case for “extended use.” The American College of Obstetrics and Gynecologists,105 the NAMS,106 and the American Heart Association160 continue to appropriately recommend against the use of HT for primary or secondary prevention of cardiovascular disease. Because cardiovascular disease is a more common cause of morbidity and mortality for women than are osteoporosis and cancer combined, efforts to identify patients at risk and recommendations on lifestyle modifications, diet, and other pharmacotherapy (i.e., beta blockers, aspirin, angiotensin-­converting enzyme inhibitors, statins) are more beneficial and probably less harmful than HT for long-­term treatment. However, for women who remain symptomatic and are unable to utilize non-­hormonal alternatives to HT, longer-­term use can and should be considered using shared decision making that takes into account the patient’s perceptions of benefit.

Cognition. Similar to the data on cardiovascular disease and HT, observational studies demonstrate a benefit of hormones, whereas RCTs demonstrate a neutral to negative effect. Thus, the timing of initiation of HT may be relevant for both cardiovascular and cognitive outcomes. While this hypothesis may be correct, a recent review by Maki79 strongly suggests that a definitive test of this hypothesis may not be feasible. KEEPS included an exhaustive cognitive battery of tests

CHAPTER 122  Menopause and Perimenopause on > 700 recently menopausal women randomized to transdermal or oral estrogen, both with intermittent progesterone, or to placebo, and found an overall neutral effect of hormones on cognition.80 KEEPS is in the process of repeating the cognitive battery on participants from the original cohort and may be able to address the timing hypothesis. Henderson et al.81 found similar neutral findings in their RCT, with a total of 567 healthy women randomly assigned to oral 17β-­estradiol 1 mg/day or placebo within 6 years of menopause or 10+ years after menopause. There were no differences in executive function, verbal memory, or global cognition when estradiol was initiated within 6 years of menopause or if begun 10+ years after menopause.

Primary Ovarian Insufficiency and Early Menopause. POI can result from genetic factors, medical conditions, iatrogenic causes, or idiopathic factors. All patients diagnosed with POI (excluding iatrogenic causes) should be evaluated with a karyotype, adrenal antibodies, FMR1 premutation, pelvic ultrasound, and bone density.161 Patients with idiopathic POI have a higher prevalence of autoimmune disorders, and all should be screened regularly for thyroid dysfunction, diabetes, and adrenal insufficiency. Ongoing screening should continue even when initial screening is negative as these conditions can present later. Other conditions such as pernicious anemia, myasthenia gravis, rheumatoid arthritis, and systemic lupus erythematous are associated with POI, and should be screened with onset of symptoms.103 HT is particularly beneficial for patients with POI or early menopause that occurs from either natural or surgical causes. These patients are at increased risk for osteoporosis, adverse cardiovascular effects, neurological disease, impaired sexual function, and psychiatric conditions, and the risk of poor outcomes increases with earlier age of loss of ovarian function.162 Estrogen replacement can prevent many of these conditions or reduce symptom severity,162 and it should be initiated immediately upon the diagnosis of POI and continued until the natural age of menopause for the highest benefit.161 HT should attempt to mimic physiological estradiol levels with estradiol-­progestin preferred over OCPs due to a higher risk of VTE and less proven benefits of bone health in OCPs. It is important to remember that younger patients with POI have a 5%–10% chance of spontaneous conception, and patients should be counseled appropriately about this risk. Those not interested in pregnancy should be on some form of contraception since HT alone does not prevent pregnancy.161

Cancer Risks and Mitigation of Risk With Hormone Therapy

Breast Cancer. In the WHI study, an additional 8 breast cancer cases per 10,000 women taking continuous combined CEE + MPA per year was observed, with a HR of 1.24 (95% CI 1.01-­1.53) after a median 5.6 years of randomized treatment.163 Somewhat surprisingly, a reduced risk of breast cancer was observed in women who had a hysterectomy in the CEE-­alone arm.164 The combined lack of cardioprotection and potential risk of breast cancer associated with HT led to widespread discontinuation of its use. Unfortunately, the reassuring message of reduced risk of breast cancer in association with estrogen-­only therapy was essentially unheard by the public. Yet, the findings raise fundamental questions about the biology of breast cancer and how hormone exposure contributes to risk. Women who were HT naïve when randomized in the WHI had an overall similar breast cancer incidence whether they took CEE+MPA therapy or placebo for up to 11 years of follow-­up; women who had previously taken HT had an overall lower incidence of breast cancer when they were randomized to placebo.165 The most recent WHI data containing more than 20 years of median follow-­up confirms that women randomized to CEE+MPA had a higher long-­term risk of breast cancer, but no increase in breast cancer mortality, and that women randomized to CEE alone had a lower risk

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of both breast cancer and breast cancer mortality.166 Two more recent RCTs that initiated HT within 6 years of FMP, with a primary outcome being the effects of HT on cardiovascular disease, showed no increased risk of breast cancer compared with placebo; however, these trials were not sufficiently powered to provide definitive data for a breast cancer outcome.84,158 The Collaborative Group on Hormonal Factors in Breast Cancer’s recent meta-­analysis found that all forms of HT, excluding vaginal estrogen, were associated with an increased risk of breast cancer, and this risk progressively increased with the duration of HT use and was higher for estrogen plus progestin than estrogen alone. These excess risks were detectable even during years 1–4 of use (estrogen-­ progestogen RR 1.60, [95% CI 1.52–1.69]; estrogen-­only RR 1.17, [1.10–1.26]), and increased incrementally with longer duration of use (years 5–14 estrogen-­progestogen RR 2.08, [2.02–2.15]); estrogen-­only RR 1.33, [1.28–1.37]).167 The French E3N-­EPIC cohort study found an increased risk of breast cancer in postmenopausal women on HT compared with non-­users (RR 1.2 [1.1–1.4]), with most risk attributable to HT containing synthetic progestins compared with micronized progesterone, the RRs being 1.4 [1.2–1.7] and 0.9 [0.7–1.2], respectively.168 For women without a uterus who do not need a progestin, the overall data favor that HT with estrogen alone is risk neutral to minimal. However, for women who require a progestin to prevent endometrial hyperplasia, breast cancer risk appears to increase with duration of use, and this risk may be mitigated by use of micronized progesterone, but currently there are no randomized clinical trials to support this notion. Breast cancer risk is a serious adverse effect and therefore counseling is recommended to be conservative (i.e., reliant on the best available RCT data) and to involve shared decision making. Although the overall risk of breast cancer attributable to menopausal HT with E+P equates to less than 1 additional case per 1000 woman-­years of use,169 risk accumulates with duration of use and is one of the principal drivers of the dictum: “lowest possible dose for shortest possible time.” KEY POINTS • The current recommendation is to use HT in the lowest effective dose for the shortest time needed to control symptoms.

Other Cancers. Unopposed estrogen is a well-­known risk factor for endometrial hyperplasia and endometrial cancer, and the major method to protect the endometrium in women taking HT with an intact uterus is the addition of progestin. Multiple studies have found an increased risk of endometrial cancer among users of ET only compared with age-­matched controls,170 but this risk does not accrue to women using vaginal estrogen alone.116 Concomitant estrogen plus progestin (or BZA) is required for women with a uterus who are taking systemic HT. Progestin may be given either continuously or sequentially. Sequential use may be more palatable for women who have adverse effects from progestin use, but it will result in continued menstruation. Sequential combined estrogen plus progestin therapy is also less effective in eliminating the risk of endometrial cancer compared with continuous combined estrogen plus progestin, which decreases endometrial cancer risk below that of never users.170 In regards to ovarian cancer risk, several meta-­analyses171,172 have shown an increased risk of ovarian cancer associated with HT use. The pooled RR of ovarian cancer was 1.29 (95% CI 1.19–1.40), and increased risk was only found for the two common types, serous and endometrioid tumors. A recent prospective Swedish population‐based matched‐cohort study included all women aged ≥ 40 years having used systemic HT between 2005 and 2012 compared with non-­users. These investigators found that current estrogen plus progestin HT use correlated with a moderately higher risk of ovarian cancer (OR = 1.38,

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PART 10  Female Reproduction

95% CI 1.18–1.62), with no increased risk among past users, and a reduced risk was observed for estrogen-­only use (OR = 0.25, 95% CI 0.22–0.29).173 These findings are in direct contradiction of those of the WHI, in which E alone but not E+P, was associated with increased risk of ovarian cancer. Current data regarding association of HT and ovarian cancer is therefore somewhat unclear. Incorporation of a woman’s background risk of ovarian cancer is recommended to be part of the risk/benefit discussion of menopausal HT for symptomatic women. Risk of VTE in patients taking HT is another potential risk, and has been evaluated in many studies, including the WHI. Women randomized to combined E+P HT had double the risk of VTE compared with those randomized to placebo; this risk fell to background levels shortly after discontinuation of HT. Many observational studies have reported similar results in women taking oral estrogen, but no elevated risk of VTE has been reported in patients taking transdermal estrogen.106 Lower doses of estrogen, and the use of oral estradiol may reduce or avert the increased risk of VTE,114 and use of vaginal estrogen has not been shown to increase the risk of VTE.116

SUMMARY In conclusion, menopausal HT remains the most effective treatment modality for severe menopausal symptoms. In addition, it protects against bone resorption and improves vaginal atrophy. It is beneficial for adverse mood and may be helpful for sleep problems, particularly during the transition. Current recommendations are to use the lowest effective dose for the shortest time needed to control the symptoms. Long-­term outcomes of women from the WHI who have now been followed for over 20 years174 confirm the overall safety of this intervention for a mean period of use of 5 to 7 years. Ongoing work will help increase understanding of the mechanisms by which menopausal changes cause symptoms, refine our ability to identify women at risk for more severe and prolonged symptoms, and target hormonal and non-­hormonal treatments that will be most effective for maintaining the vigor of women traversing this reproductive milestone.

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PART 10  Female Reproduction

80. Miller VM, Naftolin F, Asthana S, et al. The kronos early estrogen prevention study (KEEPS): what have we learned? Menopause. 2019;26:1071. 81. Henderson VW, John JAS, Hodis HN, et al. Cognitive effects of estradiol after menopause: a randomized trial of the timing hypothesis. Neurology. 2016;87:699–708. 82. Kitzman DW, Scholz DG, Hagen PT, et al. Age-related changes in normal human hearts during the first 10 decades of life. Part II (Maturity): A quantitative anatomic study of 765 specimens from subjects 20 to 99 years old. Mayo Clin Proc. 1988 Feb;63(2):137-46. 83. Duncan AK, Vittone J, Fleming KC, et al. Cardiovascular disease in elderly patients. Mayo Clin Proc. 1996 Feb;71(2):184-96. 84. Hodis HN, Mack WJ, Henderson VW, et al. Vascular effects of early versus late postmenopausal treatment with estradiol. N Engl J Med. 2016;374:1221–1231. 85. Cagnacci A, Cannoletta M, Palma F, et al. Menopausal symptoms and risk factors for cardiovascular disease in postmenopause. Climacteric. 2012;15:157–162. 86. Thurston RC, Carroll JE, Levine M, et al. Vasomotor symptoms and accelerated epigenetic aging in the Women’s Health Initiative (WHI). J Clin Endocrinol Metab. 2020;105:1221–1227. 87. Svartberg J, von Mühlen D, Kritz-­Silverstein D, et al. Vasomotor symptoms and mortality: the Rancho Bernardo study. Menopause. 2009;16:888–891. 88. Wolff EF, He Y, Black DM, et al. Self-­reported menopausal symptoms, coronary artery calcification, and carotid intima-­media thickness in recently menopausal women screened for the Kronos early estrogen prevention study (KEEPS). Fertil Steril. 2013;99:1385–1391. 89. Tseng LA, El Khoudary SR, Young EA, et al. The Association of menopausal status with physical function: the Study of Women’s Health across the Nation (SWAN): menopausal status and physical function. Menopause. 2012;19:1186. 90. Cray L, Woods NF, Mitchell ES. Symptom clusters during the late menopausal transition stage: observations from the Seattle Midlife Women’s Health Study. Menopause. 2010;17:972–977. 91. Moen M, Kahn H, Bjerve K, et al. Menometrorrhagia in the perimenopause is associated with increased serum estradiol. Maturitas. 2004;47:151–155. 92. Donnez J. Menometrorrhagia during the premenopause: an overview. Gynecol Endocrinol. 2011;27:1114–1119. 93. Blake RE. Leiomyomata uteri: hormonal and molecular determinants of growth. J Natl Med Assoc. 2007;99:1170. 94. Stewart EA, Nowak RA. Leiomyoma-­related bleeding: a classic hypothesis updated for the molecular era. Hum Reprod Update. 1996;2:295–306. 95. DeWaay DJ, Syrop CH, Nygaard IE, et al. Natural history of uterine polyps and leiomyomata. Obstet Gynecol. 2002;100:3–7. 96. Mishra GD, Cardozo L, Kuh D. Menopausal transition and the risk of urinary incontinence: results from a British prospective cohort. BJU Int. 2010;106:1170–1175. 97. Waetjen LE, Johnson WO, Xing G, et al. Serum estradiol levels are not associated with urinary incontinence in mid-­life women transitioning through menopause. Menopause. 2011;18:1283. 98. Burger HG, Dudley EC, Hopper JL, et al. The endocrinology of the menopausal transition: a cross-­sectional study of a population-­based sample. J Clin Endocrinol Metab. 1995;80:3537–3545. 99. Doufas AG, Mastorakos G. The hypothalamic‐pituitary‐thyroid axis and the female reproductive system. Ann N Y Acad Sci. 2000;900:65–76. 100. Cohen LS, Soares CN, Vitonis AF, et al. Risk for new onset of depression during the menopausal transition: the Harvard study of moods and cycles. Arch Gen Psychiatry. 2006;63:385–390. 101. Bromberger J, Kravitz H, Matthews K, et al. Predictors of first lifetime episodes of major depression in midlife women. Psychol Med. 2009;39:55–64. 102. Santoro N, Miller Valery T. Menopause and the postmenopausal state. Curr Med. 1998;1:423–428. 103. Kim TJ, Anasti JN, Flack MR, et al. Routine endocrine screening for patients with karyotypically normal spontaneous premature ovarian failure. Obstet Gynecol. 1997;89:777–779. 104. Nelson HD, Humphrey LL, Nygren P, et al. Postmenopausal hormone replacement therapy: scientific review. JAMA. 2002;288:872–881.

105. ACo O, Gynecologists. ACOG committee opinion no. 556: postmenopausal estrogen therapy: route of administration and risk of venous thromboembolism. Obstet Gynecol. 2013;121:887. 106. Schnatz PF. The 2017 hormone therapy position statement of the North American Menopause Society. Menopause. 2017;24:728. 107. Nelson HD. Commonly used types of postmenopausal estrogen for treatment of hot flashes: scientific review. JAMA. 2004;291:1610–1620. 108. Somboonporn W, Panna S, Temtanakitpaisan T, et al. Effects of the levonorgestrel-­releasing intrauterine system plus estrogen therapy in perimenopausal and postmenopausal women: systematic review and meta-­analysis. Menopause. 2011;18:1060–1066. 109. Nelson HD, Vesco KK, Haney E, et al. Nonhormonal therapies for menopausal hot flashes: systematic review and meta-­analysis. JAMA. 2006;295:2057–2071. 110. Shams T, Firwana B, Habib F, et al. SSRIs for hot flashes: a systematic review and meta-­analysis of randomized trials. J Gen Intern Med. 2014;29:204–213. 111. Bullock JL, Massey F, Gambrell JR. Use of medroxyprogesterone acetate to prevent menopausal symptoms. Obstet Gynecol. 1975;46:165–168. 112. Quella SKLC, Sloan JA, et al. Long term use of megestrol acetate by cancer survivors for the treatment of hot flashes. Cancer. 1998:165–168. 113. Prior JC, Hitchcock CL. Progesterone for hot flush and night sweat treatment–effectiveness for severe vasomotor symptoms and lack of withdrawal rebound. Gynecol Endocrinol. 2012;28:7–11. 114. Pinkerton JV. Hormone therapy for postmenopausal women. N Engl J Med. 2020;382:446–455. 115. Depypere H, Timmerman D, Donders G, et al. Treatment of menopausal vasomotor symptoms with fezolinetant, a neurokinin 3 receptor antagonist: a phase 2a trial. J Clin Endocrinol Metab. 2019;104:5893–5905. 116. Crandall CJ, Hovey KM, Andrews CA, et al. Breast cancer, endometrial cancer, and cardiovascular events in participants who used vaginal estrogen in the Women’s Health Initiative Observational Study. Menopause. 2018;25:11. 117. Cody JD, Jacobs ML, Richardson K, et al. Oestrogen therapy for urinary incontinence in postmenopausal women. Cochrane Database Syst Rev. 2012;10. 118. Bachmann GA, Komi JO, Group OS. Ospemifene effectively treats vulvovaginal atrophy in postmenopausal women: results from a pivotal phase 3 study. Menopause. 2010;17:480–486. 119. Avis NE, Brockwell S, Randolph Jr JF, et al. Longitudinal changes in sexual functioning as women transition through menopause: results from the Study of Women’s Health across the Nation (SWAN). Menopause. 2009;16:442. 120. Dennerstein L. Sexuality, midlife, and menopause. Menopause. 2008;15:221–222. 121. El-­Hage G, Eden J, Zoa Manga R. A double-­blind, randomized, placebo-­ controlled trial of the effect of testosterone cream on the sexual motivation of menopausal hysterectomized women with hypoactive sexual desire disorder. Climacteric. 2007;10:335–343. 122. Braunstein GD, Sundwall DA, Katz M, et al. Safety and efficacy of a testosterone patch for the treatment of hypoactive sexual desire disorder in surgically menopausal women: a randomized, placebo-­controlled trial. Arch Intern Med. 2005;165:1582–1589. 123. Wierman ME, Arlt W, Basson R, et al. Androgen therapy in women: a reappraisal: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2014;99:3489–3510. 124. Labrie F, Archer D, Bouchard C, et al. Effect of intravaginal dehydroepiandrosterone (Prasterone) on libido and sexual dysfunction in postmenopausal women. Menopause. 2009;16:923–931. 125. Labrie F, Archer DF, Koltun W, et al. Efficacy of intravaginal dehydroepiandrosterone (DHEA) on moderate to severe dyspareunia and vaginal dryness, symptoms of vulvovaginal atrophy, and of the genitourinary syndrome of menopause. Menopause. 2018;25:1339–1353. 126. Al-­Safi ZA, Santoro N. Menopausal hormone therapy and menopausal symptoms. Fertil Steril. 2014;101:905–915. 127. Kravitz HM, Zhao X, Bromberger JT, et al. Sleep disturbance during the menopausal transition in a multi-­ethnic community sample of women. Sleep. 2008;31:979–990.

CHAPTER 122  Menopause and Perimenopause 128. Toffol E, Heikinheimo O, Partonen T. Hormone therapy and mood in perimenopausal and postmenopausal women: a narrative review. Menopause. 2015;22:564–578. 129. Gleason CE, Dowling NM, Wharton W, et al. Effects of hormone therapy on cognition and mood in recently postmenopausal women: findings from the randomized, controlled KEEPS–cognitive and affective study. PLoS Med. 2015;12:e1001833. 130. Gordon JL, Rubinow DR, Eisenlohr-­Moul TA, et al. Efficacy of transdermal estradiol and micronized progesterone in the prevention of depressive symptoms in the menopause transition: a randomized clinical trial. JAMA Psychiatr. 2018;75:149–157. 131. Van Voorhis BJ, Santoro N, Harlow S, et al. The relationship of bleeding patterns to daily reproductive hormones in women approaching menopause. Obstet Gynecol. 2008;112:101. 132. Tietjen GE. The relationship of migraine and stroke. Neuroepidemiology. 2000;19:13–19. 133. Sulak PJ, Cressman BE, Waldrop E, et al. Extending the duration of active oral contraceptive pills to manage hormone withdrawal symptoms. Obstet Gynecol. 1997;89:179–183. 134. Yoo HJ, Lee MA, Ko YB, et al. The efficacy of the levonorgestrel-­releasing intrauterine system in perimenopausal women with menorrhagia or dysmenorrhea. Arch Gynecol Obstet. 2012;285:161–166. 135. Bair YA, Gold EB, Zhang G, et al. Use of complementary and alternative medicine during the menopause transition: longitudinal results from the Study of Women’s Health across the Nation. Menopause. 2008;15:32–43. 136. Albertazzi P, Pansini F, Bonaccorsi G, et al. The effect of dietary soy supplementation on hot flushes. Obstet Gynecol. 1998;91:6–11. 1 37. Newton KM, Reed SD, LaCroix AZ, et al. Treatment of vasomotor symptoms of menopause with black cohosh, multibotanicals, soy, hormone therapy, or placebo: a randomized trial. Ann Intern Med. 2006;145:869–879. 138. Chenoy R, Hussain S, Tayob Y, et al. Effect of oral gamolenic acid from evening primrose oil on menopausal flushing. BMJ. 1994;308:501–503. 139. Hirata JD, Swiersz LM, Zell B, et al. Does dong quai have estrogenic effects in postmenopausal women? A double-­blind, placebo-­controlled trial. Fertil Steril. 1997;68:981–986. 140. Nedrow A, Miller J, Walker M, et al. Complementary and alternative therapies for the management of menopause-­related symptoms: a systematic evidence review. Arch Intern Med. 2006;166:1453–1465. 141. Ross AC, Manson JE, Abrams SA, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab. 2011;96:53–58. 142. Gutin B, Kasper M. Can vigorous exercise play a role in osteoporosis prevention? A review. Osteoporos Int. 1992;2:55–69. 143. Dalsky GP, Stocke KS, Ehsani AA, et al. Weight-­bearing exercise training and lumbar bone mineral content in postmenopausal women. Ann Intern Med. 1988;108:824–828. 144. Fiatarone MA, Marks EC, Ryan ND, et al. High-­intensity strength training in nonagenarians: effects on skeletal muscle. JAMA. 1990;263:3029–3034. 145. Kanis J, McCloskey E, Johansson H, et al. Development and use of FRAX® in osteoporosis. Osteoporos Int. 2010;21:407–413. 146. Kanis J, Odén A, Johnell O, et al. The use of clinical risk factors enhances the performance of BMD in the prediction of hip and osteoporotic fractures in men and women. Osteoporos Int. 2007;18:1033–1046. 147. Torgerson DJ, Bell-­Syer SE. Hormone replacement therapy and prevention of vertebral fractures: a meta-­analysis of randomised trials. BMC Musculoskelet Disord. 2001;2:7. 148. Hulley S, Furberg C, Barrett-­Connor E, et al. Noncardiovascular disease outcomes during 6.8 years of hormone therapy: heart and Estrogen/progestin Replacement Study follow-­up (HERS II). JAMA. 2002;288:58–64. 149. Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA. 2002;288:321–333. 150. Berg A, Allan J, Frame P, et al. Postmenopausal hormone replacement therapy for primary prevention of chronic conditions: recommendations and rationale. Ann Intern Med. 2002;137:834–839. 151. Gizzo S, Saccardi C, Patrelli TS, et al. Update on raloxifene: mechanism of action, clinical efficacy, adverse effects, and contraindications. Obstet Gynecol Surv. 2013;68:467–481.

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152. Cummings SR, Martin JS, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med. 2009;361:756–765. 153. Neer RM, Arnaud CD, Zanchetta JR, et al. Effect of parathyroid hormone (1-­34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med. 2001;344:1434–1441. 154. Grodstein F, Manson JE, Colditz GA, et al. A prospective, observational study of postmenopausal hormone therapy and primary prevention of cardiovascular disease. Ann Intern Med. 2000;133:933–941. 155. Hulley S, Grady D, Bush T, et al. Heart, Group EpRSR. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA. 1998;280:605–613. 156. Grady D, Herrington D, Bittner V, et al. Cardiovascular disease outcomes during 6.8 years of hormone therapy: heart and Estrogen/progestin Replacement Study follow-­up (HERS II). JAMA. 2002;288:49–57. 157. Mosca L, Collins P, Herrington DM, et al. Hormone replacement therapy and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation. 2001;104:499–503. 158. Schierbeck LL, Rejnmark L, Tofteng CL, et al. Effect of hormone replacement therapy on cardiovascular events in recently postmenopausal women: randomised trial. BMJ. 2012;345. 159. Heiss G, Wallace R, Anderson GL, et al. Health risks and benefits 3 years after stopping randomized treatment with estrogen and progestin. JAMA. 2008;299:1036–1045. 160. Mosca L, Banka CL, Benjamin EJ, et al. Evidence-­based guidelines for cardiovascular disease prevention in women: 2007 update. J Am Coll Cardiol. 2007;49:1230–1250. 161. Rafique S, Sterling EW, Nelson LM. A new approach to primary ovarian insufficiency. Obstet Gynecol Clin. 2012;39:567–586. 162. Shuster LT, Rhodes DJ, Gostout BS, et al. Premature menopause or early menopause: long-­term health consequences. Maturitas. 2010;65:161–166. 163. Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA. 2002;288:321–333. 164. Anderson GL, Limacher M, Assaf AR, et al. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA. 2004;291:1701–1712. 165. Hodis HN, Sarrel P. Menopausal hormone therapy and breast cancer: what is the evidence from randomized trials? Climacteric. 2018;21:521–528. 166. Chlebowski RT, Anderson GL, Aragaki AK, et al. Association of menopausal hormone therapy with breast cancer incidence and mortality during long-­term follow-­up of the Women’s Health Initiative randomized clinical trials. JAMA. 2020;324:369–380. 167. Collaborative Group on Hormonal Factors in Breast Cancer. Type and timing of menopausal hormone therapy and breast cancer risk: individual participant meta-­analysis of the worldwide epidemiological evidence. Lancet. 2019;394:1159–1168. 168. Fournier A, Berrino F, Riboli E, et al. Breast cancer risk in relation to different types of hormone replacement therapy in the E3N‐EPIC cohort. Int J Cancer. 2005;114:448–454. 169. De Villiers T, Hall J, Pinkerton J, et al. Revised global consensus statement on menopausal hormone therapy. Maturitas. 2016;91:153–155. 170. Sjögren LL, Mørch LS, Løkkegaard E. Hormone replacement therapy and the risk of endometrial cancer: a systematic review. Maturitas. 2016;91:25–35. 171. Shi LF, Wu Y, Li CY. Hormone therapy and risk of ovarian cancer in postmenopausal women: a systematic review and meta-­analysis. Menopause. 2016;23:417–424. 172. Liu Y, Ma L, Yang X, et al. Menopausal hormone replacement therapy and the risk of ovarian cancer: a meta-­analysis. Front Endocrinol. 2019;10:801. 173. Simin J, Tamimi RM, Callens S, et al. Menopausal hormone therapy treatment options and ovarian cancer risk: a Swedish prospective population‐based matched‐cohort study. Int J Cancer. 2020;147:33–44. 174. Manson JE, Aragaki AK, Rossouw JE, et al. Menopausal hormone therapy and long-­term all-­cause and cause-­specific mortality: the Women’s Health Initiative randomized trials. JAMA. 2017;318:927–938.

123 Female Infertility: Evaluation and Management Bart C.J.M. Fauser and Frank J. Broekmans OUTLINE Introduction, 2046 History, 2046 Epidemiology, 2047 Pathogenesis and Clinical Features, 2048 Ovulatory Dysfunction, 2048 Anatomic Dysfunction, 2048 Ovarian Aging, 2049 Unexplained Infertility, 2049 Evaluation of the Female in the Infertile Couple, 2050 Assessment of the Normal Menstrual Cycle, 2050

Assessment of Anatomic Dysfunction, 2050 Assessment of Cervical Mucus–Sperm Interaction, 2051 Assessment of Ovarian Aging, 2052 From Diagnosis to Prognosis, 2052 Expectant Management of the Infertile Couple, 2053 Ovarian Stimulation, 2054 Intrauterine Insemination, 2055 In Vitro Fertilization, 2055 Summary and Future Developments, 2056

  KEY POINTS  • This chapter reviews current knowledge on the evaluation of infertility, with emphasis on the balance between timing and extent of diagnostic procedures. • Current treatment options for specific diagnostic categories are discussed, as is the management of the large category of unexplained infertility, for which the importance of the right balance between assisted reproduction technology and expectant management is acknowledged. • Recent developments in assisted reproduction are outlined.

INTRODUCTION Infertility is defined as the inability to conceive within 1 year of regular unprotected intercourse. Fecundability is the probability of achieving a pregnancy within a given menstrual cycle, and fecundity is the capacity of a couple to achieve a live birth.1 One in every six couples visits a physician because of an unfulfilled wish for children. The 1-­year period is chosen arbitrarily and is based mainly on assessment of the likelihood for pregnancy without medical intervention (Fig. 123.1). Infertility should be viewed as a problem of the couple; therefore, medical attention should focus on both partners of a couple. Chances are roughly similar for finding some form of reproductive dysfunction in the male as in the female. Abnormalities may also be found in both the male and the female partner. Still, in a considerable number of couples, no obvious abnormalities will be found during the initial infertility workup, and it is not always clear whether an observed abnormality is indeed causally related to reduced fertility. Notably, single individuals and same-­sex couples seek fertility services to fulfill their procreative goals. They may also have infertility, warranting infertility evaluation if pregnancies have not been achieved in a 1-­year period. As age of the female partner is a major contributor to infertility, shorter time periods for couples or individuals to pursue infertility evaluation and treatment are recommended (see below). Primary (as opposed to secondary) infertility involves individuals (or couples) with no previous pregnancy. Hence, for a given couple—depending on the individual reproductive history—infertility can be primary for

2046

one, and secondary for the other. Decreased fertility (referred to by some as subfertility) of a given individual may be compensated for by enhanced fertility of the partner, and the clinical challenge is to assess chances for pregnancy, either spontaneously or after infertility treatment, of a given couple. Overall, the proposed management algorithm may include distinctly different options, ranging from expectant management without intervention, to causal therapy for those with distinct forms of infertility, to assisted reproduction as an empirical approach, to increasing chances for offspring in unexplained infertility. KEYPOINTS  • Infertility is an ill-defined condition, with often no obvious causal explanation.

HISTORY Infertility care has witnessed rapid growth since the early 1960s. The first two decades were dominated by the possibilities of endocrine diagnosis and treatments and the introduction of reproductive surgical procedures. Gonadotropin and steroid hormone assays were developed for widespread clinical use, and the synthetic antiestrogen clomiphene citrate (CC) and urinary gonadotropins (human menopausal gonadotropin [hMG]) obtained from postmenopausal women containing excreted pituitary follicle-­stimulating hormone [FSH] and luteinizing hormone [LH]), and subsequently recombinant hormones became available for stimulating gonadal function in both females and males. Initial surgical approaches in the female involved wedge resection of polycystic ovaries, microsurgery of endometriotic pelvic disease and uterine myomas, adhesions, and tubal disease2 and, more recently, by endoscopic pelvic surgery and operative hysteroscopy on uterine anomalies, endometrial abnormalities, submucous myomas, and adhesions. Surgical approaches in the male were dominated by varicocele correction to improve sperm quality, although most of these initial surgical approaches have not proven efficacious after rigorous assessment.2,3 Yet, the surgical recovery of sperm from the epididymis (percutaneous epididymal sperm extraction) or testis (testicular sperm

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Probability of clinical pregnancy

CHAPTER 123  Female Infertility: Evaluation and Management

Cumulative conception rate

1.0 0.8 0.6 0.4 0.2

75th

0.6

50th

0.4

25th

0.2

5th 0

0 0

A

95th 0.8

2

4

6

8

10

12

14

–6

–4

–2

0

2

B Months Day of intercourse Figure 123.1  A, Cumulative conception rates during the first 12 months of unprotected intercourse. B, Probability of a clinical pregnancy relative to the day of intercourse. Day 0 depicts the day of the serum luteinizing hormone surge. Individual lines depict 5th to 95th percentiles of the total population. (A, from Taylor A. ABC of subfertility: the extent of the problem. BMJ. 2003;327:434–436. B, from Evers JL. Female subfertility. Lancet. 2002;360:151–159.)

extraction) has greatly changed the perspective of the azoospermic male due to obstructive and spermatogenic causes. The past two decades have been dominated by the development of assisted reproductive technology (ART), intrauterine insemination (IUI), in vitro fertilization (IVF) in 1978,4 and intracytoplasmic sperm injection (ICSI) in 1992.5 These novel therapeutic strategies, combined with complex ovarian stimulation (OS) protocols, created new possibilities for infertile couples and individuals. Recent decades have focused on improved methods of embryo cryopreservation which have enabled the large-­scale use of single-­embryo transfer in IVF, thereby reducing rates of multiple pregnancy and associated medical risks and cost.6 Other major advances include: mild stimulation protocols using lower doses of drugs, resulting in best embryos to transfer in a given cycle7,8; vitrification of oocytes (from stimulated ovaries), which opened the field for oocyte banking for egg donation programs, fertility preservation for cancer treatment patients, and pregnancy postponers9; in vitro maturation and cryopreservation of oocytes from unstimulated ovaries, which currently lacks sufficient efficiency and safety to allow for large-­scale application10; and cryopreservation of ovarian tissue, for example, cancer patients before undergoing gonadotoxic chemotherapy and subsequent transfer into the abdominal cavity, once a pregnancy is desired. Currently, over 100 babies have been born worldwide following this procedure.11 Finally, embryo selection for transfer has been aided by cleavage or blastocyst stage embryo biopsy to detect transmitted hereditary disease (preImplantation genetic testing for disease) such as Huntington’s disease, cystic fibrosis or polycystic kidney disease, or embryo aneuploidies (preImplantation genetic testing for aneuploidies).12 Overall, these advances have enabled millions of children born to infertile couples and individuals, although the cost of IVF, ICSI, TESE, and other technologies can be prohibitive, along with disparities of availability of fertility services in certain regions across the globe, still leaving challenges to be addressed. KEYPOINTS  • In  vitro fertilization and related technologies have been the significant steps forward in infertility management.

EPIDEMIOLOGY In the general population, the highest chance of pregnancy occurs in the first cycle of unprotected intercourse. The maximal probability for a clinical pregnancy is ∼30% per cycle, and chances for pregnancy are highest

TABLE 123.1  Estimated Spontaneous

Cumulative Pregnancy Rates in Normal Fertile and Infertile Couples* Monthly Fecundity Rate (%) 6 Superfertile Normally fertile Moderately subfertile Severely subfertile Infertile

60 20 5 1 0

100 74 26 6 0

CUMULATIVE PREGNANCY RATE (%) 12

24

60

93 46 11 0

100 71 21 0

95 45 0

*Rates over a given period of time, under conditions with different monthly fecundity rates. The data indicate that women with moderate fertility, which applies to many in their 30s, may have a considerable chance (≈50%) of not having achieved a pregnancy within 1 year’s time. From Evers JL. Female subfertility. Lancet. 2002;360:151–159.

when intercourse takes place 2 days before ovulation13 (see Fig. 123.1). The likelihood of pregnancy slowly decreases in subsequent cycles, generating a cumulative chance for pregnancy of 60% within three cycles, 70% within six cycles, and ∼90% within 1 year14 (see Fig. 123.1). Very fertile couples are likely to achieve a pregnancy within the first few cycles, whereas others will conceive within 12 months (Table 123.1). Moreover, a considerable proportion of couples will still conceive spontaneously after the 1-­year period or even later (Figs. 123.2 and 123.3). The 1-­year arbitrary cutoff period is to define infertility and justify an infertility workup, as the chance of finding an exclusive cause like severe male factor or tubal obstructive pathology is realistic. Infertility occurs in ∼10% to 15% of the population. Multiple societal tendencies interfering with normal fertility have been described, especially (1) early sexarche and promiscuous sexual behavior (resulting in increased chances for tubal disease due to sexually transmitted disease), (2) postponement of pregnancy and consequently an increased proportion of women older than 30 years of age with already decreased natural fertility (due to diminished ovarian reserve) when offspring is desired, (3) female lifestyle factors (especially obesity and resulting ovarian dysfunction), and (4) environmental and toxic agents (e.g., cigarette smoking inducing accelerated follicle pool depletion). Consequently, more couples will not conceive spontaneously within 1 year of unprotected intercourse and, therefore,

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PART 10  Female Reproduction Spontaneous pregnancy rates in subfertile couples

Percentage of couples

30 25

25

22

20

18 15

15

11

10

6

5

3 0– 10%

10– 20%

20– 30%

30– 40%

40– 50%

50– 60%

1

60– 70%

>70%

Predicted pregnancy rates Figure 123.2  The distribution of the probability to become pregnant among untreated infertile couples within the year after intake for infertility workup (defined as having had at least 1 year of unprotected intercourse). (Data from Eimers JM, te Velde ER, Gerritse R, et al. The prediction of the chance to conceive in subfertile couples. Fertil Steril. 1994;61:44–52.)

Likelihood of pregnancy (%)

80 70 60 50 40

yield a cause leading to infertility in approximately 23% (bilateral tubal obstruction, amenorrhea, azoospermia); in the remainder, only factors that will reduce the probability of conception (i.e., subfertility) are identified of which female age is the factor with the highest impact.19 Couples who suffer from infertility usually do not have associated complaints or symptoms. Exceptions include abnormal or absent menstrual bleeding, a history of pelvic inflammatory disease, ruptured appendix, or maldescent of one or both testes. The general rule that the more tests conducted, the more likely it is that at least one test will be abnormal by chance also holds true for infertility. It may not be easy to identify a disease state that is causally related to reproductive failure in a given couple or individual. Classic examples include conditions such as endometriosis or uterine leiomyomas, which were widely believed to induce infertility, until it was found that both these conditions are also prevalent in the general, fertile population. Similar findings apply to male infertility. Poor sperm quality (by World Health Organization [WHO] criteria) was widely believed to be the cause of infertility in many cases, until more recent studies demonstrated that diminished sperm quality is often observed in males with children.20,21 Although criteria for defining abnormal sperm have been revised repeatedly, the predictive power of sperm quality assessment should be viewed with caution in an infertile population. A clear effect upon pregnancy rates has been demonstrated only in males with severely reduced sperm density, or azoospermia.22 Several classes of disease conditions that may underlie female infertility can be identified, notably ovulatory dysfunction, mechanical dysfunction, ovarian aging, and unexplained infertility.

Ovulatory Dysfunction

30 20 10 0 0

1

2

3

4

5

6

7

8

9

10

Duration of subfertility (y) Figure 123.3  The likelihood of pregnancy in couples relative to the duration of their infertility. Note the considerable chance to conceive within the first 3 years of unprotected intercourse. For instance, the probability for a couple to become pregnant in the course of the second year of trying is still 50%. (Data from Evers JL. Female subfertility. Lancet. 2002;360:151–159.)

will visit a healthcare professional for infertility care. It appears that the prevalence of infertility in the Western world has remained unaltered over the past 50 years or may even have reduced,14–16 although more couples have been seeking access to infertility services.17 KEYPOINTS  • Postponing family building creates higher risks of infertility occurring, in relation to the female age.

PATHOGENESIS AND CLINICAL FEATURES According to textbooks, the female factor accounts for approximately 35% of infertility, the male factor for 35%, and in about 30% of cases, both male and female factors are involved. In female infertility, approximately 30% to 40% involve ovulatory dysfunction, 30% to 40% involve tubal and pelvic pathology, and 30% are attributed to “unexplained” causes, of which advanced reproductive age may be the most important contributor.18 Clinical data supporting these figures are not robust. However, differences among populations and different referral characteristics may exist. Overall, a full workup of the infertile couple will

Changes in ovarian function usually are presented by extended bleeding intervals (oligomenorrhea) or absence of bleeding (amenorrhea). Amenorrhea is usually associated with a hypoestrogenic status, whereas bleeding in oligomenorrhea may represent occasional ovulation or breakthrough bleeding due to continued estrogen exposure. In primary amenorrhea, with or without disturbed sexual maturation, one should consider rare conditions involving abnormal gonadal or uterine development due to (inherited or spontaneous) gene mutations (Chapter 117). Ovulatory dysfunction can be classified into three categories based on FSH and estradiol (E2) hormone assays in peripheral blood.23 Low FSH and E2 indicate a central origin of the abnormality at the level of the hypothalamus or pituitary gland. Hence, ovaries fail to function normally owing to insufficient stimulation by pituitary gonadotropins (WHO group 1). In contrast, the combination of high FSH and low E2 concentrations indicates that the primary defect is at the ovarian level (WHO group 3, i.e., premature ovarian insufficiency [POI]), with the ovaries failing to produce estrogens despite maximal stimulation by endogenous FSH. The third group of oligo/anovulatory patients (WHO group 2) is represented by FSH and E2 levels within the normal range constituting approximately 80% to 85% of all women presenting with ovulatory dysfunction. This heterogeneous group especially includes polycystic ovarian syndrome (PCOS) also characterized by clinical or biochemical androgen excess and/ or polycystic ovaries (see Chapter 117).24 Luteal-­phase insufficiency (short luteal phase) as an expression of ovulatory dysfunction has long been believed to be an important factor in infertility, occurring in up to 14% of infertile patients. However, controlled studies conducted to establish its role in infertility or the value of any treatment in improving pregnancy rates are currently still lacking.25

Anatomic Dysfunction Normal functioning of the reproductive tract is important for fertility. Uterine abnormalities, including congenital uterine malformations,

CHAPTER 123  Female Infertility: Evaluation and Management malformations due to fetal exposure to diethylstilbestrol, fibroids (in particular, the submucosal or cornual myomas), polyps, and adhesions related to previous intrauterine procedures, are associated with infertility or early miscarriage.26 Normal fertility may also be affected by pelvic adhesions (especially involving the ovaries and fallopian tubes) caused by pelvic inflammatory disease, complicated appendicitis, or intraabdominal surgery. Abnormal tubal function may be due to isthmic or terminal occlusion or phimosis of the fimbriae due to salpingitis. Peritubal adhesions may interfere with normal tubal motility and, hence, with gamete and zygote transport. Severely damaged tubes may fill with fluid, giving rise to hydrosalpinges. Endometriosis (see Chapter 125) is a topic of continued controversy related to disease conditions associated with infertility. It is the ectopic localization of the endometrial tissue, most commonly in the pelvis; retrograde menstruation may be the important causal factor, but metaplastic errors during Mullerian tube development may also contribute to endometrial tissue in the myometrium or rectovaginal septum.27 Severe forms of endometriosis give rise to adhesions, cyst formation, and reduced fertility. In addition, abdominal discomfort, specifically dysmenorrhea, chronic pelvic pain, and dyspareunia are frequent complaints. Limited data suggest that minimal and mild disease are prevalent in the general population, without a clear relation to infertility or pain. Moreover, positive effects on subsequent fertility of intervention studies using hormonal medication or surgical procedures are still questionable.28,29 Uterine myomas (see Chapter 126) occur in 1% to 2.5% of infertile patients without other evident cause of infertility,30 and removal has long been thought to improve subsequent pregnancy chances. A recent meta-­analysis has shown that infertility patients with uterine cavity-­distorting fibroids have poorer reproductive outcomes than infertile patients without fibroids, and removal therefore may be considered justified despite a lack of comparative trials. Cases with intramural myomas may have a poorer prospect for pregnancy occurring, but the absence of quality studies prevents advocating their removal.31 Subserosal fibroids are not associated with fertility issues.32 As with many other factors associated with infertility, comparative data on the frequency of similar abnormalities in a matched, fertile population are lacking. In addition, randomized follow-­up studies of pregnancy rates comparing intervention versus expectant management are rare, so the efficacy of treatment is uncertain.

Ovarian Aging In many societies, increased participation of females in extended education and careers has resulted in postponement of childbearing to a later age. An increased proportion of women make their first attempts to conceive when they are beyond 30 years of age, when natural fertility starts to decrease, and with a major subsequent decline in fertility after 37 years of age.33 Beyond 41 years of age, chances of having a spontaneously conceived baby have decreased on average to close to zero. At around 20 weeks’ gestation, in the female fetus, initial germ cell mitosis is arrested, and the primordial follicle pool reaches its maximum size of around 8 million per ovary (see Chapter 122). Thereafter, this stock of resting primordial follicles is gradually depleted (by the onset of growth of follicles), falling to 2 million at birth and 0.5 million at menarche (first menstrual period), with final exhaustion of the pool with the last spontaneous menstrual period. Thus, only a small proportion (∼10% to 15%) of the initial primordial follicle pool is present at the time that many women consider conceiving their first child. Menopause, which marks the definitive end of the reproductive life span, occurs at the average age of 51, with a range from 40 to 60 years of age (see Chapter 122). This indicates that exhaustion of the follicle

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pool takes 50% more time (60 years rather than 40 years) in some women than in others. The association between chronologic age and ovarian aging therefore may be complex. Indirect evidence indicates that a fixed time interval exists between the transition from normal fertility into decreased and finally into absent fertility (sterility).34 Some women at 35 years of age may already have severely compromised fertility due to poor-­quality oocytes. These women may also present with an earlier than average menopause, although the relation between the quantitative and qualitative aspects of the ovarian ageing process are far from elucidated. For other women of identical age (35 years), natural fertility is still completely normal or may even be above normal. The latter group of women is often believed to experience menopause at a later age, although the evidence is minimal. The clinical challenge is to assess the reproductive aging status in terms of fecundability of a given woman at a given age, which will help to predict her pregnancy chances more accurately than will chance assessment based on chronologic age alone. This may help the clinician in counseling the patient regarding expectant management or therapeutic strategies.34 Studies on genetics of cell cycle and immune system maintenance may potentially identify relevant factors in this context. “Early” ovarian aging can be linked to genetic as well as environmental factors (see Chapters 122 and 137). The mechanisms underlying reduced natural fertility in relation to ovarian aging have not yet been fully elucidated. It has been clearly established that the size of the cohort of growing follicles is diminished, and this coincides with an increased proportion of chromosomally abnormal oocytes. The latter is exemplified by increased rates of aneuploid (e.g., trisomy 21) pregnancy, increased rates of early pregnancy loss (most likely based on an aneuploid conceptus), and increased numbers of aneuploid oocytes and embryos with increasing female age.35 Changes in follicle cohort size can be assessed by transvaginal ultrasound or by hormone estimates in the peripheral blood (see later). Unfortunately, as explained in the aforementioned, assessment of average oocyte quality on an individual basis so far has not proven feasible.

Unexplained Infertility The indicated percentage of infertility of unknown origin is ∼30% but may be higher depending on the criteria applied. The reported incidence is very much dependent on the extent of the infertility evaluation, characteristics of the population studied, female age, and the reported incidence of other “known” factors related to infertility. Again, a clear-­cut causal relationship between an abnormal condition as established during initial evaluation and infertility is often absent. A clear tendency towards less diagnostic evaluation resulting in an increased proportion of infertility remaining “unexplained” can be observed. This implies that several factors are known that do not per se exclude the possibility of achieving a pregnancy (e.g., mild or moderate semen abnormalities, a suboptimal or negative postcoital test, mild-­stage endometriosis, elevated basal FSH or low age-­specific anti-­Müllerian hormone [AMH], or subtle uterine abnormalities like fibroids), but they only contribute to a reduction in monthly fecundity. In assessing the remaining chances for success in these unexplained infertile couples or individuals, key factors such as female age, a previous pregnancy, duration of the current infertility status, and semen quality have been identified as crucial factors for the individualized estimates of pregnancy chances.36,37 On the other hand, many processes are involved in natural fertility that cannot be tested properly in a routine clinical setting. These involve oocyte maturation and fertilization, embryo transport through the tube and uterine cavity, chromosomal constitution of embryos, apposition and implantation of embryos, and, finally, endometrial receptivity, including regulation of endometrial maturation by steroids produced by the corpus luteum,38 and gene expression variation indicative of changes in the window of implantation.39

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PART 10  Female Reproduction

Moreover, many pregnant women, under natural conditions or in an infertile population, may miscarry very early, before the occurrence of her expected, regular menses.38 KEYPOINTS  • The true key causes for female infertility are uterine/tubal pathology and oligo-­/an-­ovulation. However, age related limitations at the level of the oocyte may comprise a contribution of yet unknown magnitude. • Female age effects on fertility are likely to be related to oocyte quality. An individual specific test to assess average ovulated oocyte quality is urgently needed.

EVALUATION OF THE FEMALE IN THE INFERTILE COUPLE The extent to which basic infertility testing should be performed is extensively debated in the current era, dominated by ART. Consequently, marked differences exist with regard to the numbers and types of tests performed.40 Tests are time-­consuming and costly, and some are not performed without risks or high patient burden. According to some clinical investigators, results may have little bearing on ART outcomes. In a commercial environment where patients have to pay for services, “customers” may prefer to spend their money on treatment with some chance for pregnancy than on diagnostic procedures that will have no impact on their chances to conceive. Many tests are not clearly standardized, and therefore reproducibility is limited. The usefulness of infertility tests should be based on the proper evaluation of (1) sensitivity (the capacity of a test to detect abnormalities), (2) specificity (the chance that an abnormality is actually present if the test yields an abnormal result), (3) invasiveness, complexity, and cost of the test, and, finally, (4) the clinical utility of the test in terms of how result will influence the management or treatment options for the infertile couple or individual. Indeed, few studies have been undertaken to evaluate the practical value of tests despite their widespread use in clinical practice.41 During the initial consultation, the physician should take a full medical history from both partners. Relevant information includes duration of infertility, past sexual development, children from current or previous relationships, general health and use of medication, current or past smoking or use of drugs, including alcohol, obstetric history, previous surgery, family history, abdominal or menstrual discomfort, and sexually transmitted diseases. Moreover, the clinician should counsel a couple regarding timing of intercourse and fertility chances, the use of LH detection tests, the effects of lifestyle factors (such as excessive smoking, alcohol intake, caffeine consumption, and exercise), food intake and obesity, and occupational exposures. A general physical examination usually is advised, with focus on height, weight, and blood pressure.17,42,43 Diagnostic tests that may be performed for the evaluation of infertile couples include assessment of the normal menstrual cycle, assessment of anatomic factors, and assessment of ovarian aging status.

Assessment of the Normal Menstrual Cycle Regular menstrual periods strongly suggest normal ovulatory cycles. Ovulation and normal corpus luteum function can be assessed in different ways.25 However, the tests are not optimal in terms of accuracy and reproducibility, or in the detection of a meaningful and treatable disease condition relevant to infertility.44 Applications for mobile phones are being developed and marketed successfully aiming to monitor the fertile period more closely, which can be used both to improve pregnancy chances when desired but also to avoid pregnancy as a means of contraception. These programs improve over time, also by applying artificial intelligence using big data sources.

Menstrual Period Chart. This represents the simplest way to document the menstrual cycle pattern. The days of onset and cessation of menses should be recorded on a calendar for several months, or may be kept in one of several apps.45 A normal menstrual cycle ranges between 25 and 35 days, with a median duration of 28 days.

Basal Body Temperature Chart. Progesterone produced by the corpus luteum has many effects on the central nervous system, including changes in the body temperature set point. Consequently, daily assessment of basal body temperature (BBT) can be used to document a persistent increase in temperature (typically from 36.6°C to 37.1°C) and therefore can provide “proof” of ovulation. A rise in temperature indicates that ovulation and corpus luteum formation have taken place. The length of the second half of the cycle should be at least 10 days. Daily temperature assessments should be performed at the same time during the early morning before the activities are begun. Although performing these tests for several months can be quite stressful to women, it may be helpful for the couple to be aware of the day of ovulation, so they can time intercourse to achieve optimal pregnancy chances. However, the BBT is notoriously unreliable for the timing of ovulation, and LH surge tests and menstrual and fertile period apps are taking over fast.45 Urinary LH Surge. When LH concentrations are high in serum during the midcycle surge, sufficient quantities are excreted into urine to allow its detection. Kits are commercially available for use at home. On average, LH appears in the urine around 12 hours after onset of the LH surge in the blood, and this can accurately predict ovulation (see Chapters 117 and 120). This method may help with optimal timing of intercourse, although evidence for a beneficial effect upon pregnancy rates has so far not been found.45,46 Transvaginal Ultrasonography. The preovulatory, Graafian follicle usually attains a diameter of around 20 mm (ranging from 16 to 24 mm) before rupture and subsequent release of the oocyte. Rupture of the follicle can be established by visualizing the disappearance or decrease in size of the dominant follicle by transvaginal ultrasound. This method usually is reserved for monitoring of OS during infertility therapy or for proper timing of a postcoital test or midluteal phase progesterone assay (see the following section).

Progesterone Assay. Appropriate timing of the assessment of progesterone serum concentrations is crucial and should always be checked in retrospect, especially in women whose cycle length may vary. A level > 2 ng/mL indicates ovulation, although levels between 6 and 25 ng/mL are considered normal and are associated with chances for pregnancy. Some investigators advocate that multiple hormone assays should be performed around the midluteal phase to document insufficient corpus luteum function. The luteal phase may be reduced in length, or insufficient quantities of progesterone may be produced during the luteal phase. However, the exact role of these variations in terms of female fecundity remains a matter of debate.47

Assessment of Anatomic Dysfunction Some type of mechanical factor relevant to infertility is reported to occur in 10% to 30% of infertile couples. Several methods exist to establish abnormalities at the level of the uterus, fallopian tubes, or ovary/ampulla complex relevant for in vivo oocyte pickup. Until recently, diagnosis of mechanical abnormalities by hysterosalpingography (HSG) during the early infertility workup and by laparoscopy later in the evaluation has been the standard procedure for each patient. Currently, the tendency is toward the use of less invasive screening tests to identify women at high risk for tubal disease and the performance of more invasive diagnostic procedures only in those women

CHAPTER 123  Female Infertility: Evaluation and Management at increased risk for such abnormalities.48 The question remains as to what extent any mechanical deviation from normal truly contributes to the infertility. The clinician should take this reservation seriously, especially in cases of minor abnormalities of the uterine cavity and of presumed unilateral tubal damage.

History Taking. The guidelines of the National Institute for Clinical Excellence (NICE) in the United Kingdom advise clinicians to use the past medical history in the woman to decide whether invasive diagnostics should be applied to assess the presence of tubal pathology (especially previous deliveries, previous pelvic inflammatory disease, or previous pelvic surgery) enables the identification of such couples.49 The addition of Chlamydia antibody testing will further enhance the correct identification of couples at such low risk for anatomic dysfunction that invasive diagnostics can well be avoided. Transvaginal Ultrasonography. Today, transvaginal ultrasonography (TVS) is standard in the infertility workup, as it is simple, highly informative and allows for a test with low patient burden. With its use, several relevant abnormalities can be assessed. These comprise uterine cavity abnormalities such as thin endometrium lining, endometrial polyps, submucous myoma, and intrauterine adhesions. Secondly, the status of the myometrium can be assessed allowing for the detection of intramural or subserous myomas, adenomyosis, or congenital anomalies such as a uterine septum, a bicornate or a didelphys uterus. Only pathologic conditions of the fallopian tubes, e.g., hydrosalpinx, can be diagnosed by TVS, due to the presence of intraluminal fluid trapped in a terminally occluded salpinx. Finally, ovarian imaging will reveal information on the presence of cysts (dermoid, endometriosis), polycystic ovarian morphology, as well as the total antral follicle count (AFC), which may indicate the ovarian reserve status. In typical cases of dysmenorrhea, indirect signs of adhesions based on endometriosis can be identified between the rectum and uterus and uterus and bladder. Screening by Chlamydia Antibody Testing in Serum. Chlamydia trachomatis represents the most common sexually transmitted pathogen in the Western world, with a reported prevalence of infection of around 5% to 10%. Most of these infections are asymptomatic, but ∼10% can cause pelvic inflammatory disease.50 Chlamydia acts as an immunogen that gives rise to antibody formation. Immunoglobulin G (IgG) antibodies remain in the circulation for years and therefore can be used as a marker for past infection. IgG concentrations are reportedly elevated in 30% to 60% of women from infertile couples. The capacity of IgG antibody levels to predict tuboperitoneal disease (as assessed by laparoscopy) has been evaluated prospectively, with acceptable receiver operator characteristic (ROC) curves and the use of titers between 16 and 32.51 In infertile couples with a relatively good fertility prognosis based on the Hunault prediction rule,52 a workup strategy starting with history taking and screening for Chlamydia antibodies is an effective approach,53,54 and for many women with only a low risk, performance of more invasive tubal patency evaluating procedures can be prevented.

Hysterosalpingography. HSG represents the radiographic visualization of the endocervical canal, uterine cavity, and lumina of the fallopian tubes. Both oil-­and water-­based contrast media are used. The oil-­based medium may provide the advantage of a minor subsequent increase in spontaneous pregnancy chances,55,56 whereas the water-­based medium is faster and less painful and results in improved visualization of abnormalities. Abnormal uterine features include filling defects (related to submucous fibroids, polyps, or intrauterine adhesions), and abnormal shape (congenital uterine anomalies such as unicornuate, bicornuate, or septate uterus, uterus didelphys, fetal exposure to diethylstilbestrol, or intrauterine adhesions). In addition,

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unilateral or bilateral tubal occlusion, with or without hydrosalpinges, may be diagnosed. The occurrence of peritubal adhesions may be suspected on the basis of an abnormal intraabdominal distribution pattern of the contrast medium (pocketing, especially noted on the post-­filling x-­ray). The HSG procedure should be planned during the follicular phase of the menstrual cycle to avoid an unexpected early pregnancy. The procedure can be quite painful, and an ascending infection with acute salpingitis is the most significant complication, occurring in ∼1% to 2% of patients. Preventive antibiotic prophylaxis should be advised in all patients for whom tubal pathology has been diagnosed or suspected. HSG is reasonably accurate in diagnosing tubal occlusion; abnormal results are associated with reduced fecundity rates, especially if abnormalities are double-­sided.57,58 An important drawback of this procedure remains the subjective nature of the evaluation, which renders the reproducibility of this test rather poor. Hysterosalpingo Contrast Sonography (HyCoSy). Hysterosalpingo contrast sonography (HyCoSy) is a simple and well-­tolerated outpatient ultrasound procedure and requires the use of a contrast agent to visualize patency of the fallopian tubes. With a small balloon catheter as filling device and using a transvaginal ultrasound, the contrast agent is instilled into the uterine cavity. The contrast medium may be an agitated saline/ air mixture or a non-­iodinated contrast agent (ExEm Foam). The contrast flow from the uterine cavity into the fallopian tubes can be visualized by the operator and allows judging rapid passage through the length of the fallopian tubes, indicative of tubal patency. Although HyCoSY may be a promising alternative for HSG with regard to accuracy and effectiveness, proper assessment studies are yet to appear.59 Laparoscopy. This procedure is considered the gold standard for the diagnosis of tubal disease and intraabdominal disorders such as endometriosis and adhesions that interfere with normal fertility.57,58 Some authorities believe that laparoscopy should be performed before unexplained infertility is diagnosed. More recent data suggest that the added value of such a procedure in women without a history suggestive of tubal disease and a normal HSG is very limited.60 Laparoscopy involves the intraabdominal inspection of the internal genital organs after abdominal insufflation with CO2 of a patient under general anesthesia. Next to the reproductive organs, the appendix should be inspected for infection and the perihepatic region for adhesions suggestive of past chlamydial infections (Fitz-­Hugh-­Curtis syndrome). In experienced hands, risks associated with this technique are minimal. Attention should be focused toward standardizing the evaluation of the abdominal and pelvic cavities.

Other Techniques. Recently introduced, more sophisticated techniques that can be used to visualize intrauterine, intratubal, or intraabdominal abnormalities include ultrasound imaging of the uterine cavity after fluid lavage and endoscopic techniques such as saline infusion sonography, hysteroscopy, salpingoscopy, falloposcopy, transvaginal hydrolaparoscopy, and microlaparoscopy.61 Although some of these techniques have seemed promising, with their focus on reduced patient discomfort and costs, the usefulness of these tools in a standard infertility workup remains to be established.

Assessment of Cervical Mucus–Sperm Interaction In humans, semen is deposited in the posterior fornix of the vagina, and the cervix via its mucus secretion is believed to play an important role in allowing sperm to enter the uterine cavity. Several functions of cervical mucus include protecting sperm from the acidic milieu in the vagina, allowing the sperm to enter the cervix, supporting energy requirements for sperm motility, and filtering morphologically abnormal sperm. Before ovulation, cervical mucus is produced in large

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PART 10  Female Reproduction

quantities, allowing easy sperm access at the most appropriate moment during the menstrual cycle. The prognostic performance of cervical mucus tests, the incidence of cervical factor infertility (“cervical hostility”), and the validity of treatment options to correct disturbed cervical mucus–sperm interactions remain poor.

Postcoital Test. This test, which involves appraisal of the presence of motile sperm in cervical mucus, after intercourse during the late follicular phase of the menstrual cycle, was originally described in 1866 by Sims and was reemphasized in 1913 by Huhner (reviewed in ref 62). There is no agreement regarding the preferred interval between intercourse and the test, nor the cutoff between a normal and an abnormal test result. The threshold is 1–5 spermatozoa per high-­power magnification (i.e., 10 × 40) field. A systematic review of 11 well-­designed studies indicated that the discriminating ability of the postcoital test is poor, regardless of the applied criteria for normality.62,63 A subsequent systematic prospective evaluation indicated that inclusion of a postcoital test in routine evaluation of new couples only results in additional interventions.62 Several national and international bodies (such as the American Society of Reproductive Medicine, the European Society of Human Reproduction, and the Royal College of Obstetrics and Gynaecology) currently advise against performing the postcoital test in routine infertility evaluation. In addition, earlier tests used to evaluate the quality of the cervical mucus, along with several in vitro tests of interactions between cervical mucus and sperm, have been abandoned.

the size of the pool of primordial follicles.71,72 Compared with AFC and basal FSH, the current AMH test has unfortunately failed to significantly improve prediction of pregnancy after IVF-­ET.73,74 For the prediction of poor response to OS, especially AMH has proven quite adequate. A poor response may indicate advanced ovarian aging, with inherent reduced prospects for a live birth, but underdosing or decreased sensitivity of the ovary may also play a role. In cases where poor ovarian response is due to exhaustion of the ovarian follicle pool, the prediction of poor response has no therapeutic implications because the prognosis for successful treatment will be poor, regardless of the chosen intervention. It needs to be emphasized, however, that especially in younger patients, oocyte quality may still be quite good in spite of low numbers obtained by OS, even if this is caused by low ovarian reserve status.75 At the other end of the spectrum, the capacity of these tests to predict excessive response to ovarian hyperstimulation allows treatment schedules to be adjusted in order to mitigate the number of stimulated follicles and thereby limit the risk for ovarian hyperstimulation syndrome (OHSS).76–78 Today, initial testing of ovarian reserve may be useful especially for managing high responses,79–81 as a reduced gonadotropin dose in high responder patients will generate less oocytes with adequate IVF success rates; this approach reduces chances for developing OHSS. However, with the use of gonadotropin-­releasing hormone (GnRH) antagonists for LH peak suppression, high responders can also be managed by GnRH agonist trigger of oocyte maturation and a “freeze all” embryos strategy.

Assessment of Ovarian Aging

From Diagnosis to Prognosis

It is well established that the female partner’s age is the most prominent factor in determining chances of spontaneous pregnancy33 or after infertility therapy (such as insemination with donor sperm64 or with in vitro fertilization–embryo transfer [IVF-­ET]).65 As discussed previously, however, chronologic age has limitations in that the degree of ovarian aging varies considerably among women in the same age group. This is true for the quantitative aspects of ovarian reserve, expressed by the AFC and AMH levels. Some 35-­year-­old women may be normally fertile (i.e., have the fecundity of a 25-­year-­old), and expectant management may be the most appropriate option. In contrast, natural conception chances may already be severely compromised in other women of the same age (with fecundity of a 41-­year-­old), and the clinician would best offer the most effective treatment options without delay. The major challenge of today is to assess the magnitude of ovarian aging on an individual basis.66,67 Endocrine and ultrasound changes go along with ovarian aging. Recent efforts have focused on the clinical implications of these changes, applying test systems that aim to predict chances for pregnancy, either spontaneously or after infertility therapy. Some factors indeed may have some capacity to predict fertility chances, although studies on the added value of these tests on top of female age are either scarce or inconclusive as to their value in patient management. While initial physiologic studies in normo-­ovulatory women have emphasized that the length of the follicular phase is shortened with increasing age and with elevated early follicular phase serum FSH concentrations, baseline FSH has limited capacity to predict IVF outcome, independent of chronologic age.68 In addition, a decreased cohort of AFC can be observed by ultrasound and used as a marker of ovarian aging.69 However, a meta-­analysis on the predictive value of baseline FSH and AFC for IVF outcome concluded that their clinical value in pregnancy prediction is restricted to only a small group of patients.70 In recent years, AMH has become the marker of promise in ovarian reserve assessment. Serum levels are proportional to the number of antral follicles in the size range of 1 to 8 mm, and indirectly represent

In many couples, a clear cause for infertility remains unknown with currently available diagnostic tools. Various prospective follow-­up studies evaluated whether specific characteristics assessed upon initial evaluation could predict the chance for a spontaneous pregnancy in these couples. Three studies82–84 identified similar factors predicting pregnancy chances for couples with unexplained infertility. The Hunault prediction model36,37 based on duration of infertility, female age, previous pregnancies, sperm motility, and referral status for couples with unexplained infertility has been externally validated, demonstrating its accuracy in prognosis assessment.85 Application of this model37 to assess chances for spontaneous pregnancy will help counseling couples with unexplained infertility regarding the preferred strategy, i.e., expectant management versus initiation of (empirical) treatment. Today, the option has been further developed to estimate the changes in the prospects for an individual couple over time, thereby facilitating potentially the right timing of initiation of infertility treatment. This approach may reduce overtreatment, in that invasive therapies may be avoided in couples who have a still favorable likelihood of spontaneous pregnancy. Notably, a well-­designed cohort follow-­up study concluded that 61% of couples achieved pregnancy independent of treatment.85,86 Similar prediction models have been developed aiming to assess cumulative pregnancy changes throughout an entire IVF treatment.87 KEYPOINTS  • The purpose of the infertility work-up in the female is to identify clear causes, such as tubal pathology and/or oligo/an-ovulation. • Invasive evaluation of uterine-tubal abnormalities is best reserved for cases with a positive history (STDs and pelvic surgery), abnormalities at transvaginal ultrasonography and/or presence of Chlamydia antibodies in the serum. • Assessment of ovarian reserve status is not meaningful in terms of diagnosis or prognosis in the infertility work-up. Its value stems from the prediction of ovarian response to ovarian hyperstimulation in assisted reproduction technology.

CHAPTER 123  Female Infertility: Evaluation and Management

EXPECTANT MANAGEMENT OF THE INFERTILE COUPLE The chance that a spontaneous pregnancy will occur after unprotected intercourse for 1 year is around 90%88 (see Fig. 123.1. This cumulative pregnancy rate is highly influenced by female age. When a couple seeks evaluation of infertility, it is important first to rule out causes of absolute infertility (i.e., tubal factor, anovulation, and severe sperm abnormalities). Thereafter, the likelihood of a spontaneous pregnancy should be assessed, before any type of empirical therapy is begun. As mentioned previously, the age of the woman and the duration of infertility are the most important factors affecting the future likelihood of conception (see Figs. 123.3 and 123.4, and Table 123.2). With the use of prognostic models applied in unexplained infertile couples, a predicted chance of > 30% live birth in the year to come may urge physicians to promote expectant management as Marital fertility rates per 1000 wives

600 500 400 300 200 100 0 20

25

30

35

40

45

50

Age of wife Figure 123.4  Natural marital fertility rates as a function of age of the woman, indicated for different populations from the sixteenth until the twentieth century (Hutterites, Geneva bourgeoisie, Canada, Normandy, Tunis, Norway, and Iran). (Data from Menken J, Trussell J, Larsen U, et al. Age and infertility. Science. 1986;233:1389–1394; Schwartz D, Mayaux MJ. Female fecundity as a function of age: results of artificial insemination in 2193 nulliparous women with azoospermic husbands. Federation CECOS, N Engl J Med. 1982;306:404–406.)

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the first-­line treatment, specifically as studies have shown that compared to treatment with mild OS combined with IUI no improvement in live birth rates will be obtained. Whether in such cases direct application of IVF will make a true benefit still remains to demonstrated. Comparing different treatment options in couples with an unfavorable prognosis (< 30% predicted one year live birth) revealed that IVF did not result in improved outcomes compared to IUI with mild stimulation.89 Increasing patient pressure and the financial interests of some infertility centers have prompted starting infertility treatment prematurely. The approach of comparing spontaneous pregnancy chances with chances following intervention may aid in the proper timing to initiate therapy, which may prevent overtreatment. This approach is distinctly different from one that focuses on the highest chances for pregnancy per treatment cycle, which is often used. The definition of cost-­effective infertility care is achieving the desired end point at the lowest possible expenditure of resources.90 Analysis modeling in the United Kingdom confirmed that IVF is a cost-­effective treatment option for severe tubal factor infertility.91 Initial cost-­effectiveness studies also support the use of IUI and mild ovarian hyperstimulation before IVF is initiated, although this may apply only for couples with unexplained infertility who have a 1-­year prognosis of at best 30% for an ongoing pregnancy.92–94 Gradually, a consensus is evolving that a healthy singleton live birth per started treatment should be the preferred end point.95,96 OS in normo-­ovulatory women is inherently associated with multiple follicle development and increased chances for multiple pregnancies and should therefore be applied with great caution. In addition, a systematic evaluation documented that success rates of infertility therapies are often overstated and that the quality of relevant clinical studies is often questionable.97 Anovulation usually can be treated effectively with ovulation induction agents (such as CC, the aromatase inhibitor letrozole [LTZ], or exogenous gonadotropins) aimed at restoring normal ovarian function (see Chapter 117). In addition, mechanical factors involving the uterine cavity, tubal function, or oocyte pickup can be restored by endoscopic surgery (such as hysteroscopic myomectomy, adhesiolysis, or fimbrioplasty). The diagnosis of unexplained infertility can be made only after an appropriate diagnostic infertility workup has established the absence of any detectable abnormalities.98 Because no consensus has been reached regarding the extent of testing that should be performed during an

TABLE 123.2  Summary of Cochrane Review Concerning the Efficacy of In Vitro Fertilization

(IVF) for Unexplained Infertility Comparison

Number of Cases

IVF vs. Expectant Management Hughes 2004143a Soliman 199313 Overall

51 35 86

Ongoing Pregnancy

Rate per Couple

IVF

Expectant

29%

12%

IVF

IUI

41%

26%

IVF

IUI Stimulation

41%

37%

Odds Ratio

95% CI

3.24

1.07–9.80

1.96

0.88–4.36

1.15

0.55–2.42

IVF versus IUI Goverde 2000144 Overall

113 113

IVF vs. IUI/Stimulation Goverde 2000144 Overall

118 118

The table shows that randomized clinical trials demonstrating the efficacy of IVF as first-­line therapy in unexplained infertility are scarce and cover only limited numbers of cases. The application of intrauterine insemination (IUI) with or without stimulation as first-­line treatment is to be recommended in view of lower costs and patient discomfort. Data from Pandian Z, Bhattacharya S, Vale L, et al. In vitro fertilisation for unexplained subfertility. Cochrane Database Syst Rev. 2005;2:CD003357.

PART 10  Female Reproduction

Ovarian Stimulation Under normal conditions, a cohort of follicles is recruited for ongoing gonadotropin-­dependent development during the inter-­cycle rise in FSH. During a limited time interval (the “FSH window”), FSH concentrations rise above the threshold before declining99 (see Chapter 118). The aim of OS, with exogenous FSH as the most frequently used drug, is to intervene in mechanisms that regulate single dominant follicle development, in order to mature multiple follicles via generation of multiple oocytes for fertilization in vivo (after intercourse or IUI) or in vitro (IVF). OS in IUI cycles can be achieved using CC, LTZ, or exogenous gonadotropins. Medication is usually administered during the early or mid-­ follicular phase (cycle days 2 through 7 or 5 through 9) in daily dosages ranging between 100 and 150 mg for CC and 2.5–5 mg for LTZ. Exogenous gonadotropins are used in a fixed dose, starting with 50–75 IU/day on day 2 to 3 of the cycle. Depending on the number of dominant follicles > 12 mm that develop, this dosage can be adjusted in a subsequent cycle, using steps of 25–37.5 IU. The use of GnRH agonist cotreatment to avoid a premature LH rise generally is not recommended in IUI cycles. Along with a possible beneficial effect on the ongoing pregnancy rate, OS may lead to multiple pregnancies and OHSS, both of which should be considered preventable complications of treatment (Fig. 123.5).100 When multiple oocytes are released in vivo, the number of oocytes fertilized cannot be controlled, and a marked increase in multiple (especially higher-­order multiple) pregnancies has occurred over the past two decades.101,102 Development of more than two dominant follicles has been shown to enhance the rate of multiple pregnancies, without a gain in overall ongoing pregnancy rates.103 The results of OS are often combined with those of IUI, and few studies allow differentiation between the independent effects of the two.102–104 Specifically, when judging the efficacy of these two treatment components, several factors need to be considered. The infertility diagnosis (unexplained, male, or cervical factor), the age of the female, and the prognosis level for spontaneous pregnancy according to the Hunault model will all affect the decisions on which treatment modality to apply. Currently, initiating treatment in couples with unexplained or moderate to mild male factor infertility and a 1-­year prognosis of ≥ 30% is not advocated. If treatment is indicated, IUI in natural cycles will increase conception rates in couples with cervical factor infertility, preventing exposure to multiple pregnancy risks. In unexplained, but even in mild male factor infertility (total motile sperm count > 10 million) or minimal endometriosis, applying only IUI in the natural cycle may not be sufficiently effective over expectant management,104 implying that only the combination with OS will clearly benefit over timed intercourse. The combined use of IUI and OS with exogenous gonadotropins or CC for unexplained infertility may bypass various subtle barriers, such as minimal sperm abnormalities, sperm–cervical mucus interaction, and the timing of sperm delivery, and may further enhance fertility through the ovulation of more than one follicle. Notably, success in these treatment modalities is further affected by female age such that applying IUI with OS may be omitted in women aged > 40 years. The procedures during the treatment itself may be also considered as to their role in the achieved efficacy. OS should lead to the release of two to three oocytes maximally to maintain the proper balance between success and complications. Because of low cost and ease of administration, the antiestrogen CC could be recommended as first-­choice medication

100 Contribution of subfertility treatments to overall pregnancies (%)

infertility workup, cases of unexplained infertility involve a notoriously heterogeneous group of patients. In many cases of unexplained infertility or male factor infertility, patients undergo empirical treatment to improve pregnancy chances. Assisted reproduction encompasses various techniques used to increase the chances of pregnancy per cycle by basically bringing more gametes closer together. This goal can be achieved by OS, combined with IUI or IVF.

50

0 Singleton

A

Twin

Triplet or higher order

50

Multiple pregnancy chances per subfertility treatment (%)

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25

0 Ovarian Ovulation hyperstimulation induction Figure 123.5  Contribution of infertility treatments to overall pregnancies (A) and reported frequency of multiple pregnancy in relation to in vitro fertilization (IVF), ovarian hyperstimulation, and ovulation induction (B). The graphs show that assisted reproduction contributes considerably to the rate of multiple pregnancies in society and affects singleton rates only minimally. Also, ovarian (hyper)stimulation without access to the choice of how many embryos may be implanting contributes heavily to the totals of multiples pregnancies from infertility treatments. (Data from Fauser BC, Devroey P, Macklon NS. Multiple birth resulting from ovarian stimulation for subfertility treatment Lancet. 2005;365:1807–1816.)

B

IVF

for OS, although gonadotropins have appeared to be more effective.105 It should be noted that limited or even absent efficacy has been suggested recently106 for any drug (CC, gonadotropins, or aromatase inhibitors) that is used as monotherapy without IUI in unexplained infertility.107 In stimulated cycles, the additional use of GnRH antagonists or agonists for LH peak suppression have shown not to be cost-­effective, while the use of luteal support with vaginally applied progesterone may be of benefit but is awaiting larger trials to demonstrate a true effect. In case the total number of motile sperm inseminated is < 5 million, IUI treatment will hardly lead to pregnancies and is best abandoned. With sufficient sperm quantity, acceptable cumulative ongoing pregnancy rates after six cycles of IUI with OS can be obtained, with the highest yield in the first three cycles and stable cycle rates up until the ninth cycle. Timing of the insemination using LH surge detection in natural cycles is preferred over the use of human chorionic gonadotropin (hCG) triggering, and generally a time interval of 12 to 36 hours is optimal, depending on the triggering mode applied.

CHAPTER 123  Female Infertility: Evaluation and Management

Intrauterine Insemination IUI refers to placing sperm, after a washing procedure in the laboratory, in the uterine cavity just before ovulation. The rationale for IUI combined with OS is to increase gamete density at the site of fertilization (i.e., the fallopian tube). Hence, the classic indications for IUI are male (erectile and ejaculatory) or female (vaginismus) sexual dysfunction and cervical hostility. IUI combined with mild OS has been applied in couples suffering from male factor and unexplained infertility. Renewed interest in IUI reflects the evolution of better washing techniques (i.e., gradient or swim-­up) to improve the quality of the initial sperm sample by removing prostaglandins, infectious agents, and antigenic proteins, and nonmotile cells (leukocytes) or immotile and immature spermatozoa and thus decreased cytokines and lymphokines and subsequent reduction in free oxygen radicals.108 IUI is usually performed within 30–36 hours after hCG administration or 20–28 hours after the LH surge.109 It is performed using a small flexible catheter through which a small volume (0.3 to 0.5 mL) of culture medium containing the washed spermatozoa is delivered into the uterine cavity. When IUI is used to manage fertility problems, single rather than double insemination should be used.110 IUI combined with OS in the treatment of infertility remains a topic of ongoing debate. A recent literature review has made clear that treatment with IUI with OS as well as IUI alone probably results in a higher cumulative live birth rate than expectant management in couples with a low prognosis for natural conception. IUI in a stimulated cycle may outperform IUI in a natural cycle.104 Pregnancy chances as such may increase, but inherent associated risks include a considerable chance of multiple pregnancies, additional costs, and increased patient discomfort and chances for complications.103 A large retrospective analysis in a single European center, which assessed 1878 pregnancies attained through IUI combined with gonadotropin stimulation, reported a 16% twin and a 6% higher-­order multiple pregnancy rate111 (see Fig. 123.5). Also, a 2-­year follow-­up in a US infertility center involving 3347 OS cycles found a high multiple pregnancy rate, with an overall pregnancy rate of 30%.112 Reducing the risk for a multiple pregnancy may result from restricting the number of dominant follicles allowed at the time of ovulation trigger. From a nationwide survey in the Netherlands, twin rates appeared to be as low as 9% per pregnancy, albeit at the cost of some reduction in the overall chance of ongoing pregnancy.89,113,114 Although births resulting from assisted reproduction represent only 1% of all live births in the United States, they now account for 35% of twin births and for more than 40% of triplet and higher-­order multiple births.101 The adverse impact of multiple births on perinatal morbidity and mortality has been clearly documented and should lead to management of the infertile couple that increases chances for pregnancy without causing complications that have lifetime consequences.115,116 Cost-­effectiveness analyses so far have led to the conclusion that IUI, with or without OS, should precede IVF.93,117 These assessments were made, however, before the advent of current approaches to minimize costs and minimizing complications in IVF through minimal OS and transfer of a reduced number of embryos.95 As pregnancy chances per cycle remain low, it can be calculated that the number needed to treat for patients to benefit from any additional intervention is high. For instance, when the combination of FSH and IUI is compared with IUI alone, 31 treatment cycles are required before there would be one more singleton live birth.118 Offset against the additional pregnancy are the added costs of medications and of frequent ovarian response monitoring. When costs related to multiple pregnancies are also taken into consideration, the conclusion seems

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justified that widespread use of OS with exogenous gonadotropins combined with IUI should be discouraged. The real question remains whether society (especially patients, physicians, and health insurance companies) considers the high rate of multiple pregnancies an acceptable price to be paid for increased pregnancy chances per cycle. Thus, current international guidelines (NICE) recommend the use of IUI in the natural cycle as standard first-­line treatment for couples with unexplained or mild to moderate male factor infertility, although the efficacy of such treatment clearly may be doubted. As the risk for a multiple pregnancy may become reduced by applying CC or LTZ as an OS agent, the widespread use of FSH OS in IUI treatment may need to be reconsidered. Recent and currently ongoing studies will provide answers to the preferred treatment modality.119 Beginning treatment with CC/IUI will result in pregnancy in nearly one fourth of the couples, with minimal risk for multiple births. With IVF procedures using a high proportion of single-­embryo transfer, eliminating gonadotropin/IUI will result in pregnancies with the lowest possible risk for multiple births. Compared with conventional infertility treatment, an accelerated approach to IVF after the initial CC/IUI treatment results in a shorter time to pregnancy with fewer treatment cycles, and cost savings.120

In Vitro Fertilization IVF involves OS, oocyte retrieval, fertilization of oocytes in vitro (either spontaneously or by ICSI), and subsequent transfer of embryos into the uterine cavity. Aspects of IVF relevant to infertility management are discussed here. IVF was originally designed for treatment of bilateral tubal occlusion, but currently the “tubal factor” represents only a small proportion of indications for IVF. At present, it is applied most often in cases of mild to moderate male factor and unexplained infertility. A meta-­analysis of five relevant trials in unexplained infertility confirmed that no difference in pregnancy rates could be observed when IVF was compared with IUI, with or without ovarian hyperstimulation in treatment-­naïve infertile couples (odds ratio: 1.96; 95% CI: 0.88– 4.36).102,121 This implies that first-­line treatment may best be carried out using mild OS combined with IUI across a series of 6–9 cycles. The next step would then be IVF, although the added benefit has not been exactly assessed. Two separate studies indicated that initial treatment with IVF is not cost-­effective when compared with a conventional treatment algorithm.121–123 Many advances in IVF, such as improved cryopreservation programs, blastocyst culture, techniques of the embryo transfer, and the widened application of GnRH antagonists cotreatment may have improved the efficiency over the past decade, along with improved safety by applying single-­embryo transfer and better OHSS management.124 Tubal occlusion can give rise to hydrosalpinges. In women undergoing IVF, the presence of hydrosalpinges is associated with early pregnancy loss and poor implantation due to altered diminished endometrial receptivity. Thus, salpingectomy or at least occlusion of the isthmic part of the tubes should be offered before IVF when hydrosalpinges are present.125 Similarly, endometriosis-­associated infertility reduces pregnancy rates. IVF represents an effective means of bypassing the hostile peritoneal environment and anatomic distortion associated with this disease. Pretreatment with GnRH agonists immediately before IVF initiation appears to be beneficial. It is uncertain whether only a specific subset or all patients with endometriosis would benefit from this approach; the use of endometrial implantation markers may be helpful in this regard.28,126

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PART 10  Female Reproduction

The incidence of multiple gestations in IVF is easier to control than OS with or without IUI, because in case of IVF the occurrence of multiples is primarily dependent on the number of embryos transferred. Increased emphasis is being directed toward decreasing the number of embryos for transfer and, therefore, diminishing the chances of (higher-­order) multiple pregnancies. In a comparison of conventional maximal OS and replacement of two embryos, the application of milder OS with single-­embryo transfer resulted in the same number of ongoing pregnancies leading to term life birth after a 1-­year treatment period. Although on average one additional treatment cycle was required, the long-­term medical costs and patient burden were the same.96,127 KEYPOINTS  • In unexplained infertility with a prognosis for spontaneous ongoing pregnancy of over 30% for the subsequent year, expected management is justified as infertility treatment is not likely to yield better prospects for the couple. • In couples with unexplained infertility and a prognosis below 30% for the subsequent year, and in couples that have applied a period of 6-12 month of expected management without achieving an ongoing pregnancy, mild ovarian stimulation combined with intra uterine insemination is the preferred first line treatment. • In vitro fertilization and related technology is considered the last resort in treatment of couples with unexplained infertility. It is however the first line treatment in couples with a clear cause such as tubal pathology. In couples with oligo-/an-ovulation, failed treatment by ovulation induction may be a justified indication to apply in vitro fertilization and related technology.

SUMMARY AND FUTURE DEVELOPMENTS Infertility represents a serious health issue and is considered as a disease by WHO. Notably, most people worldwide have insufficient access to infertility and fertility care. Postponement of pregnancy beyond 30 years of age, as is observed in the Western world, is giving rise to an increased demand for infertility services, because more couples will fail to conceive spontaneously within 1 year of unprotected intercourse. The major clinical challenge remains to assess the reproductive aging status of a given woman. This will help to predict her pregnancy chances more accurately compared with assessment based on chronologic age alone. The use of multivariate models may further improve the prediction capacity of initial screening for a given couple. The duration of infertility and a woman’s age constitute the most important predictors of chances for achieving a spontaneous pregnancy. This approach should help when couples are counselled regarding the likelihood of success of expectant management, or whether (empirical) treatment should be initiated. The diagnosis of unexplained infertility can be made only after a proper diagnostic infertility workup has established the absence of any abnormalities. Because no consensus exists regarding the extent of testing that should be performed, unexplained infertility represents a notoriously heterogeneous group of patients. This condition may include couples with undiagnosed infertility and those with normal fertility for whom pregnancy, purely by chance, has not yet occurred. These patients often undergo empirical treatment to improve pregnancy chances per cycle. The inherent disadvantage of such an approach is decreased interest in the diagnostic workup, which impairs progress in our understanding of the underlying pathophysiology. When costs and prematurity of newborns related to multiple pregnancies are also taken into consideration, the conclusion is

justified that widespread use of OS with exogenous gonadotropins with or without IUI should be discouraged. With IVF, increased emphasis is directed toward decreasing the number of embryos for transfer and, therefore, diminishing the chances of (higher-­order) multiple pregnancies. Additional cohort follow-­up studies using appropriate end points, such as singleton, term delivered, and live birth rate, are required to elucidate further the optimal approach to empirical treatment of patients with unexplained infertility. Numerous innovations in ART have emerged and are currently being evaluated for its clinical efficacy and safety. However, the stakes are high and novel technologies are often introduced prematurely in everyday patient care without sufficient scientific proof of safety and efficacy. The most important recent developments in ART may be listed as follows: 1. Improved vitrification technology, allowing oocytes, embryos, and gonadal tissue to be cryopreserved with increased efficacy. 2. Blastocyst transfer; transition from the transfer of embryos into the uterus after 2 or 3 days of culture toward development up to the blastocyst stage on day 5 or 6 generating improved implantation and ongoing pregnancy rates. 3. Time laps monitoring; embryos are constantly being monitored for development potential in special incubators, using camera images and sophisticated software technology. Its clinical usefulness remains doubtful. 4. PGT-­A; preimplantation genetic testing for aneuploidy. Aneuploidy screening can now be performed on a large scale using sophisticated next-­generation sequencing of a number of cells removed from an early stage embryo before transfer, so only euploid embryos can be transferred. Potentially, this technology could also improve implantation rates, decrease miscarriage rates following IVF and therefore improve IVF success rates and time to pregnancy. However, again its clinical usefulness remains highly controversial. 5. A GnRH agonist bolus rather than hCG to stimulate final oocyte maturation at the end of the stimulation phase and prior to oocyte retrieval. This approach considerably reduced the risk for OHSS but can only be performed with GnRH antagonist cotreatment during OS. 6. Single embryo transfer; this policy initially developed in Northern Europe and is gradually being applied by the rest of the IVF community. Multiple pregnancies can be prevented by the transfer of one embryo at a time, significantly reducing pregnancy complications and improving perinatal outcomes. Supernumerary embryos can be cryopreserved and individually transferred in subsequent cycles. National data from various countries now demonstrate multiple pregnancy rates following IVF can be < 5%, with similar overall pregnancy rates. At present, in some countries approximately 50% of all children born are from cryopreserved embryos. 7. Freeze all embryos; OS for IVF gives rise to a supraphysiological endocrine milieu during the follicular phase of the cycle. Increasing evidence suggests that this may also impact endometrial receptivity and hence embryo implantation rates may be reduced. For these reasons, increasingly, IVF centers are applying “freeze all” strategies, where only embryos are transferred in subsequent cycles after freezing and thawing. The jury is still out whether this strategy will truly improve IVF outcomes. 8.  Widespread use of fertility preservation strategies in oncology patients aiming to preserve their fertility potential once cured from the primary disease. Despite the fact that this technology is more and more available, there still seems much room for growth in the area. Even today, many young cancer patients are not being made aware of this option.

CHAPTER 123  Female Infertility: Evaluation and Management 9. Nonmedical oocyte freezing; programs are offered worldwide to cryopreserve oocytes from young potentially fertile women who would like to have children later in life once circumstances are right. It is heavily debated whether this option is actually helping women by increasing their reproductive choices, or whether this development represents yet another example of exploitation of reproductive care. KEYPOINTS  • Assisted Reproduction Technology as treatment for female infertility has evolved by creating numerous technical improvements in order to make this treatment more effective, safer and more bearable for the patient. • A special note are the possibilities that this technology offers for fertility preservation strategies as well as the growing role in the prevention of hereditary disease through preimplantation genetic testing of embryos.

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PART 10  Female Reproduction

47. Fehring RJ, Schneider M. Variability in the hormonally estimated fertile phase of the menstrual cycle. Fertil Steril. 2008;90(4):1232–1235. 48. Fiddelers AA, Land JA, Voss G, et al. Cost-­effectiveness of Chlamydia antibody tests in subfertile women. Hum Reprod. 2005;20(2):425–432. 49. Coppus SF, Opmeer BC, Logan S, et al. The predictive value of medical history taking and Chlamydia IgG ELISA antibody testing (CAT) in the selection of subfertile women for diagnostic laparoscopy: a clinical prediction model approach. Hum Reprod. 2007;22(5):1353–1358. 50. Land JA, Evers JL. Chlamydia infection and subfertility. Best Pract Res Clin Obstet Gynaecol. 2002;16(6):901–912. 51. Land JA, Evers JL, Goossens VJ. How to use Chlamydia antibody testing in subfertility patients. Hum Reprod. 1998;13(4):1094–1098. 52. van Eekelen R, Scholten I, Tjon-­Kon-­Fat RI, et al. Natural conception: repeated predictions over time. Hum Reprod. 2017;32(2):346–353. 53. Mol BW, Collins JA, van der Veen F, et al. Cost-­effectiveness of hysterosalpingography, laparoscopy, and Chlamydia antibody testing in subfertile couples. Fertil Steril. 2001;75(3):571–580. 54. den Hartog JE, Lardenoije CM, Severens JL, et al. Screening strategies for tubal factor subfertility. Hum Reprod. 2008;23(8):1840–1848. 55. van Rijswijk J, Pham CT, Dreyer K, et al. Oil-­based or water-­based contrast for hysterosalpingography in infertile women: a cost-­effectiveness analysis of a randomized controlled trial. Fertil Steril. 2018;110(4):754– 760. 56. Johnson N, Vandekerckhove P, Watson A, et al. Tubal flushing for subfertility. In: The Cochrane Library. Vol. 4. Chicester, UK: John Wiley & Sons; 2020:1–46. 1–1–0003. 57. Mol BW, Collins JA, Burrows EA, et al. Comparison of hysterosalpingography and laparoscopy in predicting fertility outcome. Hum Reprod. 1999;14(5):1237–1242. 58. Verhoeve HR, Coppus SF, van der Steeg JW, et al. Collaborative effort on the clinical evaluation in reproductive medicine. The capacity of hysterosalpingography and laparoscopy to predict natural conception. Hum Reprod. 2011;26(1):134–142. 59. Exalto N, Emanuel MH. Clinical aspects of HyFoSy as tubal patency test in subfertility workup. BioMed Res Int. 2019;2019:4827376. 60. Fatum M, Laufer N, Simon A. Investigation of the infertile couple: should diagnostic laparoscopy be performed after normal hysterosalpingography in treating infertility suspected to be of unknown origin? Hum Reprod. 2002;17(1):1–3. 61. Surrey ES. Endoscopy in the evaluation of the woman experiencing infertility. Clin Obstet Gynecol. 2000;43(4):889–896. 62. Oei SG, Helmerhorst FM, Bloemenkamp KW, et al. Effectiveness of the postcoital test: randomised controlled trial. BMJ. 1998;317(7157):502– 505. 63. Oei SG, Helmerhorst FM, Keirse MJ. Routine postcoital testing is unnecessary. Hum Reprod. 2001;16(5):1051–1053. 64. Schwartz D, Mayaux MJ. Female fecundity as a function of age: results of artificial insemination in 2193 nulliparous women with azoospermic husbands, Federation CECOS. N Engl J Med. 1982;306(7):404–406. 65. Templeton A, Morris JK, Parslow W. Factors that affect outcome of in-­vitro fertilisation treatment [see comments]. Lancet. 1996;348(9039): 1402–1406. 66. Fauser BC. Follicle pool depletion: factors involved and implications. Fertil Steril. 2000;74(4):629–630. 67. Lobo RA. Early ovarian ageing: a hypothesis. What is early ovarian ageing? Hum Reprod. 2003;18(9):1762–1764. 68. Scott MG, Ladenson JH, Green ED, et al. Hormonal evaluation of female infertility and reproductive disorders. Clin Chem. 1989;35(4):620–629. 69. Chang MY, Chiang CH, Chiu TH, et al. The antral follicle count predicts the outcome of pregnancy in a controlled ovarian hyperstimulation/intrauterine insemination program. J Assist Reprod Genet. 1998;15(1):12–17. 70. Hendriks DJ, Mol BW, Bancsi LF, et al. Antral follicle count in the prediction of poor ovarian response and pregnancy after in vitro fertilization: a meta-­analysis and comparison with basal follicle-­stimulating hormone level. Fertil Steril. 2005;83(2):291–301. 71. Hansen KR, Hodnett GM, Knowlton N, Craig LB. Correlation of ovarian reserve tests with histologically determined primordial follicle number. Fertil Steril. 2011;95(1):170–175.

72. Jeppesen JV, Anderson RA, Kelsey TW, et al. Which follicles make the most anti-­Mullerian hormone in humans? Evidence for an abrupt decline in AMH production at the time of follicle selection. Mol Hum Reprod. 2013;19(8):519–527. 73. Broer SL, Mol BW, Hendriks D, et al. The role of antimüllerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertil Steril. 2009;91(3):705–714. 74. Hamdine O, Eijkemans MJC, Lentjes EGW, et al. Antimullerian hormone: prediction of cumulative live birth in gonadotropin-­releasing hormone antagonist treatment for in vitro fertilization. Fertil Steril. 2015;104(4):891–898.e2. 75. Leijdekkers JA, Eijkemans MJC, van Tilborg TC, et al. Cumulative live birth rates in low-­prognosis women. Hum Reprod. 2019;34(6):1030– 1041. 76. La Marca A, Sunkara SK. Individualization of controlled ovarian stimulation in IVF using ovarian reserve markers: from theory to practice. Hum Reprod Update. 2014;20(1):124–140. 77. Broer SL, Dólleman M, van Disseldorp J, IPD-­EXPORT Study Group, et al. Prediction of an excessive response in in vitro fertilization from patient characteristics and ovarian reserve tests and comparison in subgroups: an individual patient data meta-­analysis. Fertil Steril. 2013;100(2):420–429.e7. 78. Broer SL, van Disseldorp J, Broeze KA, et al. IMPORT study group. Added value of ovarian reserve testing on patient characteristics in the prediction of ovarian response and ongoing pregnancy: an individual patient data approach. Hum Reprod Update. 2013;19(1):26–36. 79. Lensen SF, Wilkinson J, Leijdekkers JA, et al. Individualised gonadotropin dose selection using markers of ovarian reserve for women undergoing in vitro fertilisation plus intracytoplasmic sperm injection (IVF/ICSI). Cochrane Database Syst Rev. 2018;2018(2). https://doi. org/10.1002/14651858.CD012693.pub2. 80. Nyboe Andersen A, Nelson SM, Fauser BC, García-­Velasco JA, Klein BM, Arce JC, ESTHER-­1 study group. Individualized versus conventional ovarian stimulation for in vitro fertilization: a multicenter, randomized, controlled, assessor-­blinded, phase 3 noninferiority trial. Fertil Steril. 2017;107(2):387–396. 81. Fernández-­Sánchez M, Visnova H, Yuzpe A, Klein BM, Mannaerts B, Arce JC, ESTHER-­1, ESTHER-­2 Study Group. Individualization of the starting dose of follitropin delta reduces the overall OHSS risk and/or the need for additional preventive interventions: cumulative data over three stimulation cycles. Reprod Biomed Online. 2019;38(4):528–537. 82. Eimers JM, te Velde ER, Gerritse R, et al. The prediction of the chance to conceive in subfertile couples. Fertil Steril. 1994;61:44–52. 83. Collins JA, Burrows EA, Wilan AR. The prognosis for live birth among untreated infertile couples. Fertil Steril. 1995;64:22–28. 84. Snick HK, Snick TS, Evers JL, et al. The spontaneous pregnancy prognosis in untreated subfertile couples: the Walcheren primary care study. Hum Reprod. 1997;12(7):1582–1588. 85. van der Steeg JW, Steures P, Eijkemans MJ, et al. CECERM Study Group (Collaborative Effort for Clinical Evaluation in Reproductive Medicine): pregnancy is predictable: a large-­scale prospective external validation of the prediction of spontaneous pregnancy in subfertile couples. Hum Reprod. 2007;22(2):536–542. 86. Collins JA, Wrixon W, Janes LB, et al. Treatment-­independent pregnancy among infertile couples. N Engl J Med. 1983;309(20):1201–1206. 87. Ratna MB, Bhattacharya S, Abdulrahim B, McLernon DJ. A systematic review of the quality of clinical prediction models in in vitro fertilisation. Hum Reprod. 2020;35(1):100–116. 88. Bagshawe A, Taylor A. ABC of subfertility: counselling. BMJ. 2003;327(7422):1038–1040. 89. Bensdorp AJ, Tjon-­Kon-­Fat RI, Bossuyt PM, et al. Prevention of multiple pregnancies in couples with unexplained or mild male subfertility: randomised controlled trial of in vitro fertilisation with single embryo transfer or in vitro fertilisation in modified natural cycle compared with intrauterine insemination with controlled ovarian hyperstimulation. BMJ. 2015;350:g7771. 90. Gleicher N. Cost-­effective infertility care. Hum Reprod Update. 2000;6(2):190–199.

CHAPTER 123  Female Infertility: Evaluation and Management 91. Philips Z, Barraza-­Llorens M, Posnett J. Evaluation of the relative cost-­effectiveness of treatments for infertility in the UK. Hum Reprod. 2000;15(1):95–106. 92. Steures P, van der Steeg JW, Hompes PG, et al. Intrauterine insemination with controlled ovarian hyperstimulation versus expectant management for couples with unexplained subfertility and an intermediate prognosis: a randomised clinical trial. Lancet. 2006;368(9531):216–221. 93. Van Voorhis BJ, Sparks AE, Allen BD, et al. Cost-­effectiveness of infertility treatments: a cohort study. Fertil Steril. 1997;67(5):830–836. 94. Garceau L, Henderson J, Davis LJ, et al. Economic implications of assisted reproductive techniques: a systematic review. Hum Reprod. 2002;17(12):3090–3109. 95. Fauser BC, Bouchard P, Coelingh Bennink HJ, et al. Alternative approaches in IVF. Hum Reprod Update. 2002;8(1):1–9. 96. Heijnen EM, Macklon NS, Fauser BC. What is the most relevant standard of success in assisted reproduction? The next step to improving outcomes of IVF: consider the whole treatment. Hum Reprod. 2004;19(9):1936–1938. 97. Johnson NP, Proctor M, Farquhar CM. Gaps in the evidence for fertility treatment—an analysis of the Cochrane menstrual disorders and subfertility group database. Hum Reprod. 2003;18(5):947–954. 98. Aboulghar MA, Mansour RT, Serour GI, et al. Diagnosis and management of unexplained infertility: an update. Arch Gynecol Obstet. 2003;267(4):177–188. 99. Fauser BC, Van Heusden AM. Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev. 1997;18(1):71–106. 100. Fauser BC, Devroey P, Macklon NS. Multiple birth resulting from ovarian stimulation for subfertility treatment. Lancet. 2005;365(9473):1807– 1816. 101. Hogue CJ. Successful assisted reproductive technology: the beauty of one. Obstet Gynecol. 2002;100(5 Pt 1):1017–1019. 102. Zolton JR, Lindner PG, Terry N, DeCherney AH, Hill MJ. Gonadotropins versus oral ovarian stimulation agents for unexplained infertility: a systematic review and meta-­analysis. Fertil Steril. 2020;113(2):417– 425.e1. 103. van Rumste MM, Custers IM, van der Veen F, et al. The influence of the number of follicles on pregnancy rates in intrauterine insemination with ovarian stimulation: a meta-­analysis. Hum Reprod Update. 2008;14(6):563–570. 104. Ayeleke RO, Asseler JD, Cohlen BJ, Veltman-­Verhulst SM. Intra-­uterine insemination for unexplained subfertility. Cochrane Database Syst Rev. 2020;3:CD001838. https://doi.org/10.1002/14651858.CD001838.pub6 [doi]. 105. Danhof NA, van Wely M, Repping S, et al. Gonadotrophins or clomiphene citrate in couples with unexplained infertility undergoing intrauterine insemination: a cost-­effectiveness analysis. Reprod Biomed Online. 2019. doi: S1472-­6483(19)30740-0 [pii]. 106. Bhattacharya S, Harrild K, Mollison J, et al. Clomifene citrate or unstimulated intrauterine insemination compared with expectant management for unexplained infertility: pragmatic randomised controlled trial. BMJ. 2008:337:a716. 107. Athaullah N, Proctor M, Johnson NP. Oral versus Injectable Ovulation Induction Agents for Unexplained Subfertility. Cochrane Database Syst Rev. 2002;3:CD003052. 108. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil. 1987:81:459–469.

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109. Duran HE, Morshedi M, Kruger T, et al. Intrauterine insemination: a systematic review on determinants of success. Hum Reprod Update. 2002;8(4):373–384. 110. Boomsma CM, Heineman MJ, Cohlen BJ, et al. Semen preparation techniques for intrauterine insemination. Cochrane Database Syst Rev. 2007;4:CD004507. 111. Tur R, Barri PN, Coroleu B, et al. Use of a prediction model for high-­ order multiple implantation after ovarian stimulation with gonadotropins. Fertil Steril. 2005;83(1):116–121. 112. Gleicher N, Oleske DM, Tur-­Kaspa I, et al. Reducing the risk of high-­ order multiple pregnancy after ovarian stimulation with gonadotropins. N Engl J Med. 2000;343(1):2–7. 113. Steures P, van der Steeg JW, Hompes PG, et al. Intrauterine insemination in The Netherlands. Reprod Biomed Online. 2007;14(1):110–116. 114. Custers IM, Steures P, Hompes P, et al. Intrauterine insemination: how many cycles should we perform? Hum Reprod. 2008;23(4):885–888. 115. Suri K, Bhandari V, Lerer T, et al. Morbidity and mortality of preterm twins and higher-­order multiple births. J Perinatol. 2001;21(5):293–299. 116. Stromberg B, Dahlquist G, Ericson A, et al. Neurological sequelae in children born after in-­vitro fertilisation: a population-­based study. Lancet. 2002;359(9305):461–465. 117. Goverde AJ, McDonnell J, Vermeiden JP, et al. Intrauterine insemination or in-­vitro fertilisation in idiopathic subfertility and male subfertility: a randomised trial and cost-­effectiveness analysis. Lancet. 2000;355(9197):13–18. 118. Collins J. Stimulated intra-­uterine insemination is not a natural choice for the treatment of unexplained subfertility: current best evidence for the advanced treatment of unexplained subfertility. Hum Reprod. 2003;18(5):907–912. 119. van Rumste MM, Custers IM, van Wely M, et al. IVF with planned single-­embryo transfer versus IUI with ovarian stimulation in couples with unexplained subfertility: an economic analysis. Reprod Biomed Online. 2014;28(3):336–342. 120. Reindollar RH, Regan MM, Neumann PJ, et al. A randomized clinical trial to evaluate optimal treatment for unexplained infertility: the fast track and standard treatment (FASTT) trial. Fertil Steril. 2010;94(3):888–899. 121. Pandian Z, Bhattacharya S, Vale L, et al. In vitro fertilisation for unexplained subfertility. Cochrane Database Syst Rev. 2005;(2):CD003357. 122. Soliman S, Daya S, Collins J, et al. A randomized trial of in vitro fertilization versus conventional treatment for infertility. Fertil Steril. 1993;59:1239–1244. 123. Karande VC, Korn A, Morris R, et al. Prospective randomized trial comparing the outcome and cost of in vitro fertilization with that of a traditional treatment algorithm as first-­line therapy for couples with infertility. Fertil Steril. 1999;71(3):468–475. 124. Dosouto C, Haahr T, Humaidan P. Gonadotropin-­releasing hormone agonist (GnRHa) trigger -­state of the art. Reprod Biol. 2017;17(1):1–8. 125. Kontoravdis A, Makrakis E, Pantos K, et al. Proximal tubal occlusion and salpingectomy result in similar improvement in in vitro fertilization outcome in patients with hydrosalpinx. Fertil Steril. 2006;86(6):1642–1649. 126. Lessey BA, Kim JJ. Endometrial receptivity in the eutopic endometrium of women with endometriosis: it is affected, and let me show you why. Fertil Steril. 2017;108(1):19–27. https://doi.org/10.1016/j.fertnstert.2017.05.031. 127. Min JK, Breheny SA, MacLachlan V, et al. What is the most relevant standard of success in assisted reproduction? The singleton, term gestation, live birth rate per cycle initiated: the BESST endpoint for assisted reproduction. Hum Reprod. 2004;19(1):3–7.

124 Androgen Excess Disorders in Women Zi-­Jiang Chen, Richard S. Legro, David A. Ehrmann, and Daimin Wei

OUTLINE Androgen Physiology and Pathophysiology, 2060 Androgen Biosynthesis, 2060 Regulation of Androgen Secretion, 2062 Blood Levels and Transport of Androgens, 2064 Mechanisms of Androgen Action, 2066 Clinical Manifestation of Androgen Excess, 2066 Cutaneous Manifestations, 2066 Polycystic Ovary and Polycystic Ovary Morphology, 2067 Metabolic Dysfunction, 2067 Common Androgen Excess Disorders, 2068 Polycystic Ovary Syndrome, 2068 Ovarian Neoplasms, 2070 Other Ovarian Hyperandrogenic Disorders, 2071

Congenital Adrenal Hyperplasia, 2071 Other Adrenal Hyperandrogenic Disorders, 2072 Idiopathic Hyperandrogenism, 2072 Diagnosis of Hyperandrogenism, 2072 Clinical Hyperandrogenism, 2072 Biochemical Hyperandrogenism, 2073 Treatment of Polycystic Ovary Syndrome and other Androgen Excess Disorders, 2074 Androgen Suppression, 2074 Ovulation Induction and Assisted Reproduction, 2075 Prevention of Long-­Term Complications, 2076 Summary and Future Directions, 2076

  In this chapter, we first summarize the normal physiology of androgen production in women. This is followed by a review of the pathogenesis and clinical presentation of disorders leading to androgen excess. Finally, an approach to the diagnosis, differential diagnosis, and therapy of these disorders is provided.

ANDROGEN PHYSIOLOGY AND PATHOPHYSIOLOGY Androgen Biosynthesis Androgens (from the Greek word andre-­, the stem of the word meaning “man”) comprise a group of steroid hormones that regulate the developmental process and maintain male and female characteristics by binding to their cognate receptors. Classically, four main androgens have been identified including testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and androstenedione, which are secreted from endocrine glands through similar pathways and are released into the circulation to exert a number of physiological functions (Table 124.1). More recently, other androgens, such as androsterone, have been found to play a key role in male sexual development and female hyperandrogenic disorders. Although androgens are essential during childhood, subsequently, their synthesis is important for pubertal development, sexual life, and improving fertility in males and females. Thus, androgen biosynthesis is regulated both in fetal and pubertal life in males and females.

Pathways of Androgen Biosynthesis. The early steps of steroid synthesis are shared between glucocorticoids, mineralocorticoids, and sex steroids; however, defects in the initial step will definitely affect the final product. Two possible pathways are described for androgen biosynthesis: the “classic” and the “backdoor” pathways1 (Fig. 124.1). Animal studies, analyses of human tissues, and clinical studies have

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converged to form a classic model of androgen synthesis in a zone of the primate adrenal cortex called the zona reticularis. The Classic Pathway of Androgen Biosynthesis. Cholesterol is the main precursor for the formation of all steroid end products, and the first step is transport to the inner mitochondrial membrane. Acute stimulation of androgen biosynthesis is mediated by steroidogenic acute regulatory protein (StAR), which is an active transporter of cholesterol through the inner mitochondrial membrane. Notably, any defect or mutation in StAR is accompanied by severe loss of adrenal steroidogenesis and lack of virilization.2 StAR mutations, along with all other genetic defects of androgen biosynthesis, are inherited in an autosomal recessive fashion, and both types of genetic male and female individuals can be affected.3 The first enzymatic and rate-­limiting step for the steroid cascade inside the mitochondrial inner membrane is supported by side-­chain cleavage (SCC), a mitochondrial cytochrome enzyme (P450scc) that mediates the conversion of cholesterol into pregnenolone (Preg). Two cytochrome P450 enzymes (P450scc and cytochrome P450c17) are involved. P450scc stimulates the chemical reaction with the help of StAR protein in preparation for steroid hormone biosynthesis in the adrenal glands and the gonads. In contrast, cytochrome P450c17 is important for cortisol and sex hormone biosynthesis. This enzyme is a qualitative regulator of steroid synthesis which has two distinct activities: 17α-­hydroxylase and 17,20-­lyase activity. These are essential for synthesis of cortisol, 17-­ketosteroids DHEA, and androstenedione, precursors for steroid hormones. For the production of testosterone through the classic pathway, first, pregnenolone is converted to 17-­hydroxypregnenolone (17OH-­preg) and then the 17,20-­lyase activity of P450c17 converts 17OH-­preg to DHEA, supported by cofactors P450 oxidoreductase and cytochrome

CHAPTER 124  Androgen Excess Disorders in Women

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TABLE 124.1  Major Types of Androgen Source, Functions, and Metabolism in Women Androgen Types

Site of Production

Testosterone

Ovarian stroma, adrenal zona reticularis Peripheral tissue, adrenal zona reticularis Ovarian theca, adrenal zona reticularis Ovarian stroma, adrenal zona fasciculata

DHEA-­S DHEA Androstenedione

Production Rate/Day (mg)

Adrenal (%)

Ovarian (%) Functions

0.1–0.4

25

25

3.5–20

95–100

0–5

6–8

50

20

1.4–6.2

50

50

Gonadotropin regulation, estradiol precursor, primordial follicle growth Preantral follicle development, androgen precursor, oocyte development Testosterone precursor, anti-­aging effect, improve sexual functions, endothelial proliferation Testosterone precursor, follicle growth

DHEA, Dehydroepiandrosterone; DHEA-­S, dehydroepiandrosterone sulphate. Cholesterol P450 StAR

3βHSD2

Pregnenolone

Progesterone

P450c17

3βHSD2 17OH-Prog

P450c17 +b5

Androstenedione

SRD5A1

17OH-DHP

AKR1C2/4

Testosterone

17OHAllopregnanolone

P450c17 P450aro

Estrone

Androstenedione HSD17B3/5

17βHSD3/5 3βHSD2

Allopregnanolone

P450c17

17, 20-lyase

3βHSD2

17βHSD3/5 Androstenediol

Dihydro-prog

Red 3αHSD AKR1C2/4

P450c17

17OH-Preg

DHEA

5αRed1

5αRed2

DHT

HSD17B6

Androstenediol

P450aro Estradiol Classical pathway

Backdoor pathway

Figure 124.1  Outlines of classic and backdoor pathway of androgens biosynthesis. Adrenal and gonadal pathways are combined in the figure. The left hand column indicates the Δ5 classic pathway, in which steroids retain the double bond between carbons 5 and 6 in cholesterol’s B-­ring. The StAR mediates the transport of cholesterol to the inner membrane of mitochondria, where P450 facilitates to cleave off the side chain and resultant pregnenolone (Preg) formation. Δ4 steroids are converted from Δ5 steroid by using 3βHSD2 (HSD3B2) within the adrenal and gonadal tissue. Progesterone (Prog) is converted into 17OH-­prog in the zona fasciculate and gonads by P450c17. 17OH-­prog and 17OH-­preg is converted to androstenedione and DHEA by human P450c17 activity, followed by testosterone production in stepwise manner. DHEA is converted into androstenediol and then androstenedione to testosterone by the activity of 17βHSD3. Right hand side in red arrows indicates the backdoor pathways of adrenal biosynthesis. In alternative backdoor pathway, progesterone is converted into neuroactive steroid, allopregnanolone. Note that in the alternative pathway, 17OH-­prog is 5α, 3α reduced and then converted into androstenedione. Androsterone is the principle androgen in fetal circulation which is converted into androstenediol by 17HSD17B3/5 and then further oxidized by HSD17B6 to yield into potent androgen DHT.5-­αRed2, 5α-­reductase type 2; AKR1C2/4, aldo-­keto reductase family 1, member C2/4; Aro, aromates; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; HSD3B2, 3β-­hydroxysteroid dehydrogenase type 2; Preg, pregnenolone; Prog, progesterone; StAR, steroidogenic acute regulatory protein.

b5. P450c17 is encoded by a single copy gene on chromosome 10q24.3, and mutations within this gene can inhibit either all functions or selectively the 17,20-­lyase activity of the resulting protein.4 DHEA is a C19 steroid and androgen precursor, which is then either converted to androstenedione (Δ4A) or androstenediol and finally to testosterone by the enzymes 3β-­hydroxysteroid dehydrogenase type 2

(HSD3B2) and 17β-­hydroxysteroid dehydrogenase (HSD17B3) that exhibit tissue-­specific expression. Approximately 50% of testosterone comes from direct secretions from steroid-­producing endocrine glands, whereas the remaining 50% is produced by different organs including the adipose tissue, liver, and skin which contain 3β-­HSD type 1, 17β-­HSD type 5, and P450arom enzymes.

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PART 10  Female Reproduction

Testosterone is further converted to the most potent androgen, dihydrotestosterone (DHT), by 5α-­reductase type 2 (SRD5A2). Two isoforms of 5α-­reductase are expressed in different tissues; however, 5α-­reductase type 2 is the more abundant form in the gonads. With regard to the degree of androgen action at the androgen receptor (AR), DHT is the most potent androgen followed by testosterone, whereas weak stimulation of AR has been observed by androstenedione and androstenediol.2 The Backdoor Pathway of Androgen Biosynthesis. For the synthesis of DHT, an alternative “backdoor” pathway was first disco­ vered in the tammar wallaby pouch young and then was identified in mouse pups.5 It provided evidence that testosterone is the androgen in the circulation, while DHT exerts its actions locally on genital skin. This pathway bypasses the usual intermediate steroids DHEA, androstenedione, and testosterone. This alternative backdoor pathway diverges from the classic pathway with further conversion of 17-­hydroxyprogesterone (17OH-­prog) to 17OH-­dihydroprogesterone (17OH-­DHP; 5α-­pregnane-­17α-­ol-­3,20-­dione), 17OH-­allopre­ gnanolone (17OH-­ Allo; 5α-­pregnane-­3α,17α-­ol-­3,20-­dione), andro­ sterone, and androstanediol (3αDiol) to DHT. The stepwise conversion of 17OH-­prog to DHT is supported by the enzymes 5α-­reductase type 1 (SRD5A1), AKR1C2/4, P450c17, HSD17B3/5, and AKR1C2/4. This novel alternate pathway to DHT thus circumvents the usual steroid intermediates and is thus considered as “the backdoor pathway.” The backdoor pathway plays an important role in a number of hyperandrogenic disorders including PCOS,6 congenital adrenal hyperplasia (CAH),7 some virilized female newborns with P450 oxidoreductase deficiency,8 and mini puberty of infancy.9

Regulation of Androgen Secretion

Organ-­Specific Androgen Secretion Regulation. In women, the ovaries and adrenal glands are the prime source of androgen secretion stimulated by luteinizing hormone (LH) and adrenocorticotropic hormone (ACTH), respectively. ACTH is a 39-­amino acid peptide synthesized and secreted by the anterior pituitary (Fig. 124.2). Both corticotropin-­releasing hormone and arginine-­vasopressin work synergistically to regulate the synthesis of ACTH. The secretion and levels of adrenal androgens are controlled by intraglandular paracrine and autocrine mechanisms, rather than direct negative feedback by the pituitary gland. Circulating androgens are predominantly produced in the ovaries, adrenal glands, and from peripheral tissue conversion. Androgen precursors, including DHEA, DHEAS, and androstenedione, and the active androgen testosterone are the major androgens secreted from the adrenal glands. Two potent bioactive androgens, testosterone and DHT, directly bind to the receptors, whereas DHEA, DHEAS, and androstenedione are pro-­androgens that exert androgenic effects after conversion to testosterone and DHT. DHT, the most potent androgen, circulates at very low levels due to its production from local metabolism within tissues. The synthesis and production of androgens vary by age; for example, DHEAS levels are high in newborns and then precipitously decline until adrenarche, which commences approximately at 6–8 years of age. High levels of DHEA and DHEAS are the hallmark of adrenarche. Increased levels of DHEAS are noted prior to the increase in the circulating concentrations of estrogens and androgens, whose synthesis and increasing blood levels are directly associated with puberty. Circulating levels of DHEAS are high during the second decade of life, and higher levels are noted in males than in females. However, no significant increase in the concentration of cortisol and ACTH has been observed during this period, indicating that adrenarche is not dependent on activation by the pituitary axis. The following sections describe the organs specific for androgen production and metabolism.

Hypothalamus CRH

Anterior pituitary ACTH

Adrenal gland

Adrenal androgens and estrogen

Kidney 5-alpha reductase

DHT Figure 124.2  Schematic outlines of regulation for androgen secretion by adrenal structure. ACTH, Adrenocorticotropin; CRH, corticotrpin-­ releasing hormone; DHT, dihydrotestosterone.

Ovary. The principal source of ovarian androgens is theca interna cells and the ovarian stroma of the developing follicles. The mechanism of regulation of androgen secretions is similar to Leydig cell regulation in the testes. The combined activity of LH and follicle-­stimulating hormone (FSH) is required to stimulate ovarian steroidogenesis, with LH stimulating the theca-­ interstitial (thecal) cells to produce androgen precursor for subsequent conversion by ovarian granulosa cells under the action of FSH (Fig. 124.3). In the adult human ovary, progesterone and estradiol are produced from the androgen precursor cholesterol under the control of LH and FSH. The production of progesterone occurs during the luteal phase, while estrogens are produced in the follicular phase. Human granulosa cells convert cholesterol to pregnenolone (due to lack of P450c17) which is further converted into progesterone and then ultimately to androstenedione. Androstenedione secretion from the theca cells represents approximately 50% of circulating androgens in women. Androgenic precursors (especially androstenedione) are produced by thecal cells under LH stimulation and aromatized to estrogen by FSH-­ stimulated granulosa cell aromatase activity. During the preovulatory phase, the high level of LH inhibits the activity of 17,20-­lyase and 17α-­ hydroxylase, thereby ultimately reducing testosterone and androstenedione concentrations. The growth of antral stage follicles requires stimulation of granulosa cells via FSH action. Increased androstenedione and estradiol secretion accompanies the development of the dominant follicle. Androgens are obligate intermediates for the synthesis of estradiol and play an important role in follicle selection, follicle recruitment, and ultimately follicular growth (see Chapters 118 and 119). Therefore, the overall functional activity of the ovarian follicle structure is highly associated with the level of estradiol. High

CHAPTER 124  Androgen Excess Disorders in Women

LH ±

+ Cholesterol StAR SCC

2063

THECA CELL

3β Pregnenolone 17α-hydroxylase 3β 17-HydroxyPregnenolone 17,20-lyase 3β Dehydroepiandrosterone

Progesterone 17α-hydroxylase 17-HydroxyProgesterone 17,20-lyase

+ + – –

Androstenedione 17β-HSD5 Testosterone

FSH – –

+

Aromatase Estrone 17β-HSD1

GRANULOSA CELL

Estradiol

5α-R DihydroTestosterone

Inhibin IGF

IGF

Insulin Figure 124.3  Outline of the organization of the major steroid biosynthetic pathways in the small antral follicle of the ovary, depicted according to the two-­gonadotropin, two-­cell model of ovarian steroidogenesis. Luteinizing hormone (LH) stimulates androgen formation within theca cells via the steroidogenic pathway common to the gonads and adrenal glands. Follicle-­stimulating hormone (FSH) regulates estradiol biosynthesis from androgen by granulosa cells. Long-­loop negative feedback of estradiol on gonadotropin secretion does not readily suppress LH at physiologic levels of estradiol and stimulates LH under certain circumstances. Androgen formation in response to LH appears to be modulated by intraovarian feedback at the levels of 17-­hydroxylase and 17,20-­lyase, both of which are activities of cytochrome P450c17. The quantitative importance of androstenedione formation from 17 OH-­Prog (dotted arrow) in the intact follicle is unknown. Androgens and estradiol inhibit (minus signs) and inhibin, insulin, and insulin-­like growth factor-­1 (IGF-­1) stimulate (plus signs) 17-­hydroxylase and 17,20-­lyase activities. Pertinent enzymes are italicized.3β, Δ5isomerase-­3β-­hydroxysteroid dehydrogenase type 2; 17βHSD, 17β-­hydroxysteroid dehydrogenase types 1 and 5; 5α-­R, 5α-­reductase; scc, side-­chain cleavage; StAR, steroidogenic acute regulatory protein. (Modified with permission from Ehrmann DA, Barnes RB, Rosenfield RL. Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev. 1995;16:322–353. Copyright © 2007 The Endocrine Society.)

levels of androgens suppress ovulation and cause the development of the histologic and gross anatomic features of the polycystic ovary (PCO, see below). Ovarian thecal androgen production is regulated by the functional activity of LH. Desensitization in part is mediated by downregulation of the LH receptor binding sites by LH itself (homologous desensitization). The main product obtained from the theca interna and stroma cells of the ovary is androstenedione, which ultimately is transformed into testosterone by the activity of theca and granulosa cells; however, the ability of the granulosa cells to synthesize testosterone is relatively high compared with theca cells. These androgens transport to the granulosa cells by the process of diffusion through the basal membrane of follicles, and inside the granulosa cells, the androgen precursor is aromatized to 17β estradiol by the stimulatory action of FSH on the aromatase enzyme activity. FSH in combination with estradiol increase the division of granulosa cells and ultimately the volume of the antral cavity. Regulation of androgen secretion is a complex process in which different hormones and growth factors are involved that mediate ovarian functional activity that ultimately increases steroidogenesis by activation of P450c17 (see Fig. 124.3). The activity of P450c17 is inhibited by estrogen through a negative feedback mechanism using both paracrine and autocrine routes. These inhibiting activities are balanced by the action of different hormones and growth factors including insulin, insulin-­like growth factor-­1 (IGF-­1), and inhibin that ultimately amplify P450c17 action (see Fig. 124.3 and see Chapter 119).

Adrenal Cortex. The adult human cortex is composed of three layers: the zona glomerulosa (ZG) for mineralocorticoid production, the zona fasciculata (ZF) for glucocorticoid production, and the zona reticularis (ZR) for androgen production. ZG and ZF play an important role during fetal life, while the ZR production of steroid hormones begins slowly after birth and becomes active at adrenarche, approximately at 6–8 years of age. Differentiation of the centripetal progenitor cells into ZR, then ZG, and finally again conversion into ZR cells underlies the development of the adrenal zones. In terms of androgen production, the activity of ZR reaches a maximum at the age of 30 years and then declines thereafter. The activity of ZR is low for HSD3B2 and high for P450C17-­20 lyase, and sulfonyl-­transferase (SULT2A1), ultimately yielding massive production of DHEA and DHEAS.10 Adrenal 17-­ketosteroid is an important androgen whose secretion commences in mid-­childhood and reaches its maximum at adrenarche. Adrenarche is characterized by a change in the pattern of the adrenal secretory response to ACTH. ACTH elicits cortisol production prior to adrenarche, although both cortisol and 17-­ketosteroid secretion begins after adrenarche. Factors contributing to this change at adrenarche are nutritional status, somatic growth, secretion of leptin, insulin, and IGFs; however, further investigation is needed to highlight the role(s) of each of these factors. There are marked increases in the production of 17OH-­preg and DHEA that cause increased transformation of DHEAS, the predominant androgen secreted by the adrenal gland. DHEAS is a unique secretory product released from the ZR at 3.5–20

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PART 10  Female Reproduction

∆5-pathway

∆4-pathway

Cholesterol StAR Side chain cleavage Pregnenolone

21

3β

Progesterone

17αhydroxylase

DHEA-SO4

17-HydroxyPregnenolone

PAPSS

Dehydroepiandrosterone

11,18 11

Aldosterone

17αhydroxylase

3β

b5 17, 20lyase SULT

DOC

17-HydroxyProgesterone

21

b5 17, 20lyase

3β

Cmpd S

11

Cortisol

Z.glomerulosa

Androstenedione

Z.fasciculata Z.reticularis

17βHSD5 Aromatase

Testosterone Estrone Figure 124.4  Outline of the organization of the major steroid biosynthetic pathways in the adrenal cortex. The area within the dotted square contains the core steroidogenic pathways, of which the left column shows the Δ5-­pathway and the right column shows the Δ4-­pathway, also utilized by ovarian theca cells. The top yellow row shows the pathway to aldosterone; the middle blue row shows the zona fasciculata pathway to cortisol. The lower green row shows the zona reticularis steps to DHEA-­sulfate (SO4) and other 17-­ketosteroids. The dotted box encompasses steps common to all zones and the gonads. Dotted pathways are considered to be relatively minor. Cmpd S = 11-­deoxycortisol; the 11-­deoxy intermediate to aldosterone (deoxycorticosterone) is not shown. The steroidogenic enzymes are italicized. Cytochrome P450 enzyme steps are: side-­chain cleavage (scc); 17α-­hydroxylase/17,20-­lyase; 21-­hydroxylase (21); 11β-­hydroxylase/18-­ hydroxylase-­dehydrogenase (11,18); and aromatase. Non-­P450 enzyme steps are steroidogenic acute regulatory protein (StAR); Δ5-­isomerase-­3β-­ hydroxysteroid dehydrogenase type 2 (3β); 17β-­hydroxysteroid dehydrogenase type 5 (17βHSD5); and sulfotransferase 2A1 (SULT). Clinically relevant electron and sulfate transfer enzymes are, respectively, P450-­oxidoreductase (POR), cytochrome b5 (b5), and 3′-­phosphoade nosine-­5′-­phosphosulfate synthase 2 (PAPSS). (Modified with permission from Rosenfield RL. Identifying children at risk for polycystic ovary syndrome. J Clin Endocrinol Metab. 2007;92:787. Copyright © 2007 The Endocrine Society.)

mg per day in reproductive age women. Changes during adrenarche are highly associated with the development of the ZR and the adrenal zone that produces large amounts of DHEA and DHEAS (Fig. 124.4).2 The activity of 3β-­HSD and cytochrome b5 increases the production of DHEA that is further converted into DHEAS in the adrenal gland by the action of high zonal expression of SULT2A1. SULT2A1 (steroid sulfotransferase or cholesterol sulfotransferase) is highly expressed in the cytoplasm of adrenocortical cells in the reticularis and its substrates include pregnenolone, 17α hydroxypregnenolone, and DHEA. During fetal development, these steroids are used as substrates resulting in high levels of sulfated products that are then transported to the placenta for further biosynthesis. However, the predominant substrate in adults is DHEA which ultimately is transformed into DHEAS. The quantity of DHEAS varies across the life span, however, although it is the dominant androgen released from the adrenal gland. Similarly, in the ovary different factors, including insulin and IGFs, contribute to the production of adrenal steroids in response to ACTH. Infusion of insulin to hyperandrogenic women modestly potentiates the 17-­ketosteroid response to ACTH in a manner that is compatible with increases in 17-­hydroxylase and 17,20-­lyase activities. In vitro studies have directly shown that insulin and IGFs upregulate adrenal 17-­hydroxylase, 17,20-­ lyase, and 3β-­HSD activities.

Blood Levels and Transport of Androgens Serum concentrations of androgens and intermediate products in their biosynthesis and metabolism are shown in Table 124.2. Androgens in women are mainly derived from the adrenal gland and ovary. While serum DHEAS levels usually reflect adrenal androgen production, testosterone, androstenedione, and DHEA are secreted by both the ovary and the adrenal gland. Among circulating androgens, testosterone is the most important biologically and clinically because of its relatively high serum concentration and potent effect on target organs. Androgens exist in the blood in conjugated or unconjugated forms. Over 96% of plasma testosterone binds to carrier proteins including sex hormone-­binding globulin (SHBG) and albumin. SHBG is a glycoprotein synthesized in the liver and secreted into the circulation.11 It has a high binding affinity for testosterone, leading to a small fraction of total testosterone as free and bioavailable. The binding of testosterone to albumin is of much lower affinity, and therefore SHBG concentration is the major determinant of the testosterone remaining free and biologically active in the blood. SHBG serum levels are influenced by metabolic and hormonal factors in various physiological and pathological conditions. Therefore, they not only change (usually decreased) in metabolic diseases such as obesity, type 2 diabetes mellitus, and non-­alcoholic fatty liver disease, but also in thyroid disorders, pituitary diseases, and breast and

0.0289 (nmol/L)

25–40 50–180

0.0347 (nmol/L)

A c.934G>A c.977C>A c.977C>T c.983G>A c.879+1G>A c.966_969dupCCTG c.905_906delGT c.931delT c.958delC c.967_979del13

Exon 6 c.661A>T c.682G>T c.739C>T c.748A>T c.755C>T c.769C>T c.653-387G>A c.653-1G>A c.652+14C>T c.789delC c.653-6_6534delTCC

6

7

8

9

SAND

Exon 10 c.1096-1G>A c.1096-1G>C c.1095+6G>A c.1103dupC c.1155dupA c.1249dupC c.1242_1243insA c.1244_1245insC c.1163_1164insA c.1189delC c.1193delC c.1214delC c.1249delC c.1265delC c.1236_1239dupGGCC

Exon 7 c.834C>G c.845dupC c.798delC

EXon5 c.607C>T

10 11 12 13 14

PHD1

L

L Amino acid number

EXon 4 c.463G>A c.517C>T c.463+2T>C c.489dupC c.515_516ins13 c.540delG

Exon 2 c.173C>A c.202A>G c.230T>C c.232T>A c.232T>C c.239T>G c.238G>T c.247A>G c.254A>G c.260T>C c.269A>G c.274C>T c.278T>G c.290T>C c.132+1G>C c.93_94insT c.205_208dupCAGG c.132+1_132+3delGTGinsCT c.267_275delCTATGGCCG

PRR

Exon 9 c.1072C>T c.995+5G>T c.1067_1071dupGGCCC c.995+3_995+5delGAGinsTA T c.1053_1060delGGCAGAGG

Nuclear localization

300 Protein-protein interaction; DNA binding

PHD2

Exon 12 c.1411C>T c.1400+1G>A c.1450G>A Exon 13 c.1513delG c.1516delG Exon 14 c.1616C>T c.1638A>T c.1567-2A>G

C

L

400 Histone code readers; central tolerance

Exon 11 c.1322C>T c.1336T>G c.1347C>A c.1370dupG c.1295_1296insAC c.1296delGinsAC c.1314_1326del13ins2 c.1344delCinsTT c.1344delC c.1314_1326del13ins2 c.1296delGinsAC c.1344delCinsTT

C

L 200

2227

500

]

Coactivator of nuclear receptors

Fig. 132.3  The autoimmune regulator (AIRE) protein with its functional domains and reported mutations. AIRE harbors four major subdomains: the homogeneously staining region or caspase recruitment domain/homodimerisation domain (CARD/HSR, amino acids 1–105); the SAND domain (for Sp100, AIRE-­1, NucP41/75 or NucP41/75, DEAF-­1, amino acids 181–280); and two plant homeodomain (PHD)–type zinc fingers (amino acids 296–343 and 434–475). In addition, the AIRE protein contains four LXXLL domains that are found on coactivators of nuclear receptors (amino acids 7–11, 63–67, 414–418, 516–520) and a nuclear localization signal (amino acids 100–189). AIRE acts in a multiprotein complex and was early recognized as a transcription factor, as it localizes to the cell nucleus. The functions of the different domains of AIRE are shown in the boxes. Founder and common mutations are shown in blue, including c.254A>G (Persian Jewish), c.415C>T (Sicilian), c.607C>T (Sardinian), c.769C>T (Finnish), and c.967-­979del13 (Norwegian, British, North American). Dominant negative mutations are shown in red. Gross deletions and gross insertions are not included. (Slightly modified from Bruserud O, Oftedal BE, Wolff AB, et al. AIRE-­mutations and autoimmune disease. Curr Opin Immunol. 2016;43:8–15.)

channel regulator expressed in the terminal alveoli, is associated with bronchiolitis,30 as is BPIFB1, a protein involved in innate immunity in the airways.31 Another member of this protein family, BPIFA2, was recently found to be associated with dry mouth and “Sjøgren-­like” symptoms in patients with APS-­1.32 Reactivity to surface receptors is also seen, such as the calcium-­sensing receptor, which can result in hypoparathyroidism.33 KEY POINTS  • Autoimmune polyglandular syndrome type 1 is defined by the presence of two of the three manifestations––adrenal insufficiency, chronic candidiasis, and hypoparathyroidism––or the presence of a disease-­causing variant in the autoimmune regulator gene. Interferon type I autoantibodies are a highly specific diagnostic biomarker. Inheritance is autosomal recessive or dominant; the latter forms are generally milder, with similarities to autoimmune polyglandular syndrome type 2.

Clinical Features Typically, APS-­1 manifests itself in childhood with chronic mucocutaneous candidiasis followed by hypoparathyroidism and adrenal insufficiency.34 However, in recent years we have come to appreciate that the presentation is highly variable, even within the same family.19,35,36 A major explanation is that screening for interferon autoantibodies and the development of next-­generation sequencing have allowed diagnosis of patients with atypical disease, patients with late debut disease, and sometimes patients who are lacking the diagnostic dyad. The prevalence of APS-­1 is approximately 1:100,000 in most countries, and epidemiological surveys are scarce. In some populations with founder mutations, higher prevalence is observed, including Finns (1:25,000), Sardinians (1:14,000), and Persian Jews (1:9000). The natural history is highly variable, with between one to over 20 different components in an individual patient. Some features, such as chronic diarrhea, keratitis, autoimmune hepatitis, periodic rash with fever, or

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PART 11  Multisystem Endocrine Disorders

TABLE 132.2  Characteristics of Autoimmune Polyendocrine Syndrome Type 1 Components

Clinical Features

Diagnosis

Key Autoantigens

Chronic mucocutaneous candidiasis Primary adrenal insufficiency

Chronic recurrent candidiasis of mucous membranes, skin and nails Hyperpigmentation, salt craving, hyponatremia, hyperkalemia Early menopause (7 mmol/L, HbA1c >48 mmol/mol Decreased pituitary hormones

IA-­2

Low vitamin B12, abnormal FBC and iron levels Increased fat in stools Abnormal liver function tests Decreased fecal elastase Peribronchiolar infiltrates on computed tomography, respiratory failure Interstitial nephritis Clinical signs

Intrinsic factor and parietal cell (H+/K+-­ATPase) Tryptophan hydroxylase CYP1A2 and AADC

Tudor domain-­containing protein 6

KCNRG and BPIFB2

Nephritis Vitiligo

Reduced kidney function, proteinuria Ranging from patchy to total nonsegmental distribution

Alopecia

Ranging from patchy to total or universal distribution Ranging from small pits on some teeth to lack of enamel Reduced vision

Clinical signs

Aquaporin-­2, HOXB7, NFAT5 Tyrosinase, tyrosinase-­related proteins, AADC, transcription factors (SOX9, SOX10) Tyrosine hydroxylase

Clinical signs

Protein phosphatase 4

Ophthalmological signs of retinitis

Interphotoreceptor retinoid-­binding protein

Extreme light sensitivity

Clinical signs Clinical signs Clinical signs

Enamel dysplasia Retinitis Keratitis Nail dystrophy Skeletal metaplasia

AADC, Aromatic L-­amino acid decarboxylase; ACTH, adrenocorticotropic hormone; CYP1A2, cytochrome P450 family 1 subfamily A member 2; BPIFB2, bactericidal/permeability-­ increasing fold-­ containing B1FSH protein, follicle-­ stimulating hormone; FBC, full blood count; FSH, follicle-­ stimulating hormone; HbA1c , glycated hemoglobin; HOXB7, human homeobox B7 transcription factor; IA-­2, protein tyrosine phosphatase-­like protein islet antigen-­512; KCNRG, putative potassium channel regulator; LH, luteinizing hormone; MHC, major histocompatibility complex; NALP-­5, NACHT, LRR and PYD domains-­containing protein 5; NFAT5, nuclear factor of activated T-­cell 5 transcription, SOX, SRY-­related HMG-­box; TSH, thyroid-­stimulating hormone.

severe constipation, may dominate the initial picture before any of the major components develop.36 Thus, a low threshold for autoantibody testing or sequencing of AIRE is recommended.

Chronic Mucocutaneous Candidiasis Chronic superficial infection with Candida albicans is usually the first of the major components of APS-­1 to appear.34 It affects mucous membranes, skin, and nails, but never causes deep, systemic Candida infections. In mild forms only angular cheilitis is seen, but in more severe cases the whole mouth is involved, with white or grey plaques of yeast and hyperkeratosis. In the atrophic form the mucosa is erythematous with leukoplakic areas or nodules that may develop into squamous cell carcinomas; this is a significant cause of death, especially if the patient smokes and overuses alcohol.37,38 Candidiasis may also only be confined to the esophagus, causing problems with swallowing caused by overgrowth or strictures, sometimes requiring dilation. A reddish and

watery rash may affect the skin of the hands and face. The fingernails may be affected and females may suffer from genital candidiasis. The diagnosis is based on the signs and symptoms, as well as culture of C. albicans. It is very important to treat oral candidiasis to prevent cancer development, which includes practicing good oral hygiene and refraining from smoking and excessive alcohol. This is also important because the majority of patients develop enamel hypoplasia of the permanent teeth,19 and many have a mild Sjøgren-­like syndrome with reduced saliva production.32 Infrequent bouts of Candida can be effectively treated with azole preparations such as fluconazole and ketoconazole, but with regular use there is a risk of drug resistance. Thus, for recurrent and chronic cases, treatment with nystatin and amphoterizin B in combination is an alternative, with pulsed prophylaxis with either of these drugs; alternatively, chlorhexidine mouthwash can be applied. A biopsy should be taken if mucositis with an ulceration fails to respond to treatment within 2 weeks. Angular cheilitis can be treated

CHAPTER 132  Autoimmune Polyglandular Syndromes with azole, chlorhexidine, or amorolfine creams. Candida esophagitis usually heals with the discussed treatment regimen in 1 to 2 weeks. If not, endoscopy should be considered. Female genital candidal infection usually responds to a short course of vaginal fluconazole.

Hypoparathyroidism Hypoparathyroidism is the second most common major manifestation and can be the first endocrine component. The combination of hypocalcaemia, hyperphosphatemia with normal or low parathyroid hormone, and normal plasma creatinine is diagnostic. Symptoms of hypocalcaemia may be vague in the beginning, with muscle cramps during infections, intermittent mild paraesthesia, and clumsiness. Frank hypocalcemia, sometimes with seizures, can be triggered by a febrile illness and fasting. APS-­1 should be considered in every patient with acquired hypoparathyroidism because it may be the only component in some patients. Half of patients harbor autoantibodies against NALP5,29 and some also have autoantibodies against the calcium-­ sensing receptor.33 Hypoparathyroidism is treated with vitamin D derivatives, calcium, and magnesium. Alphacalcidol (T½ 2 days) and calcitriol (T½ 1 day) are recommended because of their relatively short half-­lives. Calcium supplementation in divided doses of 100 to 500 mg elementary calcium outside meals is suggested, preferably calcium citrate, as it helps calcium stay in solution in the urine. Likewise, a daily supplement of magnesium and cholecalciferol should be given to avoid deficiencies and to cover other functions of vitamin D. Therapy is aimed at maintaining plasma calcium in the lower half or slightly below the normal range (total 2.10–2.30 mmol/L, ionized 1.05–1.15 mmol/L), urinary calcium in the normal range, and plasma magnesium in the upper half of its normal range (0.85–1.00 mmol/L). Hypercalcemia may damage the kidneys, particularly if the patient is not well hydrated. Serum calcium should be checked quarterly and when symptoms indicate hypo-­ or hypercalcemia. Patients with hypoparathyroidism and adrenal insufficiency may develop hypercalcemia as a result of sodium deficiency and/or dehydration. Increase in glucocorticoid dose tends to cause hypocalcaemia, and vice versa. Increased calcium intake or additional alphacalcidol or calcitriol may be required if glucocorticoid doses remain high for more than 24 hours.

Adrenal Insufficiency Adrenocortical insufficiency most commonly appears after mucocutaneous candidiasis and hypoparathyroidism, typically between 5 and 15 years of age, but may appear later. The prevalence in different published cohorts is around 70%.19,37 The characteristic symptoms are fatigue, salt craving, hypotension, weight loss, and an increased pigmentation of the skin and mucous membranes. Biochemically, symptomatic patients display low basal plasma cortisol, elevated adrenocorticotropic hormone (ACTH), low aldosterone, and elevated plasma renin activity. Hyponatremia is common, sometimes in combination with hyperkalemia. Of note, hypocortisolism and hypoaldosteronism may appear dissociated years apart. Just like in the more common form of autoimmune Addison disease, most patients display autoantibodies against 21-­hydroxylase.6,39 In addition, many APS-­1 patients also have antibodies against side-­chain cleavage enzyme.39 Autoantibodies can be present years before biochemical or clinical evidence of adrenal insufficiency and call for intensified follow-­up of adrenal function. Adrenal insufficiency should be diagnosed before a potentially fatal adrenal crisis develops. Replacement therapy with hydrocortisone and fludrocortisone should be individualized to provide optimal well-­being and safety in the patient’s life situation, including incidents of sickness and stress.

2229

All patients should be provided with a steroid acute card; a common European card is available for adults and children. The patient and her/his close family should be equipped with syringes for self-­injection of hydrocortisone. Glucocorticoid treatment influences calcium and glucose levels. Thus, treatment of hypoparathyroidism and diabetes mellitus may have to be adjusted. For details, see Chapters 39 and 52.

Gonadal Insufficiency Primary ovarian insufficiency manifests particularly early, and some patients have primary amenorrhea, with either a complete failure of, or arrested, pubertal development. Others develop premature menopause, with a median age of 16 years.40 Adrenal insufficiency was more common in APS-­1 patients with ovarian insufficiency then those without.40 The biochemical findings are typically elevated plasma follicle-­ stimulating hormone and luteinizing hormone and low estrogen levels. Ovarian insufficiency is associated with presence of autoantibodies against side-­chain cleavage enzyme and NALP5 and are typically present before clinical symptoms develop.39 Estrogen replacement should be started at pubertal age, with gradually increasing doses to maintain a normal pace of female development. Periodic gestagen should be added at the appropriate stage. There are reports of cases where pregnancies have occurred even several years after menopause, but patients should be encouraged not to delay plans if they wish to have a child. Storing of ovarian tissue may be an option for future pregnancies. Testicular insufficiency does occur, albeit more seldom and mostly at an adult age, with a maximum prevalence of approximately 25%, as seen in the Finnish series.39 The blood-­testis barrier may protect the Leydig cells from an autoimmune attack, but lack of AIRE may affect fertility by disrupting scheduled apoptosis of testicular germ cells.41 Recently, male APS-­1 patients were found to harbor autoantibodies against transglutaminase-­4.42 Aire knockout mouse models display severe prostatitis; but so far there are no reports of autoimmune prostatitis in APS-­1.

Other Endocrinopathies The prevalence of diabetes mellitus varies between different populations, from a few percent to one-­third, being more common in countries and populations with high prevalence of T1DM. The presence of autoantibodies against the IA-­2 tyrosine phosphatase-­like protein, but not glutamic acid decarboxylase (GAD), is predictive for the development of diabetes.39 Thyroid autoantibodies are common, and hypothyroidism is relatively common after puberty, reaching a prevalence of one third by middle age.37 Graves disease is very rare. Autoimmune hypophysitis is seen with lack of different pituitary hormones, including growth hormone, which may need replacement to ensue normal growth.35

Gastrointestinal Manifestations Autoimmune gastritis with pernicious anemia is a common entity that affects approximately 30% by middle age,37 associated with autoantibodies to parietal cells and/or blocking intrinsic factor. Vitamin B12 and iron status should be checked, and replacement given if indicated. A definite diagnosis can be made by gastroduodenoscopy and biopsy. Celiac disease has been reported in a few patients43 but is uncommon. Conversely, autoimmune enteropathy is common and manifests with chronic or recurrent diarrhea and steatorrhea, as well as periodic or chronic constipation. Absence of enterochromaffin cells is typical, and patients have autoantibodies against tryptophan hydroxylase, the rate-­ limiting enzyme in serotonin biosynthesis.44 Aberration in serotonin levels could explain changes in intestinal motility. Paneth cells are also affected, and levels of defensins are decreased,45 perhaps explaining the alterations in the microbiome observed in these patients.46 Exocrine

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PART 11  Multisystem Endocrine Disorders

pancreatitis occurs in a small percentage of patients, usually associated with insulin deficiency. Typically, subnormal levels of secretin-­ stimulated lipase are found. Replacement of pancreatic enzymes may improve diarrhea, and immunosuppression has been reported to improve exocrine pancreatic function.47,48 Malabsorption can seriously disturb replacement therapy with steroids, vitamin D preparations, and calcium, and requires close follow-­up. Autoimmune hepatitis is a serious complication that is reported to affect around one fifth of the patients and usually manifests in childhood.37 After careful scrutiny, a recent survey from the National Institutes of Health revealed that approximately 40% of patients are affected by autoimmune hepatitis.49 Severity ranges from slightly elevated plasma alanine amino transferase levels to fulminant hepatitis. High levels should prompt liver biopsy. Autoantibodies against CYP1A2 correlate with the presence and severity of the autoimmune hepatitis.35 CYP2D6, the autoantibody marker for isolated autoimmune hepatitis type 2, is not seen in APS-­1.50 If a biopsy reveals active hepatitis, immunosuppressive therapy with steroids, azathioprine, or other immunosuppressants is indicated.16,49

Ectodermal Manifestations There are several ocular manifestations associated with APS-­1, such as chronic keratitis, dry eye, ptosis, cataract, iridocyclitis, retinal detachment, and optic atrophy. Keratitis appears early and can be the first manifestation, and can lead to blindness if not treated appropriately. Symptoms include intense photophobia, blepharospasm, and reduced lacrimation. The etiology is presumably autoimmune, and treatment consists of immunosuppression with local glucocorticoids, or topical cyclosporin A. Sometimes corneal transplantation is warranted. Retinitis is rarer and linked to the presence of autoantibodies to interphotoreceptor retinoid-­binding protein.51 Alopecia is the most frequent skin manifestation and is usually more severe than in isolated cases. Approximately 40% are affected by middle age. Loss of all hair on the head (alopecia totalis) or even the whole body (alopecia universalis) is not uncommon. It can sometimes be reversible, especially when immunosuppressive therapy is given for other components. Alopecia correlates with the presence of autoantibodies against tyrosine hydroxylase, which is highly expressed in hair follicles.52 Vitiligo is also common, and its prevalence approached one-­third of the Finnish cohort.37 It is of variable extent and distribution, but tends to include most of the skin. The contrast to surrounding normal skin may fade, yet the patients often feel it to be disfiguring on the face and hands. Vitiligo correlates with autoantibodies against transcription factors SOX9 and SOX10.53 Periods with maculopapular, morbilliform, or urticarial rash with fever come early.36,37 Skin biopsies have reveal lymphoplasmacytic vasculitis.43 Punctate nail dystrophy is common, without evidence of Candida infections.34 Enamel hypoplasia of permanent teeth is one of the most common manifestations of APS-­1 and is present in 70% to 80% of patients.19 It can be mild, with transverse grooves or rows of pits alternating with normal enamel. In severe cases, all enamel is hypoplastic. The enamel defects predispose to further damage and caries. Extensive dental repair is often necessary and should be promptly performed. Even this manifestation is probably autoimmune, and recently autoantibodies against protein phosphatase-­4, involved in odontogenesis, have been detected in APS-­1 patients.54

Other Manifestations Aplasia or hypoplasia of the spleen occurs in up to 20% of patients37,55 and renders them susceptible to bacterial infections. Vaccination for Pneumococcus pneumonia, Haemophilus influenzae, and meningococcus

is recommended. Autoimmune hemolytic anemia,37,43 hypoplastic anemia,35,37 and autoimmune thrombocytopenia56 have been reported in a few patients. Autoimmune bronchiolitis is an underappreciated life-­threatening component of APS-­1. Careful pulmonary investigation in a single center revealed that approximately 40% of patients had pneumonitis.57 Lung biopsy revealed interstitial lymphoid infiltrates around bronchioles. Patients display autoantibodies against the BPIFB2 and KCNRG proteins. Beneficial effects of steroid and rituximab treatment have been reported. Reversible metaphyseal dysplasia with growth retardation and severe progressive myopathy have been observed in isolated cases.

Natural Course and Mortality The overall mortality is increased,58 but the course of the disease varies widely. Some patients have very mild disease and are not diagnosed before adult age, whereas others develop multiple and severe manifestations early on. Life expectancy depends on the severity of the disease, the quality of management, including psychosocial support, and the ability of the patient and family to assume responsibility. There is an increased risk of death from oral/esophageal squamous cell carcinoma, acute adrenal and hypocalcemic crises, complications such as pneumonitis, hepatitis, and renal failure, and accidents.19,35,37,57,59 A recent Finnish survey revealed that standard mortality rates were increased approximately ten times, more so for younger age groups. The cumulative mortality rate up to 60 years of age was over 80%, compared with less than 10% for the general population.59

Diagnosis and Management The traditional diagnostic criterion of the diagnostic dyad remains valid34 (Table 132.2), but leaves many cases unrecognized. Some patients never develop more than one of these components, or the components manifest at a late stage. Initial presenting components can be chronic diarrhea, keratitis, periodic rash with fever, severe constipation, autoimmune hepatitis, pneumonitis, alopecia, or vitiligo34,37 Any of these “minor” components in a child should alert clinicians to look for other manifestations and check autoantibodies directed against interferon-­omega or -­alpha, which are positive in almost all patients. We recommend securing the diagnosis by sequencing AIRE. Every patient should, in addition to a local doctor’s follow-­up, be seen by appropriate specialists, particularly an endocrinologist and an oral specialist, at least once annually, and know to contact them immediately when new symptoms appear. Ideally, the follow-­up should be sufficiently centralized so that the responsible specialists have adequate experience with several APS-­1 patients. The goal is to safeguard the patient’s physical and psychosocial well-­being, as well as recognize and treat components at an early stage. Patient education to avoid and treat acute complications such as adrenal and hypocalcemic crises is important. All patients should be equipped with an acute care card or bracelet and steroids for self-­injections and be well informed about components that might develop in the future. KEY POINTS  • Autoimmune polyglandular syndrome type 1 often develops in childhood. Manifestations other than the main can appear first, such as intermittent fever with rash, hepatitis, intestinal malabsorption, keratitis, enamel hypoplasia, alopecia, vitiligo, and premature ovarian insufficiency. Treatment is replacement of missing hormones, antifungal treatment, and immunosuppression for some components. Mortality is increased because of adrenal insufficiency, hypocalcemic crises, complications owing to organ-­specific autoimmunity (liver, lungs), and carcinoma of the mouth and esophagus.

CHAPTER 132  Autoimmune Polyglandular Syndromes

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TABLE 132.3  Immune Dysregulation Polyendocrinopathy Enteropathy X-­Linked and Immune

Dysregulation Polyendocrinopathy Enteropathy X-­Linked-­like Syndromes Gene

Manifestations

Pathway

Inheritance

FOXP3

IPEX––T1DM, enteropathy, eczema, cytopenias, nephritis, autoimmune hypothyroidism, recurrent infections Type 1 diabetes, thyroiditis, enteropathy, eczema, cytopenias, recurrent infections Enteropathy, hemolytic anemia, pneumonitis, lymphadenopathy, and hypogammaglobulinemia, autoimmune thyroid disease Enteropathy, hemolytic anemia, splenomegaly, T1DM CMC, thyroiditis, T1DM, eczema, enteropathy T1DM, enteropathy, celiac disease, autoimmune thyroid disease, hemolytic anemia, autoimmune thrombocytopenia Growth hormone insensitivity, thyroiditis, enteropathy, eczema, cytopenias, recurrent infections Developmental delay, dysmorphic features, autoimmune hypothyroidism, hepatitis, enteropathy Enteritis, pneumonitis, infections

Defective IL-­2 signaling and Treg function

X-­linked

Defective IL-­2 signaling and Treg function

Autosomal recessive

Defective CTLA-­4 function in Tregs

Autosomal dominant, haploinsufficiency, dominant negative effect Autosomal recessive

CD25 (IL2R) CTLA4 LRBA STAT1 STAT3 STAT5b ITCH BACH2

Defective CTLA-­4 cycling in the cell increases lysosomal degradation Inhibited Th17 cell differentiation Increased Th17 cells, reduced Foxp3+ Tregs Defective IL-­2 signaling pathway

Autosomal dominant, gain of function Autosomal dominant, gain of function Autosomal recessive

Reduced TGF-­β–mediated Foxp3 expres- Autosomal recessive sion Defective immunoglobulin class switching, Autosomal dominant, haplo-­ lymphocyte maturation defect insufficiency

CMC, Chronic mucocutaneous candidiasis; CTLA-­4, cytotoxic T lymphocyte antigen-­4; Foxp3, forkhead box P3; IL, interleukin; IPEX, immumodysregulation polyendocrinopathy enteropathy X-­linked; T1DM, type 1 diabetes mellitus; TGF, transforming growth factor; Th, T helper; Treg, T regulatory cell.

IMMUMODYSREGULATION POLYENDOCRINOPATHY ENTEROPATHY X-­LINKED Immumodysregulation polyendocrinopathy enteropathy X-­ linked (IPEX; OMIM 304790) is an exceedingly rare syndrome characterized by early-­onset T1DM, autoimmune enteropathy with diarrhea and malabsorption, and dermatitis.60,61 Eosinophilia and elevated IgE levels are common, and some patients develop renal disease with membranous glomerulonephritis or interstitial nephritis. Other manifestations are alopecia, autoimmune thyroiditis, hepatitis, exocrine pancreatitis, and different autoimmune cytopenias.62 Many of the components overlap with APS-­1 but in general develop much earlier. IPEX is caused by mutations in the FOXP3 gene, located on the X chromosome, which is a key transcription factor needed for Treg development. IPEX patients develop a number of autoantibodies, some of which overlap with those found in APS-­1. Examples are glutamic acid decarboxylase and islet cell antibodies associated with T1DM. Many develop autoantibodies against harmonin and villin,63 proteins involved in anchoring intestinal villi, also expressed in the proximal tubules of the kidney. Intriguingly, they also display autoantibodies against interferon-­ omega and a number of nuclear receptors, including HNFA1, mutations of which cause maturity-­onset diabetes of the young.64 High-­ throughput DNA sequencing technology has uncovered other unique monogenic syndromes with endocrine manifestations. Examples are loss-­of-­function mutations in genes such as CD25, which encodes the receptor for IL-­2, as well as STAT5b,65 ITCH,66 BACH2,67 CTLA-­4, and LRBA, and gain-­of-­function mutations in STAT168 and STAT3,69 all of which affect Treg function (Table 132.3). KEY POINTS  • Immumodysregulation polyendocrinopathy enteropathy X-­linked is caused by mutations in the forkhead box protein 3 gene and lack of functional T regulatory cells. The disease starts early, often in infancy, with components such as type 1 diabetes, enteritis, and dermatitis. Eosinophilia, elevated IgE, and autoantibodies against villin and harmonin are common. Bone marrow transplantation is often needed.

AUTOIMMUNE POLYGLANDULAR SYNDROME TYPE 2 Definition As originally described, Schmidt syndrome comprises the occurrence of Addison disease together with autoimmune thyroid disease and/ or T1DM and has been regarded as synonymous with APS-­2 (OMIM 269200). Addison disease was the index disorder in originally separating APS-­1 from APS-­2 and has been regarded as an essential component.7 A broader definition has since developed, with the description of multiple autoimmune diseases in patients with autoimmune thyroid disease, T1DM, or Addison disease. Attempts to subdivide patients have been made, such as those with “incomplete” forms of APS-­2 or additional types like APS-­3 (autoimmune thyroid disease plus another organ-­specific autoimmune disease excluding Addison disease) and APS-­4 (Addison disease plus another organ-­specific autoimmune disease excluding autoimmune thyroid disease and T1DM), but no classification has been universally adopted. The picture is rendered still more complex by the existence of subclinical autoimmune disease and the temporal evolution of the various components, and the past division of autoimmune diseases into organ-­specific or non–organ-­specific is artificial. In our view, the term APS-­2 should be reserved for the occurrence of at least two autoimmune endocrinopathies––Addison disease, autoimmune thyroid disease, T1DM––in the same patient.16 There is an increased risk of developing another autoimmune disease with any of these components in isolation, and so APS-­2 represents a dense and distinctive clustering of autoimmunity within a wider set of disease associations. The key features are summarized in Table 132.4.

Pathogenesis APS-­2 is primarily caused by genetic polymorphisms controlling antigen presentation and immunoregulation, which are associated with susceptibility to multiple autoimmune diseases. Tissue-­specific gene polymorphisms, genetic heterogeneity in susceptibility to the underlying components, and environmental factors, which are also associated with these diseases, determine the exact pattern of autoimmunity in an individual patient. Although APS-­2 is often multigenerational, the

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PART 11  Multisystem Endocrine Disorders

TABLE 132.4  Characteristics of Autoimmune Polyendocrine Syndrome Type 2 Components

Clinical Features

Diagnosis

Autoantigens

Primary adrenal insufficiency

Type 1 diabetes mellitus

Hyperglycemia, lack of insulin

Primary ovarian insufficiency

Early menopause (7 mmol/L, HbA1c >48 mmol/mol Elevated FSH and LH, low estrogen

21-­hydroxylase

Autoimmune thyroid disease

Hyperpigmentation, salt craving, hyponatremia, hyperkalemia Hypothyroidism and Graves disease

Autoimmune gastritis type A on biopsy

Intrinsic factor and parietal cell (H+/ K+-­ATPase) Transglutaminase-­2

Gastritis Celiac disease Exocrine pancreatitis, Vitiligo Alopecia Others (95% of multiple endocrine neoplasia type 1 patients and is the first manifestation of disease in the majority of patients. Most centers recommend subtotal parathyroidectomy of at least three and a half glands because typically there is synchronous or asynchronous involvement of all four parathyroid glands. Cinacalcet may be used for those in whom surgery has failed or is contraindicated.

Pancreatic Tumors The incidence of clinically apparent pancreatic NETs in patients with MEN1 varies from 30% to 80% in different series, although microscopic islet tumors are found in almost all MEN1 patients. Malignant pancreatic NETs remain the leading cause of premature death in MEN1 patients.15-­17,30,31 MEN1-­ associated pancreatic NETs (Table 133.1) may produce excessive amounts of hormone (e.g., gastrin, insulin, glucagon, or vasoactive intestinal polypeptide [VIP]) and are associated with distinct clinical syndromes, although some (e.g., those secreting pancreatic polypeptide [PP]) may not be associated with clinical manifestations or may be nonsecretory (i.e., nonfunctioning) (see Fig. 133.1).1 Indeed, nonfunctioning NETs are now reported to be the most common pancreatic NET in MEN1.4,15-­17,31,32 MEN1 patients may have

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more than one synchronous pancreatic NET, and it cannot be assumed that tumor visualization on imaging studies correlates with the site of hormone excess in functioning tumor syndromes (e.g., gastrinoma, insulinoma).1,16 The goal of treatment of MEN1 pancreatic NETs is to reduce the morbidity and mortality associated with these tumors, which may be associated with hormone hypersecretion (e.g., gastrin, insulin) and/or their malignant behavior (i.e., development of metastases).31 Surgery is generally recommended for functioning pancreatic NETs associated with localized disease (i.e., within the pancreas), as it offers a potentially curative approach.1,4,17 For nonfunctioning pancreatic NETs, surgery is typically reserved for localized tumors greater than 2 cm, as the risk of metastasis and death is observed to increase significantly above this size.1,4,15-­17,33 Indeed, when evaluated as a single group, the risk of metastases in MEN1 duodenopancreatic NETs is associated with size greater than 2cm and age greater than 40 years, and surprisingly not with hypersecretion (or nonsecretion) of hormones.34

Gastrinoma. Zollinger and Ellison initially described two patients in whom non–β islet cell tumors of the pancreas were associated with recurrent peptic ulceration and marked gastric acid production, and gastrin was subsequently extracted from such tumors. The association of recurrent peptic ulceration, marked gastric acid production, and non–β islet cell tumors of the pancreas is referred to as the Zollinger– Ellison syndrome (ZES).1 ZES patients may also suffer from esophageal symptoms (e.g., related to esophageal stricture, or Barrett’s esophagus), as well as diarrhea and steatorrhea. The diagnosis is established by demonstration of a raised fasting serum gastrin concentration in association with increased basal gastric acid secretion.1 Occasionally, intravenous provocative tests with either secretin or calcium infusion are required to distinguish patients with ZES from other causes of hypergastrinemia, which include achlorhydria, antral G-­cell hyperplasia, Helicobacter pylori infection, renal failure, hypercalcemia, and use of proton pump inhibitors (PPIs).1 Investigations that may help to localize the gastrinomas include ultrasonography, endoscopic ultrasonography (EUS), CT, MRI, selective abdominal angiography, or somatostatin receptor scintigraphy (SRS).1 In addition, the combined use of intraarterial calcium injections with hepatic venous gastrin sampling has been shown to regionalize the gastrinomas, while the selective arterial secretagogue injection test may also help localize the tumor.1 However, MEN1-­ associated gastrinomas frequently occur with concomitant pancreatic NETs, most frequently nonfunctioning tumors, and this failure to distinguish between tumor types may have confounded earlier studies. Thus, it is likely that in many MEN1 patients with ZES, large (>2 cm) pancreatic tumors that were assumed to be gastrinoma were instead nonfunctioning NETs, and that the gastrinomas, which occurred within the duodenal wall, were unrecognized.35 Moreover, in patients with MEN1, ZES does not appear to occur in the absence of primary hyperparathyroidism, and hypergastrinemia has also been reported to be associated with hypercalcemia. The successful treatment of the primary hyperparathyroidism with restoration of normocalcemia may ameliorate the clinical symptoms and biochemical abnormalities in approximately 20% of patients.1 Thus, the diagnosis of ZES may be difficult in some MEN1 patients. Gastrin-­secreting tumors (gastrinomas) are reported to occur in 20% to 60% of MEN1 patients (see Fig. 133.1), and approximately 20% of patients with gastrinomas will have MEN1.1,22 The majority of MEN1-­associated gastrinomas occur as multiple small tumors (20 times the upper limit of normal), a pancreatic NET greater than 2 cm, synchronous liver metastases, multiple concurrent NETs, and gastroduodenoscopy suspicious of gastric NET, each contributing to the reduced survival.18 KEY POINTS  • Gastrinomas occur in 20% to 60% of MEN1 patients, and when untreated are associated with the Zollinger–Ellison syndrome and increased mortality. Insulinomas occur in 10% to 30% of MEN1 patients, typically presenting at under 40 years of age, and are the first manifestation of disease in 10% of MEN1 patients.

KEY POINTS  • Clinically apparent nonfunctioning pancreatic neuroendocrine tumors occur in 15% to 55% of patients and are the leading cause of premature death in MEN1. The risk of malignancy is partially dependent on tumor size, and most centers recommend surgery for tumors over 2 cm, although some advocate lower thresholds.

Treatment. The aims of treatment are to control symptoms and/or sequelae associated with hypergastrinemia (e.g., ZES) and to reduce the mortality associated with the development of metastatic disease.39 However, the inability to accurately predict disease course makes decisions regarding the optimal treatment challenging, with some centers recommending medical treatment and others favoring early surgical intervention.36,39 Medical treatment of MEN1 patients with ZES is directed toward reducing basal acid output to less than 10 mmol/L, and this may be achieved by high-­dose parietal cell H+/K+-­adenosine triphosphatase (ATPase) inhibitors (i.e., PPI therapy including omeprazole or lansoprazole). Some patients may also require additional treatment with the histamine H2 receptor antagonists cimetidine or ranitidine.1 The introduction of acid-­suppressive therapies has markedly reduced the morbidity and associated mortality from the direct effects of hypergastrinemia, such that the overall prognosis of gastrinoma in MEN1 patients is now generally favorable, with 5-­, 10-­, and 20-­year survival rates of approximately 80% to 95%, approximately 65% to 95%, and approximately 60% to 90%, respectively,18,36 However, the effects of medical treatment on disease course (i.e., tumor growth, risk of metastases) and long-­term consequences with continuous use for greater than 10 to 20 years are not known. The value of somatostatin analogues (SSAs), which have been used in a limited number of patients for the treatment of MEN1-­associated gastrinoma, remains uncertain. The benefits of surgery for the majority of MEN1 patients with multiple duodenal gastrinomas are not established. For example, extensive duodenal resection aiming to remove all gastrinomas and thereby achieve persistent postoperative biochemical remission (i.e., eugastrinemia) is rarely possible, and duodenal-­preserving procedures

are associated with persistent or recurrent hypergastrinemia in 60% to 100% of patients.1,17,35 However, some studies have reported improved remission rates of 75% to 100%, with short-­to medium-­term follow up,35 following extensive surgical approaches including: duodenotomy with local removal of tumors in the duodenal wall, coupled with enucleation/resection of tumors in the pancreatic head and peripancreatic lymph node removal; partial pancreaticoduodenectomy; and pancreas-­preserving duodenectomy.1,35,36 Whipple pancreatoduodenectomy has been reported to achieve a greater than 65% cure rate but is associated with a higher operative mortality and long-­term complications, which include weight loss, diabetes mellitus, and malabsorption, such that it is reserved for patients with large tumors, and total duodenopancreatectomy is very rarely performed because of the high risk of long-­term sequelae (i.e., brittle insulin-­dependent diabetes).1 Laparoscopic surgery is not recommended, while total gastrectomy is rarely undertaken and only employed in very limited situations (e.g., a persistently noncompliant patient). Currently, most centers undertake nonsurgical management for the majority of MEN1 patients with duodenal gastrinomas, although some specialized centers with experience in preoperative and intraoperative imaging (e.g., transillumination of the duodenum) and regionalization advocate surgery for gastrinoma early in the disease course.1 However, surgery is recommended for the rare patients (90

Per- and Polyfluoroalkyl Substances (PFAS)a

∼100

Polybrominated Diphenyl Ethers (PBDEs)b

68–100

Polychlorinated Biphenyls (PCBs)c

>95

1,1-Dichloro-2,2-Bis(p-Chlorophenyl) Ethylene (DDE)

100

BPA

>95

Triclosan

>85

Parabens

>90

Phthalatesd

100

Perchlorate

100

aPerfluorinated

Source of Contamination

Effects

Degradation of lead-­based paint and the contaminated dust that it forms. Diet is a significant source of blood mercury, especially in some seafood species. Some manufacturing can produce atmospheric mercury that enters the air and ultimately the food chain. Used to repel oil and water from clothing, furniture, and food packaging such as pizza boxes and fast-­food containers. Firefighting foams, cleaners, some paints, roof treatments, and hardwood floor products. Persist and bioaccumulate in environment and have long half-­lives in humans. Used as flame retardants in foam, furniture, electronic equipment, and high-­impact plastics. There are many individual congeners that have different exposure profiles in the population. Prevalent in the built environment, especially in dust. Persist and bioaccumulate in environment and have long half-­lives in humans. Production was banned in the 1970s, but they are still used in a number of commercial and personal applications. They were used as dielectric fluids, wood treatments, printing ink, and in many other products. Because of their persistence and ability to bioaccumulate, they are still widely present in the environment. Metabolite of the insecticide dicloro-diphenyl-trichloroethane (DDT). Because of previous extensive and wide use, and because of its relatively long environmental half-­life, DDE is ubiquitous in human samples. Can linings, epoxy resins, some plastics, thermal receipts, others

Effects on brain development and IQ. Multiple endocrine and toxic effects. Effect on brain development, learning, and memory. Effects on thyroid and other endocrine and toxic effects.

Antimicrobial soaps and other products, clothing, kitchenware, body washes, and some cosmetics. These are esters of parahydroxybenzoic acid. Present as preservative in many consumer products including nail polish, hair spray, lipstick, fragrance, body wash, sunscreen, etc. Different phthalates are used in different applications including personal care products, PVC plastics, some flooring, car products, insect repellents, etc.

Widely present in foods including fruits and vegetables, beer and wine, and other products. It is FDA approved for use in food packaging.

Many different congeners. Linked to thyroid disease as well as several cancers.

Many different congeners. Some interact with estrogen, androgen, and thyroid hormone actions. Metabolites may be most important.

Many different congeners. Some interact with estrogen, androgen, and the actions of thyroid hormones. Metabolites may be most important.

Interacts with estrogen signaling. Associated with hormone-­related cancers and precocious puberty. Interacts with estrogen receptor and at higher doses also interacts with the androgen and thyroid hormone receptor. Linked to a variety of chronic diseases including obesity, cardiovascular disease, and some cancers. Appears to interact with thyroid hormone through several mechanisms. Class of chemicals with estrogenic activity.

Can interact with androgen and thyroid hormone systems. Associated with lack of masculinization in boys including reduced anogenital distance and lower sperm count in men. Blocks iodide uptake into the thyroid and reduces serum thyroid hormone levels in animals. Linked to lower thyroid hormone levels in some women, and to negative cognitive impacts on children.

chemicals including perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). diphenyl ethers. Many different congeners based on bromine number and placement. cPolychlorinated biphenyls. Many different congeners based on chlorine number and placement. dThere are several variations of chemical structure for phthalates. BPA, bisphenol A; FDA, Food and Drug Administration; PCBs, Polychlorinated biphenyls. Data are derived in part from Woodruff TJ, Zota AR, Schwartz JM. Environmental chemicals in pregnant women in the United States: NHANES 2003-­2004. Environ Health Perspect. 2011;119(6):878–885 and from Calafat AM, Ye X, Wong LY, Bishop AM, Needham LL. Urinary concentrations of four parabens in the U.S. population: NHANES 2005-­2006. Environ Health Perspect. 2010;118(5):679–685. bPolybrominated

CHAPTER 137  Endocrine-­Disrupting Chemicals and Human Health 1899, just a few years after they were first synthesized in the laboratory, a condition called chloracne (a painful skin disease) was noted in PCB production workers. In 1936, workers in a production plant also were affected by chloracne and liver disease, and some subsequently died. Animal studies showed that high doses of PCBs can cause liver damage. In the late 1960s, it was reported that PCBs were found at high concentrations in Baltic Sea animals, and, in 1968, a human population was accidently exposed to high levels of PCBs as a result of rice-­oil contamination in Japan.52 This was followed by studies showing that PCBs were found throughout the environment and were bioaccumulating in a variety of animals including fish. In 1972 Sweden banned the use of PCBs in situations where environmental contamination was likely. In the United States, Congress banned all manufacturing, processing, and distribution of PCBs in 1979. That same year there was another serious accident in which PCBs contaminated rice oil, this time in Taiwan (using the same manufacturing equipment that produced the accident in Japan).53 Until the 1990s, the general theory was that PCBs were toxic because they interact with the aryl hydrocarbon receptor (AhR),54 thereby acting like dioxin. Of the 209 different PCB congeners, some are “dioxin-­like”, and some are not. This concept led to categorizing PCB mixtures in terms of their “toxic equivalence” (TEQ)55; that is, their ability to exert dioxin-­like effects. However, in 1990, there was a report in nonhuman primates and in cell culture that nondioxin-­like PCB congeners could affect dopamine levels in specific brain areas.56. This spawned research focused on congener-­specific effects of PCBs on health outcomes including cognitive function. It is now well known that background exposure to PCBs in pregnant women is associated with lower IQ as well as other cognitive deficits in their offspring.29 Moreover, these studies were done 20 years or more after the ban on PCB production, demonstrating that these environmentally persistent chemicals have long-­lasting impacts on human health. Today the Food and Drug Administration (FDA) considers PCB contamination in food to be “unavoidable” and set limits in 2010 for PCBs in various foods. For example, the acceptable limit of total PCBs in milk or cheese is 1.5 mg/kg (fat, not volume).57 Considering that the half-­life of PCBs in the human body is measured in years,58 these FDA limits do not appear to be health protective. Different PCB congeners appear to interact with different systems to impact brain development. For example, some PCB congeners can be hydroxylated by the P450 enzyme CYP1A1 and interact with the thyroid hormone (TH) receptor.59,60 There are other PCB congeners that can displace T4 from the serum transport protein, transthyretin.61,62 Still others can activate liver enzymes that increase T4 clearance from serum.63 Finally, some PCB congeners can interact with the ryanodine receptor, an important component of calcium signaling.64 Again, an important lesson is that early recognition of toxicity was related to very high exposures and acute symptoms including death. However, the assumption that toxicity was limited to high exposure levels was again mistaken, and this caused a delay in the recognition of more widespread toxicity. We now have very good mechanistic data demonstrating that specific PCB molecules (congeners) can directly interact with, for example, the thyroid system at low concentrations.65 In addition, other PCB congeners can interact directly with the ryanodine receptor to influence intracellular calcium signaling at very low concentrations.64 Both of these pathways are important for brain development and may well help explain much of PCB’s effects on cognitive function.

The Lessons of Diethylstilbestrol (DES) DES is a relatively potent nonsteroidal estrogen that was prescribed to pregnant women from the 1940s to the 1970s ostensibly to prevent

2309

miscarriages based on the belief that spontaneous abortion was the result of a decrease in estrogen levels (now shown to be a consequence, not a cause). DES was marketed using more than 200 names, and during that time tens of millions of women and their offspring worldwide were exposed to this drug. The first indication of a problem occurred when DES was linked to a rare form of vaginal cancer, vaginal clear cell adenocarcinoma, in a small number of young women exposed in utero.66 It was subsequently shown to be associated with a number of reproductive problems in the DES-­exposed daughters including increased uterine fibroids, endometriosis, breast cancer, miscarriage, preterm delivery, and ectopic pregnancies later in life, as well as reproductive tract malformations.66 Moreover, data have demonstrated abnormalities in adult male offspring of women taking DES during pregnancy.67–69 A critical lesson from the DES experience—and one that could not have been gained without formal records of exposure and the astute perception of a physician—was that exposure to a synthetic estrogen during fetal development could increase the risk of various adverse effects in the adult offspring, decades after the chemical has disappeared from the body. We know from many population-­based endocrine studies that developmental hormone exposure can have lifelong impacts. For example, serum T4 levels at age 2 are associated with verbal IQ in adulthood.70,71 Thus, the findings of DES exposure during fetal development are consistent with our understanding of the impacts of various hormones on development and the manifestation in adulthood of developmental endocrine interference. The effects of DES exposure shown in humans have been reproduced in animal studies; in fact, some effects were first observed in rodent studies and were then discovered in humans. Thus, DES is an example of an estrogenic agent that produces adverse effects in the human population. Not only do these studies demonstrate a proof of principle for developmental exposures to estrogenic compounds leading to birth defects, but they also demonstrate the temporal dissociation between exposure and manifestation of effects— the so-­called “developmental origins of health and disease” (DOHaD).72 An important lesson common among the three examples is that the risk of adverse outcomes of chemical exposures was initially underestimated in each example. This is also a common feature in a broader analysis of human health effects of chemical exposures.73,74 This failure to recognize the risk is due in part to a reliance on insensitive measures of chemical effects. In the case of lead and PCB exposures, there was reliance on measures of acute toxicity. In the example of DES, it was a reliance on measures of adverse outcome in the mother, not in the fetus. Therefore, as independent research progressed, more sensitive measures of adverse outcome were implemented. In the case of DES, the recognition that fetal exposure could lead to adverse outcome 20 years later was a novel and unexpected insight. These are not just lessons of academic interest but are chronicles of avoidable harm to fellow humans on a population level and over generations. KEY POINTS  • We have highlighted the history of 3 chemicals that illustrate important features of EDCs: Lead, Polychlorinated Biphenyls (PCBs), and Diethylstilbestrol (DES). In each case, the risk to human health was dramatically underestimated because the outcome measures evaluated were insensitive. Generations of people have been damaged by these mistakes.

Windows of Susceptibility Hormones are well known to produce effects on development during very specific developmental times (“windows”). For example, the “critical period” of TH action was initially viewed as the early postnatal

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period.75 However, it is now recognized that there are different critical periods of TH action for different development events and cognitive domains.76,77 Likewise, testosterone acts during specific times to affect male phenotypic development,78 which is very different from the effects of testosterone in the adult.79 Moreover, the effect of androgens in adulthood can depend on the action of testosterone during development.80 Considering this background of information about critical periods of hormone actions on different organ systems, it is not surprising that EDCs would be found to have different effects when exposures are studied at different life stages. Awareness of the developmental effects of chemical exposures has been heightened by studies of DES exposures (above) and of fetal alcohol exposure.81 Focus on EDC action has been, for the most part, limited to specific windows of sensitivity including in utero exposures, neonatal exposures, and adult exposures. It is not clear what mechanisms account for a sensitive window of exposure to EDCs and thus how many sensitive time points there might be across the lifespan, largely because this issue is not well understood for hormones either. Nonetheless it is likely that preconception, preimplantation, early development, puberty, aging, as well as pregnancy itself are particularly sensitive times for the adverse effects of environmental exposures. Thus, it is clear that an approach is needed that includes not only a lifespan approach but also examination of effects across generations. There are now multiple examples of EDC effects that are heritable across not just one generation but also two, three, and four generations.82,83 Thus exposures during pregnancy may lead to increased disease susceptibility that may last for generations. Everyone is exposed across their lifespan starting with fertilization. Actual exposures depend on where one lives and one’s lifestyle, such that exposures within and between chemicals can be quite variable. However, it is not just the level of exposures that are key to causing health concerns but perhaps more importantly when the exposure occurs. It is now absolutely clear that in utero and/or early in life, when tissues are forming, they are very sensitive to the adverse health effects of EDC exposures leading to an increase in noncommunicable diseases. There are two reasons for this sensitivity to the action of EDCs. First, development is a highly sensitive and orchestrated process controlled by hormones and growth factors. They act by controlling gene expression to stimulate the development of specific tissues. Thus, any EDC that interferes with endogenous hormone signaling will upset the normal control of gene expression leading to the development of a tissue that may “look” normal but that will not function normally. This phenomenon, first observed in the nutrition field, is now guiding research in the environmental chemical field and is called the DOHaD.72 The second reason for this sensitivity to EDCs is that the enzymes that metabolize chemicals are not fully developed so the EDCs are active for longer than “normal times” along with a lack of DNA repair and a lack of blood/brain/organ barriers.84 Because tissues develop at different times, the effects of EDCs on tissue development will depend on tissue specificity and timing of their development. Data in support of this DOHaD concept are sufficiently robust and repeatable across species to support incorporation into clinical practice.85 The difficulty arises in that the exposure in utero may not result in increased disease until years or decades later, making it difficult to link the exposure to the disease. Thus, the clinical focus needs to be on reducing the risk of adult disease by limiting exposures before and during pregnancy and early childhood. Exposures to EDCs occur across the lifespan. Adult exposures to EDCs can also cause health effects, but those effects typically remain only while the exposure exists; for example, the effects are reversible, and they may require higher concentrations due to the fact that the tissues are fully formed.

Integrating Mechanistic Information to Interpret Exposures and Health Outcomes Mechanistic information from experiments using animal models or cell-­based/biochemical studies provide important information to support epidemiologic data linking chemical exposures to human health outcomes at both the individual and population level. This body of research is highly multidisciplinary—from cell-­ free and cell-­ based experiments, to animal models across most vertebrate classes and some invertebrates, including the use of various genetic strains of animals. This of course requires high-­quality research in these various domains and this level of information is not available for a large number of chemicals. A good example is BPA for which there are well over 1000 publiations.86 Mechanistic research shows that exogenous chemicals can interact with biological systems much the way drugs do—by interacting with hormone receptors, biosynthetic enzyme systems, catabolic and metabolic pathways, and with various transport processes.23 The regulation of hormone action is highly complex, and the consequences of interfering with hormone action are likewise highly complex. Mechanistic research since the mid-­1990s has provided insight into the many ways in which chemicals can and do interfere with hormone action. We present here examples that provide some insight into these issues.

MECHANISMS OF EDC ACTIONS Chemicals can interfere with hormones anywhere along the chain of events that control hormone action, from synthesis, release, and transport of the hormone to hormone effects on receptor function or on hormone degradation and elimination (metabolism).34 In general, this means that chemicals can either interfere with the delivery of the hormone to and from its target tissues and cells or interfere with the action of the hormone on the receptor—or both. There are situations in which EDCs can interfere with the delivery of some hormones to specific target cells; in this case the effects are tissue-­specific. Likewise, there are known cases in which EDCs must be metabolized before they can act on a hormone receptor, and this metabolism occurs only in certain tissues.59,87 Thus, there are few rules about EDC actions that can be generalized to all cells and all organs of the body and all EDCs. Hormone receptors have a high affinity for their natural ligand, but they often have a much lower affinity for endocrine disruptors—with some exceptions. However, it is important not to confuse affinity (ability to bind) for the receptor with potency (dose at which a given effect occurs) or efficacy (maximum effect). It is clear from clinical chemistry that differences in affinity do not always translate to differences in potency or efficacy. Recently, for example, it was shown that mutations in the gene coding for the estrogen receptor alpha (ESR1) that are associated with estrogen insensitivity in patients alter the effects of ligand binding without affecting affinity.88 This can be true for exogenous ligands. For example, the endocrine disruptor BPA has the same potency as 17β-­estradiol in pancreatic beta cells,89 although its affinity for endoplasmic reticulum the estrogen receptor alpha (ERalpha) is much lower compared to that of 17β-­estradiol. The way in which BPA exerts the same effect on the ER as the natural estrogen in one cell type is not known, but it should be emphasized that affinity measurements usually are made in completely artificial circumstances that do not reflect the environment of the cell. Moreover, these studies also emphasize that endocrine disruptors can act on a hormone receptor in one tissue type without affecting the receptor in another tissue type. Thus, just like the hormones they mimic or disrupt, the effects of endocrine disruptors will be receptor, tissue, and coregulator specific. Some endocrine disruptors

CHAPTER 137  Endocrine-­Disrupting Chemicals and Human Health actually have an affinity that is similar to or greater than that of the natural ligands. An example of this is tributyltin (TBT), which has an affinity for the nuclear proteins, Retinoid-X-Receptor (RXR) and Peroxisome Proliferator-Activated Receptor gamma (PPAR(gamma symbol)) in the low nanomolar range. Indeed, it is the most potent agonist known for these receptors.90 See Table 137.2 for a list of examples of EDCs and how they interact with hormone systems.

DOSE-­RESPONSE CHARACTERISTICS OF EDCS A significant challenge in the area of EDCs is to understand fully the dose-­ response characteristics of EDCs on various elements of hormone action in a manner that provides useful insight into the potential for human health effects. Within this context, the concepts of homeostasis and compensation need to be considered carefully. The term “homeostasis” refers to the property of a dynamic system that maintains “internal constancy.” The term “compensation” is related in that when a homeostatic system is perturbed, various elements within the system are activated or inhibited to compensate for the perturbation. For example, when glucose levels rise, insulin is secreted and glucagon secretion is inhibited such that glucose levels fall again. But homeostatic systems can reset in the face of chronic perturbation, as illustrated by baroreceptor resetting in the face of even short-­term changes in mean arterial pressure.91 Thus, in the face of chronic (lifetime) exposure to low doses of environmental chemicals, we need to be cautious about using the concepts of homeostasis and compensation when no studies have been designed to inform us about whether—or how—homeostatic endocrine systems may change in the face of these chronic exposures. These issues are particularly important when considering the potential adverse effects of low-­dose exposure to the fetus compared to the adult. Fetal development is a time when disruption of hormone action may not be reversible because early developmental events become the substrate for subsequent events (see “Windows of Susceptibility”). In addition, the fetus may have fewer homeostatic and compensatory mechanisms to ameliorate effects of EDCs. Finally, a variety of hormone receptors are much more abundant during development than in the adult, and this may also serve to make the fetus much more sensitive to chemical interference.92 Thus, adults may be both less sensitive to EDC actions and more able to compensate—if homeostatic set points do not shift with chronic lifetime exposure. Considering this, it is important to recognize that the field of “endocrine disruption” is focused on a novel area within the established fields of both endocrinology and toxicology, and the dose-­ response characteristics observed in short-­term, high-­dose toxicity studies may bear little resemblance to the dose-­response characteristics in real life. Moreover, because endogenous hormones exhibit nonlinear dose responses, it is expected that environmental chemicals interacting with hormone systems will also exhibit nonlinear responses. These nonlinear dose responses can take several forms.13 In its simplest form, because hormones act on receptors, which are limited in number, the response itself is “saturable.” That means that there is a dose of hormone—or endocrine disruptor—beyond which there is no further response on a measured outcome. Hormones and EDCs can also produce nonmonotonic dose responses. Technically, the term “nonmonotonic” simply means that the slope of the curve changes sign (positive to negative or vice versa) over the dose-­response range.93 For example, when fetal mice are exposed to low or high doses of DES, a synthetic estrogen, their adult prostate weights are relatively low. However, intermediate doses of DES produced significantly heavier prostates.94 Research also suggests that nonmonotonic responses can be extended to the population level. For example, individuals in the highest quartile of environmental exposure to 1,1-Dichloro-2,2-Bis(p-Chlorophenyl) Ethylene (DDE) (an estrogenic Dichlorodiphenyltrichloroethane

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[DDT] metabolite) have decreased body mass index (BMI) and blood triglyceride levels compared to individuals in the third quartile.95 Moreover, women exposed to the lowest and highest doses of dioxin had no changes to the age at which they entered menopause, whereas those women exposed to intermediate doses showed an increased risk of early menopause.96 Finally, low or high free T4 in first trimester women were both associated with IQ deficits in the offspring.97 The mechanisms underlying these effects have not always been defined, but it is important to recognize that these dose-­response characteristics are fully within the realm of our understanding of hormone action and endocrine disruption.93

Chemical Exposures in the Human Population If the human population is being affected by exposures to EDCs, then those exposures must be sufficient in abundance and timing to impact human health outcomes. The CDC manages a biomonitoring program in part to characterize human exposures to environmental chemicals in a nationally representative manner. The fourth national report on human exposures to environmental chemicals was published in 2009,98 and this information is updated regularly on the CDC website (http://www.cdc. gov/exposurereport/). Although the fourth national report makes it clear that the presence of a chemical in the human population does not mean that it causes disease, it is also clear that the US population is simultaneously exposed to a large number of chemicals with a wide range of chemistries and biological actions, and that most of these chemicals are not tested for safety. This is true for the general population as well as for subgroups that might be expected to be particularly vulnerable, including pregnant women35 and newborns, and people living near areas of intense pollution. In fact, in a 2005 study of cord blood samples, nearly 300 chemicals were identified, with each cord blood sample containing at a minimum more than 100 different chemicals.99 This means that babies are born pre-­polluted, and it is likely that some of those exposures will cause adverse effects that could lead to increased sensitivity or susceptibility to disease later in life. Thus, the human population is exposed to a great many environmental chemicals throughout the life cycle, and most of these chemicals are not tested for safety. It should also be noted that the chemicals included in the NHANES biomonitoring program are only a subset of chemicals to which the human population is exposed. The reason for this is that analytical methods for identifying chemicals are not available for the large number of chemicals licensed for commerce (more than 70,000), or even the several thousand high-­production volume chemicals. In addition, there are estimated to be about 10,000 synthetic chemicals approved as food additives, the majority of which include little or no toxicity data.100 Thus, we do not know the complete picture about human exposures to anthropogenic chemicals, which increases the difficulty in linking exposures to human health. Nonetheless, it remains important to know that everyone is exposed to uncharacterized chemicals throughout his or her lifespan. An important lesson that is reflected in the experience of lead toxicity, PCBs, and DES is that ambient low-­dose exposure and/ or exposures during critical periods of development can produce adverse effects that go unnoticed without considerable and costly study. Furthermore, these adverse effects can be predicted and mechanistically understood by experimental studies in animals as well as cell-­free and cell-­based biochemical and molecular studies. Therefore, considering this large and largely unregulated chemical exposure pattern, studies designed to identify relationships between chemical exposure and human health outcomes particularly need to be incorporated into a logical framework quickly so that the findings can reliably inform patient care and public health policy.101

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KEY POINTS  • The human population is heavily contaminated with industrial chemicals, many of which have not been tested for health effects and very few of which have been tested for activities other than acute toxicity. In fact, chemical measurements of cord blood demonstrate that babies are born pre-­polluted with at least one hundred known chemicals. • Regulatory agencies have not evolved to test the ability of chemicals to interact with endocrine systems, nor to test the ability of chemicals to produce chronic disease. • EDCs can produce effects in animal studies at concentrations consistent with human exposures and with concentration-­response characteristics that do not fit the regulatory assumption that the “dose makes the poison.”

ENDOCRINE SYSTEMS: DISEASE TRENDS AND EFFECTS OF EDCs This section focuses on the state of the science linking EDC exposures to human disease. There are a number of lessons that we have learned from this work, which should be considered when reviewing data from human health studies. These are highlighted as follows: • Outcome measures linked to EDC exposures in humans occur at exposure concentrations lower—often considerably lower—than predictions based on regulatory studies. • EDC effects in the human population depend on genetic background creating a heterogeneity in responses that require large population studies using a genomic and statistical approach. • EDC effects are almost certainly the result of mixtures of EDCs, as that is what humans are exposed to, and also are a result of low environmentally relevant concentrations that are difficult to measure that require highly sensitive assays of mixtures. • The effects will be a result of “multiple hits” across the lifespan with the most sensitive exposure window during development (in utero and early life), which will require a lifespan approach. • EDC effects may occur after a long latent period including effects transmitted across multiple generations (transgenerational), requiring long-­term prospective studies. • Effects will vary depending on other environmental stressors: medications, stress, socioeconomic status, infections, and microbiome, which will require the analysis of confounders. • Effects can vary according to sex, tissue, dose, and route of exposure. Indeed, effects noted at high doses may be different from those at lower doses. • Epidemiological studies identify potentially avoidable risks to individual health much like smoking, drinking and dietary choices. This can be applied to adults as well as pregnant women and children.

Disease Trends and Effects of EDCs Temporal trends in the incidence and prevalence of disease in the human population can provide circumstantial evidence for a causal relationship between chemical exposure and human disease.13 Although these trends do not constitute proof of causation, they are important elements of the evidence. Chronic disease has increased during the past 50 years and is now more prevalent than infectious disease worldwide, with the exception of COVID-­19.102 Public agencies around the world have invested heavily in research on EDCs in large part because the human population manifests increased chronic diseases such as obesity, diabetes, infertility, and certain cancers.103,104 increased incidence of anomalies of male reproductive development105 and various aspects of fertility. In addition, there are also clear

increases in the incidence and prevalence of neurobehavioral disorders including attention deficit (AD),106 autistic spectrum (ASD),107 and measures of cognitive function. One of the most visible changes in the human population is the increased incidence and prevalence of obesity,108 especially in the pediatric population,109 with attendant increases in type 2 diabetes and heart disease. The incidence of some cancers is also increasing, including cancers of the brain, breast, male and female reproductive tract, and thyroid.110,111 A disease trend that is increasing during just a few decades and that cannot be accounted for by changes in diagnostic criteria is important evidence for a role of environment in disease because genetic drift does not occur during such a short time frame. EDCs play an important role in the etiology of such disease trends. The Endocrine Society’s Second Statement on EDCs,23 which provides an overview of the effects of EDCs on many of the most prevalent human diseases in 2015 along with a shortened executive summary.112 This review states, “The Endocrine Society’s first Scientific Statement in 2009 provided a wake-­up call to the scientific community about how environmental EDCs affect health and disease. Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans—especially during development—may lay the foundations for disease later in life”.

Obesity. The prevalence of obesity has increased dramatically both in the United States and in many countries worldwide since the mid-­ 1990s13 such that there are currently more than one billion overweight adults, with 400 million being classified as obese; 20 millions of these are children. Obesity is an intractable problem in that nearly 90% of those who lose weight gain it back within a year.113 Obesity is a complex endocrine condition caused by an interaction between genetics and environment with the environmental component consisting of nutrition, drugs, behavior and some EDCs. Conventional wisdom posits that obesity is an energy-­balance disorder resulting from excess calories and decreased exercise. However, both the increased incidence during the past few decades and data showing that only a subset of people become obese suggest that there is more to obesity than this simple explanation. Moreover, the incidence of obesity among infants is also increasing, which would appear to counter the energy-­balance hypothesis. Hormones such as estrogens, androgens, glucocorticoids, insulin, and thyroid hormones play important roles in controlling adipose tissue development, metabolism and satiety, and body-­weight regulation is also sensitive to environmental chemicals and drugs that can affect these hormonal pathways. Indeed, some drugs include a side effect of increasing weight gain, such as some antipsychotics114 and antidiabetic drugs such as Avandia.115 The fact that there are drugs with side effects of weight gain provides proof of the principle that the endocrine systems controlling weight gain and metabolism are sensitive to perturbation. Events occurring in early life—during pregnancy and early childhood—may increase the risk of obesity.116 There are considerable data linking altered development to increased susceptibility to disease later in life including obesity (DOHaD). Although the initial focus of these studies was on altered nutrition during development, it is now clear that there also are EDCs that can cause altered developmental programming, likely because of altered epigenetic regulation of gene expression, potentially leading to weight gain later in life. These EDCs have been called obesogens.117 It is key to note that exposures to obesogens during development can permanently alter programming of the system regulating body weight, producing an altered “set point” for adipose tissue formation, regulation of satiety, and metabolic rate that

CHAPTER 137  Endocrine-­Disrupting Chemicals and Human Health persists throughout life. Tributyl Tin, for example, induces an increase in fat cell number and these fat cells are abnormal in their response to hormones including impaired glucose uptake and insulin signaling.118 In fact, even after body weight reduction, there are long-­term changes in a profile of hormones that support obesity.119 In addition, EDC exposure can trigger changes in the hypothalamus that play a particularly important role in feeding behaviors. Improper hypothalamic programming may adjust metabolic “set points” in adolescents and adults, and these adjustments may help explain differences between the eating behaviors of lean and obese individuals.120 Thus, the obesogen hypothesis offers an explanation for both the increase in obesity rates and also the differences between lean and obese individuals. The obesogen hypothesis was developed in the early 2000s in response to the startling rise in the incidence and prevalence of obesity in the human population, especially in children.121 Although a relatively new field of study, there is now strong evidence in experimental animals showing that developmental exposure to a variety of environmental chemicals, including those that activate PPARγ such as TBT, and estrogenic chemicals such as BPA, some organochlorine and organophosphate pesticides, and chemicals acting through other mechanisms such as elements of air pollution, lead, perfluoroctanoic acid, phthalates, and nicotine, can lead to weight gain later in life.13 There are now about 50 chemicals and chemical classes classified as obesogens.122 These animal studies provide significant evidence for a role of EDCs in affecting specific elements of body weight regulation, including appetitive behavior and metabolism. An important example is that of TBT, which is a potent activator of the nuclear receptor PPARγ, a key receptor involved in the development of new adipocytes. Human studies are more difficult to perform and interpret in part because humans are exposed to complex chemical mixtures and are more genetically variable as well as variable in their environmental conditions.123 Nonetheless, animal studies have produced data that are concordant with epidemiological data linking exposures to obesogens during development to increased body weight in infants and later in life. Some of the strongest associations between exposures during pregnancy and obesity in their offspring include smoking, BPA, persistent organic pollutants, DDT/DDE, per- and polyfluoroalkyl substances (PFAS), and air pollution.120 It has been proposed that the confluence of developmental programming of metabolic set points by obesogens in association with continued obesogen exposures throughout the lifetime, overconsumption of processed foods containing added sugars, and EDCs from the packaging materials, together with decreased physical activity throughout life, creates an important combination that is driving the obesity epidemic.124,125 The obesogen hypothesis proposes both a mechanism for the increased epidemic of obesity and also a solution. If obesity results in part from continuous exposures to obesogens from development and later, then for the first time there is actually a path for individuals and for physicians to reducing the risk of obesity by reducing these exposures. Thus, rather than waiting for obesity to occur and trying with poor success to intervene and cause weight loss, we may be able to reduce the risk of overweight and obesity from the beginning.

Diabetes. Type 2 diabetes—especially in the pediatric population—is becoming recognized as a global epidemic.126 Diabetes has increased dramatically since the mid-­1990s to 347 million in 2011.13 And, parallel to the incidence of obesity, there has been a significant increase in childhood type 2 diabetes, something that was rare just a few decades ago. Although type 2 diabetes can occur by itself, 70% of the risk associated with it is attributed to weight gain, indicative of the direct link between increased adiposity and insulin resistance. In animal studies,

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many chemicals that cause weight gain also disrupt glucose management. EDCs including phthalates, BPA, some flame retardants, arsenic, some persistent organic pollutants such as PCBs, and pesticides have been linked to the development of type 2 diabetes in both animal studies and in human epidemiologic studies.120,127 Overall, there are significant data in both animal and human studies that support concern about the relationship between chemical exposures and the production of type 2 diabetes. In contrast, there is little information about the link between EDC exposures and type 1 diabetes. However, there are environmental chemicals that exhibit both EDC activity and immunotoxicity, which would be prime candidates for EDC effects on this hormonally and immune-­mediated, largely childhood, disease.

Endocrine Disruptors, the Epigenome, and Developmental and Transgenerational Effects Parents pass on their genes to their children and in so doing pass on various traits associated with those genes. The combination of all genes in a species is referred to as the “genome,” and “genomic” studies refer to those studies designed to understand how various patterns of genes are controlled; but cells in the body can pass on heritable traits to their cellular progeny without altering their genome. Certainly, EDCs can also act across the lifespan just as hormones do. However, there is a major difference in effects noted after developmental exposures vs adult exposures. The ability of endocrine disruptors to alter the normal hormonal control of development is perhaps the most significant consequence of exposure, because developmental effects will occur at lower doses than are required for effects in adults. Additionally, the effects of exposure to endocrine disruptors during development will remain throughout life and may have a long latency between exposure and disease outcomes; whereas, in many cases, exposures in adults remain only while the chemical is present. These differences in sensitivity to EDCs and persistence of effects are related to the epigenetic state of the organism. During development, a single cell—the ovum—will divide, multiply, and differentiate into many cell types and tissues and ultimately will become a person. Development from this perspective is a process of “fate restriction”—permanently turning on or turning off different combinations of genes required for a cell to be a functional cell in the liver, kidney, brain, etc. The traits a cell passes on to its cellular progeny are controlled by “epigenetic” mechanisms. Epigenetics is broadly defined as those heritable changes not dependent on genetic sequences, and it is these epigenetic processes that define and control tissue development by controlling gene expression. Thus, a major route by which hormones act during development is by changing the epigenome and the combination of genes that can or cannot be expressed. The fact that endocrine disruptors can influence epigenomic mechanisms during the times when tissues are forming is likely the mechanism whereby development (in utero and early childhood, when tissues are developing) is a sensitive window for EDC action. It is also the reason that developmental exposure leads to effects long after the exposure is gone and may occur after a long latent period across the lifespan. In adults, the tissues are already formed, and the epigenetic marks are relatively stable; thus, EDCs may not act on the epigenome but may act directly on genomic pathways—effects that are more easily reversible when exposure is controlled. Endocrine disruptors have also been shown to produce transgenerational effects as a result of their ability to alter epigenetic processes. This issue first arose with studies in which an antiandrogenic pesticide (vinclozolin) was administered to developing mice at a single time during development—when the testis was in a critical period of development. Not only did vinclozolin produce adverse effects on the developing testis, but also this was transmitted through four generations of

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mice. This effect is likely caused by epigenetic changes that were transmitted with high fidelity from one generation to the next via the germ cells (sperm). A number of endocrine disruptors have now been shown to influence epigenetic mechanisms and to produce effects in several generations of animals.129 Grandmother exposures may be affecting the F2 and subsequent generations.

Reproductive System. Because the reproductive system was an early focus of studies on chemical toxicities in the 1930s and 1940s, it should be no surprise that there is abundant literature on EDCs that interfere with estrogen or androgen actions. In addition, the field of reproductive endocrinology has significantly matured both in terms of our understanding of the medicinal chemistry associated with estrogen and androgen receptors (ARs), but also in terms of the role of sex steroids in development, in puberty, and in the adult. Thus, there is a significant amount of information now about environmental chemicals and different aspects of reproduction, as discussed in the following text. Male Reproduction. One of the most significant concepts in this field is that of the testicular dysgenesis syndrome (TDS).130 Skakkebaek et al. proposed that poor semen quality, testis cancer, undescended testis, and hypospadias are symptoms of one underlying entity caused by defects in androgen action during fetal development. One of the most compelling observations in support of this hypothesis is a report of a novel AR mutation in a family that exhibits all of the hallmark characteristics of the TDS.131 This mutation (c.2214T>G;p.il3738Met) produces an AR protein with only 50% of the transcriptional potency of the wild-­type protein, suggesting that a 50% reduction in androgen action can produce all of the elements of TDS. However, the implication is that a reduction in AR action for any reason may also contribute to TDS. Importantly, each of these symptoms is increasing in incidence and prevalence, suggesting an environmental component.132 In addition, a number of EDCs to which the human population is chronically exposed have antiandrogenic actions, including phthalates, BPA, and some pesticides.133,134 In addition, evidence generated from controlled animal studies indicates that males exposed to estrogens and antiandrogens develop hypospadias, undescended testis, and low sperm counts.130 Given the number of estrogenic and antiandrogenic chemicals to which the human population is chronically exposed, it is reasonable to posit that there is a significant contribution of EDCs to the increased incidence of TDS symptoms. These are described as follows. Sperm Counts and Fertility. Infertility, as defined by the inability to conceive after frequent and unprotected sexual intercourse for 12 months, currently affects 15% of couples worldwide.135 There has been a significant effort to associate infertility to sperm counts, and to further associate sperm counts to environmental stressors including EDCs. Since the mid-­ 1990s, there has been a growing recognition that sperm count and sperm quality are declining in humans across the globe. The first report of this decline was published in 1992,136 and since then the original data have been reanalyzed and confirmed. Although there was early and reasonable skepticism about these observations and conclusions, new prospective data have been added on a regional basis, which also demonstrate declines in sperm counts that vary by geographic location. At this time, there is general scientific consensus that sperm counts have declined throughout the past 50 years in the human population, and that there are likely to be both genetic and environmental contributors to this decline. A causal link between exposures to EDCs, especially those that act via the AR or those that decrease testosterone production, and sperm counts and fertility is plausible, and there is evidence that several pesticides as well as polybrominated diphenyl ethers are involved. It is possible that adult sperm production is sensitive to current environmental

exposures because it is an ongoing process. However, it also appears that adult sperm counts are sensitive to developmental programming, especially to EDCs with antiandrogenic properties like phthalates. Phthalates can reduce serum androgens in rodents and have been linked to reduced anogenital distance in humans, a marker of fetal testosterone levels.137 In animal models, there are strong and consistent data showing effects of EDCs, including BPA, vinclozolin, dioxin, and phthalates, on several aspects of male reproduction, including sperm counts. Indeed, all these chemicals also cause reduced sperm counts and fertility across four generations, indicating transgenerational effects in animal models.138 These new data are significant, as they suggest that exposures during pregnancy may lead to toxic effects in grandchildren and great-­grandchildren. Because of the lifelong sensitivity of sperm production to EDCs, along with the fact that there are hundreds of EDCs with varying exposures throughout the lifespan, it will be difficult to ferret out the exact contribution of individual EDC exposures to sperm counts and fertility. Nonetheless, data support a role for EDCs impacting both sperm counts and fertility and possibly the secular decline in these measures globally. Hypospadias and Cryptorchidism. Normal differentiation and growth of the male external genitalia require normal androgen production and an adequate tissue response to the hormone. Hypospadias can be defined as a consequence of the abnormal development of urethral spongiosum tissue, with a defect of the ventral aspect of the prepuce and the skin of the distal shaft. There are many anatomic variants exhibited; minor forms are characterized by an ectopic urethral opening on the glans penis or more proximally, whereas the more severe forms present a proximal urethral opening even at the perineum. The different forms of hypospadias may represent phenotypes that are linked to different risk factors,139 and this heterogeneity may be important in identifying specific causes. The incidence of male urogenital abnormalities has increased during the past few decades.13 Early estimates of hypospadias incidence may have been underreported in some countries, depending on the way the data were gathered. However, in the case of the more severe proximal phenotype, where surgical correction is required, the reporting metrics have not changed, and incidence has clearly increased in the United States, Australia, and many European countries.132 There is increasing circumstantial evidence that at least some forms of hypospadias and other TDS symptoms are caused by environmental factors. One thread of evidence for this is that idiopathic partial androgen insensitivity syndrome can produce symptoms of TDS including hypospadias,140 even in cases where the androgen system appears to be working normally. Despite this, there is no clear relationship between hypospadias and individual EDCs. Given the heterogeneous nature of hypospadias and the large mixture of chemicals known to produce this defect in animals, including those chemicals to which humans are exposed, it should be expected that relationships of interest will be difficult to identify. Like hypospadias, cryptorchidism presents with different degrees of severity, from palpable undescended testes to abdominal testes that are not palpable. Epidemiologic studies with well-­defined criteria for identifying the incidence of cryptorchidism have shown very large differences in the incidence between countries and increasing trends in Denmark and the United Kingdom.132 Genetic associations with the presence of cryptorchidism do not account for a large proportion of cases.141 In addition, genetic mouse models have shown important mechanisms of male genital development and provide insight into the mechanisms by which cryptorchidism and hypospadias may be triggered. Normal androgen action and tissue response are essential for penile development and testicular descent. However, estrogens can interfere with normal male genital development by downregulating

CHAPTER 137  Endocrine-­Disrupting Chemicals and Human Health insulin-­like factor 3 and stimulating ATF3, leading to cryptorchidism and hypospadias, respectively. Therefore, endocrine disruptors that alter the estrogen-­androgen ratio or act as hormone mimics (agonists or antagonists) can cause maldevelopment in animals, which is highly plausible in humans. Interestingly, BPA exposure is associated with poor outcomes in in vitro fertilization.142 Female Reproduction. Conception rates in both Danish and US women have declined 44% since 1960.143 There are limited prevalence data on women’s environmental health diseases including menstrual cyclicity, pubertal development, endometriosis, uterine fibroids, or polycystic ovarian syndrome (PCOS); however, it is clear that these diseases are common. Indeed, PCOS is the most common endocrine disorder in women of reproductive age, although, because the definition of PCOS is still under debate, diagnosis is variable and actual prevalence is unclear, which results in an estimated occurrence between 3% and 15% depending on criteria used (see Chapter 126).144 Uterine fibroids (leiomyomas) are common with estimates that they occur in 25% to 50% of all women; however, the effects of EDCs have not been examined in detail for possible impact on these diseases.145 Interestingly, the incidence of endometriosis is thought to be in the range of 10% to 15% for reproductive-­age women (see Chapter 127).146 As noted earlier, studies of DES in humans and animals establish the principle that estrogenic EDC exposure during development can produce adverse effects on women’s reproductive health. In addition, data from animals models consistently show that developmental or adult exposure to some EDCs including organochlorine pesticides, heavy metals, phthalates, BPA, and dioxin can alter ovarian development including follicle formation, follicle growth, and disruption of steroid hormone levels.147 Studies in humans show that adult exposure to some of these EDCs is associated with increased risk of miscarriage, time to pregnancy and reduced size for gestational age.147 For example, BPA has been associated with infertility, shortened gestation, and increased preterm birth in women. These observations are concordant with those in animals showing that BPA affects the morphology and function of the oviduct, ovary, and hypothalamic-­pituitary-­ovarian axis.148 Puberty. Puberty is a complex physiological process involving central activation of the hypothalamic-­ pituitary-­ gonad axis. Two hundred years ago, the average age of puberty was around 17 years old, and this declined and stabilized in the 1950s at about 13 years of age.149,150 However, the age of puberty has declined again during the past few decades; in girls, the average age of pubertal onset is 10 years, and in boys it is around 11.5 years.151 Improved nutrition is reasonably proposed to contribute to these changes, especially as the average BMI has increased and the relationship between BMI, leptin, and puberty onset is well known (see Chapter 104). However, the incidence of precocious puberty in girls less than 8 years old has increased in general and is geographically variable, indicating that environmental factors can affect both the average age of puberty onset and the incidence of precocious puberty.152 There are multiple reports that focus on a role of EDCs as an explanation for early puberty.153 Because of the role of sex steroids in the onset of puberty, it is reasonable to propose that exposures to a variety of EDCs during development or early life may contribute to the earlier onset of puberty.154 There is convincing evidence from animal studies that prenatal and/ or neonatal treatment with EDCs that act via the estrogen receptor can accelerate pubertal development in females, whereas estrogenic chemicals tend to delay puberty onset in males. EDCs acting by other mechanisms, which are shown to have effects in some animal studies, include lead, styrene, methoxychlor, phthalates, and BPA. Indeed, the data linking estrogenic EDCs to early puberty in animal models

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are strong. There are some epidemiologic studies linking exposures to EDCs (DDT/DDE, dioxins, PCBs, PBBs, some flame retardants, hexachlorobenzene, BPA, lead, and cadmium);155 however, overall epidemiologic evidence for a role of EDCs in early puberty is still limited.156 The importance of changes in puberty is partly a result of the effects of puberty on other health outcomes where early puberty is associated with reproductive tract cancers, depression and eating disorders, diabetes, and cardiovascular disease.153 Note also that obesogens can increase weight leading to an increase in leptin and puberty advance,157 and that early puberty is a risk factor for breast cancer.158 Endometriosis. Endometriosis is an estrogen-­ dependent disorder, but the exact etiology is unclear. The strongest data linking EDCs to endometriosis are those of dioxin causing endometriosis in primates,150 which is supported by experiments in rodents. Although the mechanism for the dioxin effects on endometriosis is unclear, there are data showing that dioxin decreases circulating estradiol and also causes its degradation, and that dioxin reduces the expression of the progesterone receptor159 as well as acting as an immunosuppressant. Because of the role of the sex steroids in reproduction, it is biologically plausible that EDCs that mimic or antagonize sex steroids will play a role in the multicausality for female reproductive disorders.160 KEY POINTS  • Secular trends in chronic human disease and disorders represent evidence of the impact of EDC exposures on human health in part because these outcomes are endocrine in nature. • Obesity and diabetes are epidemic in the United States and the world and evidence indicates that a variety of chemicals contribute to these human health problems. • Infertility and male and female reproductive disorders represent significant human health issues today that are being exacerbated by chemical exposures.

Cancer A large amount of research during the past several decades has established the involvement of endogenous estrogens and androgens in cancers of reproductive organs and the thyroid.161 However, the possible contribution of environmental chemicals that interact with these hormone systems has only recently received attention for two primary reasons. First, cancers of the breast, endometrium, ovary, testis, prostate, and thyroid glands continue to rise among populations of Western countries, and more recently also among Asian nations. Second, the involvement of the synthetic estrogen, DES, in vaginal cancers and breast cancer has heightened concerns that a multitude of other hormonally active chemicals in everyday use can cause or increase the risk of these diseases. Most research into associations with endocrine disruptors has been performed with breast, prostate, and testis cancers, whereas other hormone-­related cancers such as endometrial, ovarian, and thyroid cancers have received less attention. Breast cancer is the most common endocrine cancer and is the second leading cause of cancer death. Its incidence has increased since the mid-­1980s in almost all environmentalized/ Westernized countries13 (see also Chapter 128). The genetic and metabolic aspects of breast cancer are responsible for up to 27% of breast cancer, with the other 70% or more related to environmental factors.162 It is clear that excess estrogen across the lifespan is an important risk factor for breast cancer. Thus, there is an increased risk of breast cancer among current and recent users of oral contraceptives.163 This has manifested in the effects on increasing breast cancer in offspring of mothers exposed to DES, the effects of hormone replacement therapy on increased breast cancer

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incidence, and the association of early puberty or increasing age of first pregnancy with increased breast cancer. It is also clear that the development period is a sensitive time for EDCs to affect breast development, and effects can lead to cancer or precancerous lesions later in life. However, in contrast to most tissues, the breast continues developing until adulthood and even changes slightly with each menstrual cycle. Polycyclic aromatic hydrocarbons are a class of chemicals that have both genotoxic/mutagenic properties as well as endocrine properties. Other EDCs that alter mammary gland development in animal models by affecting the organization of the tissue include BPA, dioxin, PFOA, the fungicide vincolozolin, polybrominated diphenyl ether (PBDE), nonophenol, and atrazine.164 Genistein, a plant estrogen found in soy products, can protect against carcinogen-­induced mammary gland tumors in some animal studies, canceling out the protective effect of tamoxifen in others, and can actually cause ductal hyperplasia in others depending on the timing and dose. Clearly, assessing the role of EDCs in breast cancer will remain highly complex. For decades environmental health scientists studied the potential link between adult exposures to a variety of EDCs to breast cancer, but the results were inconsistent. After the animal data showed the importance of developmental exposures in increasing susceptibility to mammary cancer in animals, epidemiologists started to examine developmental exposures and breast cancer. However, because of the long latency to develop cancer, these studies are expensive and time consuming, requiring assessment of developmental exposures and follow-­up for 4 to 5 decades to assess the incidence of breast cancer. A classic study examined exposure to DDT and its metabolites during childhood and throughout life and showed that there was no association of exposures in adults with breast cancer but a high correlation of exposure around puberty with adult-­onset breast cancer.165 This study provided the data that focused epidemiological studies on the in utero and childhood exposures as the sensitive window of exposure leading to increased susceptibility to breast cancer later in life. Overall, the growing literature linking developmental exposure to EDCs and later development of breast cancer is particularly strong.164 Singly and in combination, these EDCs—many of which are present in everyday products—may be contributing significantly to the public health burden of breast cancer. Thus, it is important to prioritize strategies to reduce risk in addition to improving breast cancer therapies.

Thyroid Disruption physiology.166

TH plays an important role in development and adult Therefore, it is important to recognize that there are a large number of chemicals that can interfere with thyroid function167,168 and/or with TH action.169 These different kinds of chemicals may interact to affect TH action during development. For example, perchlorate can reduce iodide uptake and in the face of low dietary iodine can affect thyroid function and TH levels.170 In addition, a large number of chemicals can increase the clearance rate of TH, similar to that of phenobarbital.171 Some chemicals also appear to interact directly with receptors for TH172,173 and may produce effects that are not fully consistent with hypo-­or hyperthyroidism.174 Finally, there are chemicals that can displace THs from serum transport proteins,175 and others that may interfere with cellular transport proteins such as MCT8 or OATP1c1, although this is less well-­studied. However, because epidemiologic studies investigating the relationship between chemical exposure and the thyroid uniformly evaluate serum hormone levels, it will be a challenge to link exposure to the combination of chemicals that can impact thyroid function and TH action to human disease. The neurobehavioral impacts of TH insufficiency are so well known that it is reasonable to consider the possibility that environmental chemicals may play

a role in the increased incidence and prevalence of neurobehavioral disorders. This may best be apparent for PCBs.29 Likewise, environmental chemical interference with TH action may contribute to adult diseases including cardiovascular, obesity, and metabolic syndrome. These relationships are much less well understood. Given the variability in clinical manifestations of thyroid disease in adults, this will be a challenging topic.

Neurobehavior Neurodevelopmental disorders have increased in prevalence during the past several decades, and the environment is likely to play a role.176–178 Since the 1970s, there have been dramatic increases in previously rare neurodevelopmental disorders. For example, in the 1970s the prevalence of autism spectrum disorders (ASD) was estimated to be between 4 and 5 in 10,000 children,179 but today this value is estimated to be 1 in 54 children.180 There is significant debate about the role of broadening definitions of ASD related to this increase181 or the role of other factors that may mask a real increase.182 Similar trends have been observed for other neurobehavioral problems such as ADHD (attention-­deficit hyperactivity disorder), learning disabilities, and childhood and adult depressive disorders. Predominant among these disorders are attention-­deficit disorders (ADDs)—with or without hyperactivity—with a worldwide, pooled prevalence estimate of about 5.3%.106 These are difficult trends to attribute unequivocally to the environment, although it appears clear that the increases are not fully attributable to artifacts related to more aggressive diagnosis and reporting and may involve genetic susceptibility. KEY POINTS  • Secular trends in the incidence of some cancers (e.g., pediatric brain cancer), neurobehavioral disorders and thyroid dysfunction represent additional impacts of EDCs on the human population. • In each case, scientific evidence from mechanistic studies support conclusions that associations between chemical exposures and these disease trends are causal. Moreover, these population studies represent evidence that can be employed in the clinic.

EMERGING ISSUES Endocrine Disruptors, the Epigenome, and Transgenerational Effects Parents pass on their genes to their children, and in so doing pass on various traits associated with those genes. The combination of all genes in a species is referred to as the “genome,” and “genomic” studies refer to those studies designed to understand how various patterns of genes are controlled. But, cells in the body can pass on heritable traits to their cellular progeny without altering their genome. Certainly, EDCs can also act across the lifespan just as hormones do. Epigenetics is broadly defined as heritable changes not dependent on genetic sequences, and it is these epigenetic processes that define and control tissue development by controlling gene expression. Endocrine disruptors have also been shown to produce transgenerational effects as a result of their ability to alter epigenetic processes. This issue first arose with studies in which an antiandrogenic pesticide (vinclozolin) was administered to developing mice at a single time during development—when the testis was in a critical period of development. Not only did vinclozolin produce adverse effects on the developing testis, but also this was transmitted through four generations of mice.128 This effect is likely to be caused by epigenetic changes that were transmitted with high fidelity from one generation to the next via the germ

CHAPTER 137  Endocrine-­Disrupting Chemicals and Human Health cells (sperm). A number of endocrine disruptors have now been shown to influence epigenetic mechanisms and to produce effects in several generations of animals.129 Grandmother exposures may be affecting the F2 and subsequent generations. Notably, in a mouse model, deleterious effects of EDCs are corrected in the mammalian germline by epigenomic reprogramming.183 These deeply concerning and contradictory observations in animal studies with profound implications for the human population warrant further study.

Increasing Exposures Worldwide There are no longer pristine areas worldwide without environmental pollutants.184 EDCs are everywhere; they are present in food, nature, and humans, and there is global transport through ocean, air currents, plants, and animals. Indeed, the Faroe Islands, off the coast of Iceland and Norway, which because of its isolated location should be one of the most pristine places in the world, has a human population that is severely affected by EDCs via the eating of contaminated whale blubber and meat.

Expanded List of Diseases Related to EDCs Until recently, the focus of EDC research was on chemicals that affected the estrogen, androgen, and thyroid pathways, and diseases that result from their disruption. Endocrine disruption is no longer limited to these pathways, as chemicals have also been shown to interfere with metabolism, fat storage, bone development, glucose homeostasis, glucocorticoid regulatory pathways, and immune systems.184 It is possible that all the endocrine systems will be impacted by environmental chemicals. Indeed, even the known EDCs may not be representative of the full range of relevant structures and properties of chemicals that may have endocrine-­disrupting properties. Because only a limited number of chemicals in commerce have been tested for endocrine-­disrupting activity, it is likely that there are many more chemicals with endocrine-­ disrupting activity than currently believed. In addition, metabolites or environmental transformation products and products formed during waste treatment are usually not examined for endocrine-­disrupting activity. Thus, research is needed to identify other EDCs.

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stripping, open burning, shredding, and melting of the e-­waste under conditions where the workers are highly exposed to the toxic chemicals via air emissions, water, and soil contamination as well as direct physical exposures.185

Green Chemistry Green chemistry, also called sustainable chemistry, is a new field that encourages the design and development of chemicals using principles that minimize the use and generation of toxic chemicals. Paul Anastas coined the term “green chemistry,” and it consists of 12 principles that he developed.97 Indeed green chemistry focuses on prevention of toxicity by producing the next generation of chemicals without toxicity. A joint project by green chemists and environmental scientists focused on the development of test methods (TIPED)98 to detect endocrine activity in new chemicals being developed before their release into the environment. Green chemistry offers an alternative to chemical development that has the potential to reduce the number of new toxic chemicals released into the environment and also to develop nontoxic alternatives to chemicals currently in use. KEY POINTS  • There are two categories of emerging issues that are important to monitor. The first category is that the current chemical mixture in the environment is being recycled in ways that contaminate workers and the general population. Thus, exposure routes are being identified that were not recognized previously. In addition, chemicals that have been banned (e.g., PCBs) or heavily regulated (e.g., lead) continue to pose health risks both because of their widespread use before being banned and because of their long-­term effects on human health. • A second category of emerging issue is that there is a nascent movement to produce safer chemicals. The traditional strategy is to produce and employ chemicals that have not been proven to cause harm and often are as toxic as the chemical it replaces. Proof of harm requires decades after deployment, harming generations of people. The revised strategy is to avoid the production and use of chemicals that exhibit any mechanistic signs of biological effects. This strategy will gain traction as innovation occurs.

Mixtures The focus of research identifying and characterizing the effects of EDC in animal models and in human populations has been on linking exposure to a single chemical at a single exposure leading to a single disease. However, humans are exposed to mixtures of chemicals throughout their lifetimes, and indeed each chemical has the potential to affect several endocrine diseases. DES is a good example of one exposure leading to many sequelae. A developmental “estrogenization syndrome” has been proposed.96 In addition, it is now clear that exposures to many chemicals at low levels that individually have no effect can actually produce significant effects because of the additivity of their individual effects.134 Although mixture studies are difficult to develop in animals and are expensive and complicated to interpret statistically in human studies, there is an urgent need to expand research into the effects of mixtures of EDCs both during development and across the lifespan.

E-­Waste Modern society has developed many electronic devices that are scrapped when broken or obsolete, including computers, televisions, phones, audio components, and refrigerators. Because these electronic components contain many endocrine-­disrupting toxicants including lead, cadmium, brominated flame retardants, plastics, and plasticizers, they can involve significant risk to workers and communities that recycle or incinerate or otherwise dispose of the components. Although the majority of e-­waste ends up in landfills, recycling involves chemical

CONCLUSION AND GUIDANCE FOR CLINICIANS The human population is heavily contaminated with environmental chemicals. Most of those chemicals have not been studied for their impact on human health, but many have been shown to contribute to chronic disease prevalence. This observation has two important implications. The first is that every patient a clinician sees will have a certain burden of toxic chemicals. The second is that the current clinical and scientific evidence can and should be included in clinical recommendations. It is important to recognize that EDC effects on human health are observed at the population level. Thus, these data can inform clinicians about relative risk, just as other health-­related domains such as smoking, exercise and nutrition inform clinicians about recommendations for health care. There are several lines of evidence that support concern that chemicals in the environment are producing adverse health effects in the human population by interfering with hormone action. These are in part articulated and referenced more fully in the UNEP/WHO State of the Science of EDCs.13 This evidence includes observations that there are a high incidence and increasing secular trends of many endocrine-­related disorders in humans; that endocrine-­related effects in wildlife populations are prevalent; and that laboratory studies have identified chemicals with endocrine-­disrupting properties that can account for some of

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these observations in humans and wildlife. Considering the evidence, the Endocrine Society has released a Scientific Statement84 that articulates the concern for the public health impact of chemical exposure. In addition, the American College of Obstetricians and Gynecologists in collaboration with the American Society of Reproductive Medicine has released a statement calling for a reduction in exposure to these chemicals and advising clinicians to be aware of the sources of contamination in women of reproductive age.85 The Royal Society of Obstetricians and Gynecologists likewise released a statement providing advice on handling the potential consequences of chemical exposures (http://www. rcog.org.uk/files/rcog-­corp/5.6.13ChemicalExposures.pdf), as has the International Federation of Gynecologists and Obstetricians.186 The overriding message from all these society statements is that clinicians must play a larger role in providing information to their patients regarding how to avoid exposure to toxic agents. Although it is not expected that health care professionals be experts in toxic chemicals, they can still provide important information via literature provided to patients, specific focus on environmental health sciences in birth preparation classes and referring patients to specialists when a hazardous exposure is suspected. The noted reports provide information regarding key chemical exposures, sources of exposures at home, work, and in the community, and clinical implications of exposure. With the help of health care professionals, patients can become more aware of the risks to their health and the health of their children, including those unborn, and they can thus take action to reduce their body burden of toxic chemicals, thereby potentially improving overall health throughout the lifespan. An example of an exposure history is available at http://prhe.ucsf.edu/prhe/clinical_resources.html. Websites that contain useful information include: www.healthandenvironment.org, www.ewg.org, www.cehn.org, and www.prhe.ucsf.edu. Simple steps that patients can take include the following: avoid smoking and secondhand smoke, avoid fast foods and processed foods, limit foods that are high in animal fat, limit fish that are known to contain PCBs and mercury (shark, swordfish, king mackerel, and tilefish), do not microwave items in plastic containers, be mindful of ingredients in cosmetics and skin care products, do not use pesticides around the home, keep the home free of dust that contains lead, pesticides, and flame retardants, use nontoxic cleaning products (www.prhe.ucsf. edu/prhe/tmlinks.html#cleaningproducts), choose plastics carefully to avoid BPA and products made with soft PVC, use VOC-­free and water-­based paints, use a digital thermometer, wash all fruits and vegetables, and eat organic and fresh products when possible.

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55. Kodavanti PR, Kannan N, Yamashita N, et al. Differential effects of two lots of aroclor 1254: congener-­specific analysis and neurochemical end points. Environ Health Perspect. 2001;109:1153–1161. 56. Seegal RF, Bush B, Shain W. Lightly chlorinated ortho-­substituted PCB congeners decrease dopamine in nonhuman primate brain and in tissue culture. Toxicol Appl Pharmacol. 1990;106:136–144. 57. FDA. In: FDA US, ed. Code of Federal Regulations, Title 21 Part 109.3. Washington, DC: US Govt; 2010. 58. Gao Q, Ben Y, Dong Z, Hu J. Age-­dependent human elimination half-­ lives of dioxin-­like polychlorinated biphenyls derived from biomonitoring data in the general population. Chemosphere. 2019;222:541–548. 59. Giera S, Bansal R, Ortiz-­Toro TM, Taub DG, Zoeller RT. Individual polychlorinated biphenyl (PCB) congeners produce tissue-­and gene-­ specific effects on thyroid hormone signaling during development. Endocrinology. 2011;152:2909–2919. 60. Gauger KJ, Giera S, Sharlin DS, Bansal R, Iannacone E, Zoeller RT. Polychlorinated biphenyls 105 and 118 form thyroid hormone receptor agonists after cytochrome P4501A1 activation in rat pituitary GH3 cells. Environ Health Perspect. 2007;115:1623–1630. 61. Grimm FA, Lehmler HJ, He X, Robertson LW, Duffel MW. Sulfated metabolites of polychlorinated biphenyls are high-­affinity ligands for the thyroid hormone transport protein transthyretin. Environ Health Perspect. 2013;121:657–662. 62. Hamers T, Kamstra JH, Sonneveld E, et al. Biotransformation of brominated flame retardants into potentially endocrine-­disrupting metabolites, with special attention to 2,2′,4,4′-­tetrabromodiphenyl ether (BDE-­47). Mol Nutr Food Res. 2008;52:284–298. 63. Hood A, Klaassen CD. Differential effects of microsomal enzyme inducers on in vitro thyroxine (T(4)) and triiodothyronine (T(3)) glucuronidation. Toxicol Sci. 2000;55:78–84. 64. Sethi S, Morgan RK, Feng W, et al. Comparative analyses of the 12 most abundant PCB congeners detected in human maternal serum for activity at the thyroid hormone receptor and ryanodine receptor. Environ Sci Technol. 2019;53:3948–3958. 65. Gilbert ME, Rovet J, Chen Z, Koibuchi N. Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology. 2012;33:842–852. 66. Newbold RR. Lessons learned from perinatal exposure to diethylstilbestrol. Toxicol Appl Pharmacol. 2004;199:142–150. 67. Troisi R, Palmer JR, Hatch EE, et al. Gender identity and sexual orientation identity in women and men prenatally exposed to diethylstilbestrol. Arch Sex Behav. 2020;49:447–454. 68. Titus-­Ernstoff L, Troisi R, Hatch EE, et al. Birth defects in the sons and daughters of women who were exposed in utero to diethylstilbestrol (DES). Int J Androl. 2010;33:377–384. 69. Klip H, Verloop J, van Gool JD, et al. Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study. Lancet. 2002;359:1102–1107. 70. Oerbeck B, Sundet K, Kase BF, Heyerdahl S. Congenital hypothyroidism: influence of disease severity and L-­thyroxine treatment on intellectual, motor, and school-­associated outcomes in young adults. Pediatrics. 2003;112:923–930. 71. Heyerdahl S, Oerbeck B. Congenital hypothyroidism: developmental outcome in relation to levothyroxine treatment variables. Thyroid. 2003;13:1029–1038. 72. Heindel JJ, Skalla LA, Joubert BR, Dilworth CH, Gray KA. Review of developmental origins of health and disease publications in environmental epidemiology. Reprod Toxicol. 2017;68:34–48. 73. Gee D. Late lessons from early warnings: toward realism and precaution with endocrine-­disrupting substances. Environ Health Perspect. 2006;114(suppl 1):152–160. 74. Gee D, Krayer von Krauss MP. Late lessons from early warnings: towards precaution and realism in research and policy. Water Sci Technol. 2005;52:25–34. 75. Schwartz HL, Ross ME, Oppenheimer JH. Lack of effect of thyroid hormone on late fetal rat brain development. Endocrinology. 1997;138:3119–3124. 76. Rovet JF. The role of thyroid hormones for brain development and cognitive function. Endocr Dev. 2014;26:26–43.

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