The Pituitary [4 ed.] 0128134216, 9780128134214

Table of contents :
Front Cover
Title Page
Copyright
Dedication
Contents
List of Contributors
Preface
I: Hypothalamic–Pituitary Function
1 Pituitary Development
Introduction
The Pituitary Gland
Formation of Rathke’s Pouch
Glandular or Endocrine Gland Development
Signals Controlling Pituitary Development
Cell Differentiation
Tissue Architecture and Cell Networks
Pituitary Cell Cycle Control
Progenitors and Stem Cells
Corticotrophs
Melanotrophs
Gonadotrophs
Somatotrophs
Lactotroph Differentiation
Thyrotrophs
Perspectives
Acknowledgments
References
2 Hypothalamic Regulation of Anterior Pituitary Function
Introduction
Anatomy and Histology of the Hypothalamic–Pituitary Unit
The Hypothalamus
Pituitary Anatomy
Cellular Composition of the Anterior Pituitary
Hypothalamic Factors Regulating Pituitary Function
Thyrotrophin-Releasing Hormone
Corticotrophin-Releasing Hormone
Growth Hormone–Releasing Hormone
Somatostatin
Gonadotrophin-Releasing Hormone
Hypothalamic Prolactin Regulators
Prolactin-Inhibitory Factors
Prolactin-Releasing Factors
Summary
References
3 Adrenocorticotrophin
Introduction
Corticotroph Biology
Embryology
Adult Anatomy
Molecular Signals in Development
Intrinsic Signals
Extrinsic Signals
Non-ACTH Peptides Within Corticotrophs
ACTH and Related Peptide Expression Outside of Corticotrophs
Ectopic ACTH-Secreting Tumors
Corticotroph Neoplasms
Proopiomelanocortin Gene
POMC Gene Structure
POMC MRNA Transcription, Splicing, and Polyadenylation
POMC Biosynthesis and Processing
Glycosylation of POMC
C-Terminal Amidation of POMC
N-Terminal Acetylation of POMC
Proteolytic Processing of POMC
Proteolytic Processing Enzymes
Tissue Specificity of POMC Processing
POMC Mutations Leading to POMC Deficiency
Regulation of the HPA Axis
POMC Gene Regulation
Ontogeny of ACTH Regulation
Fetal and Neonatal Regulation of ACTH Secretion
Regulation of ACTH Secretion During Adrenarche, Puberty, and Adulthood
Hormonal and Pharmacological Regulators of ACTH
Corticotrophin-Releasing Hormone
CRH Stimulation of ACTH Secretion
Modulators of CRH Release
CRH Receptors
AVP
AVP Stimulation of ACTH Secretion
Modulators of AVP Release
AVP Receptors
Synergism Between CRH and AVP
Cytokines
Oxytocin
Glucocorticoids
Other Hormones and Pharmacological Regulators of ACTH Secretion
Physiological Regulation of ACTH Secretion
Secretion Dynamics of ACTH In Vivo
Circadian Regulation of ACTH Secretion
Physiological Regulators of ACTH Secretion
Hypoglycemia
Exercise
Starvation
Hypotension and Hypovolemia
Surgical Stress
Brain Death
Critical Illness
Cosyntropin Administration
High-Dose (HDT=250μg) Versus Low-Dose (LDT=1μg) Cosyntropin Test
Psychological and Emotional Stress
Effects of Secreted POMC-Derived Peptides in Physiology and Disease
ACTH
Adrenal Gland
Melanocortin 2 Receptor—the ACTH Receptor
Familial Glucocorticoid Deficiency Syndrome Type 1
Familial Glucocorticoid Deficiency Syndrome Type 2
Other Causes of Familial Glucocorticoid Deficiency Syndrome
ACTH-Independent Activation of ACTH Receptor Pathways
Nonadrenal Actions of ACTH
α-MSH
Skin
Melanocortin 1 Receptor
Melanocortin 5 Receptor
Brain
Melanocortin 4 Receptor
Melanocortin 3 Receptor
β-Endorphin
Additional Potential Melanocortin Actions
Measurement Assays for ACTH (RIA, IRMA, LC/MS, etc.)
References
4 Growth Hormone
Introduction
Growth Hormone Gene Structure
Somatotroph Development and Differentiation
GH Synthesis
Neuroendocrine Control of GH
Hypothalamic Hormones
Growth Hormone-Releasing Hormone
Somatostatin
GHRH and SRIF Interaction in Regulating GH Secretion
GH Autoregulation
Physiologic Factors Affecting GH Secretion
Aging
Gender
Sleep
Exercise
Stress
Nutritional and Metabolic Regulation
GH-Binding Proteins
Peripheral GH Action
GH Receptor
Insulin-Like Growth Factors (IGFs)
IGF-Binding Proteins
GH Action
Role of GH/IGF-1 in Growth and Development Throughout the Lifespan
Bone Acquisition
Bone Loss
GH and Metabolism
Lipids
Body Composition
Carbohydrate Metabolism
Protein Metabolism
Muscle Strength and Exercise Performance
GH and Reproduction
GH/IGF-1 and Cardiovascular Function
Effects of GH and IGF-1 on Cardiac Structure and Function
GH Therapy in Congestive Heart Failure
GH as a Biomarker of CVD
GH Effects on Renal Function
Tests of GH Secretion
Integrated 24-Hour GH Concentrations
Evaluation of GH Hypersecretion
Evaluation of GH Deficiency
Single GH and IGF-1 Measurements
Provocative Tests
Insulin-Induced Hypoglycemia (Insulin Tolerance Test)
Clonidine
l-Dopa/Propranolol
Arginine/GHRH
Ghrelin Mimetics
Glucagon
Approach to Provocative GH Testing
Variability of GH Assays
Variability of IGF Assays
Clinical Use of GH
GH Therapy in Childhood
Idiopathic Short Stature
Turner Syndrome
Children Born Small for Gestational Age
Chronic Renal Insufficiency
SHOX Gene Deficiency
GH Therapy in Adults
Adult GHD Syndrome
Etiology
Diagnosis
GH Replacement Therapy
Effects of GH Replacement Therapy
Hypopituitarism
Thyroid Hormone
Gonadal Steroids
Glucocorticoids
GH Replacement in Acromegaly
GH in the Healthy Elderly
GH Abuse by Athletes
Testing for GH Doping in Athletes
Complications of GH Treatment
Decreased IGF-1 Levels
Protein–Calorie Malnutrition, Starvation, Anorexia Nervosa
Diabetes Mellitus
Laron Syndrome
References
5 Prolactin
Introduction
Historical Overview
Cell of Origin
Lactotroph Ontogeny
Prolactin Gene
Pit-1
Estrogen
Ets
Other Transcription Factors
Signaling Pathways That Converge Upon the PRL Promoter
Hormone Biosynthesis
Prolactin Protein
Structural Characteristics and Posttranslational Modifications
Proteolysis
Macroprolactin
Placental, Decidual, and Lymphoblastoid Forms
Hormone Secretion: Biochemistry
Measurement of Prolactin
Assays and Bioassays
Clinical Testing
Artifacts
Hook Effect
Macroprolactin
Physiology
Metabolic Clearance and Production Rates of Prolactin
Hormone Secretion Patterns
Changes in Prolactin With Age
Changes in Prolactin Levels during the Menstrual Cycle
Changes in Prolactin Levels During Pregnancy
Changes in Prolactin Levels With Postpartum Lactation
Changes in Prolactin Secretion With Stress
Neuroendocrine Regulation
Prolactin-Inhibiting Factors
Dopamine
Gonadotrophin-Associated Peptide
γ-Aminobutyric Acid
Prolactin-Releasing Factors
Thyrotrophin-Releasing Hormone
VIP and Peptide Histidine Methionine (PHM)/PHI
Serotonin
Opioid Peptides
Growth Hormone-Releasing Hormone
Posterior Pituitary, Oxytocin, and Vasopressin
Gonadotrophin-Releasing Hormone
Renin–Angiotensin System
Other Neuroactive Peptides and Neurotransmitters
Histamine
Acetylcholine
Prolactin Short-Loop Feedback
Prolactin Action
Prolactin Receptor
PRLR Signal Transduction
Female Reproductive Tissues
PRL Effects on Breast
Galactorrhea
PRL and Breast Cancer
PRL Effects on Gonadotrophin Secretion
PRL Effects on the Ovary and Fertility
Clinical Effects of Hyperprolactinemia on Menstrual Function
Male Reproductive Tissues
Clinical Effects of PRL in Males
Carbohydrate Metabolism and Adiposity
Adrenal Cortex
Calcium and Bone Metabolism
Immune System
Acknowledgments
References
6 Thyroid-Stimulating Hormone
Introduction
Ontogeny of Thyrotroph Cells
TSH Subunit Genes
TSH β-Subunit Gene Structure
α-Subunit Gene Structure
TSH BIOSYNTHESIS
TSH transcription
TSH translation
TSH glycosylation
TSH folding, combination, and storage
Regulation of TSH Biosynthesis
Hypothalamic Regulation of TSH Biosynthesis
Peripheral Regulation of TSH Biosynthesis
TSH Secretion
Ontogeny of TSH Levels
Patterns of TSH Secretion
Regulation of TSH Secretion
Hypothalamic Regulation of TSH Secretion
Peripheral Regulation of TSH Secretion
Action of TSH
TSH Receptor Gene
TSH Receptor Structure
Determinants of TSH Receptor Binding
Signal Transduction at the TSH Receptor
TSH actions
Effects of TSH on Thyroid Gland Development and Growth
Effects of TSH on Thyroid Cell Morphology
Effects of TSH on Iodine Metabolism
Effects of TSH on the Synthesis of Thyroid Hormone
TSH-Induced Receptor Desensitization
Extrathyroidal Actions of TSH
TSH Measurements
Free TSH β- and α-Subunit Measurements
Provocative Testing of TSH
Drugs and TSH Levels
Drugs that Decrease Serum TSH Levels
Drugs That Increase Serum TSH Levels
Disorders of TSH Production
Acquired TSH Deficiency
Congenital TSH Deficiency
Acquired TSH Excess
Congenital TSH Excess
References
7 Gonadotrophin Hormones
Introduction
Development, Embryology, and Histology
Gonadotrophic Cells in the Pituitary
Molecular Basis of Gonadotroph Development
Biochemical Structure and Molecular Biology of LH and FSH
Hormone Structure
The α-Subunit
The LHβ Subunit
The FSHβ Subunit
Synthesis and Posttranslational Processing of LH and FSH
Ontogeny and Physiology of LH and FSH Secretion
Fetal Life
Postnatal Life and Childhood Years
Gonadotrophin Secretion During the Menstrual Cycle
Aging and Gonadotrophins
Biologic Functions of LH and FSH
Roles of LH and FSH in the Male
The Roles of LH and FSH in the Female
Gonadotrophin Receptors
Assay Systems for the Measurement of Gonadotrophins
Radioimmunoassays for LH and FSH
Improvements in LH and FSH Immunoassays
Bioassays for LH and FSH
Hypothalamic Regulation of LH and FSH
GnRH Neuronal Development
GnRH Secretion
GnRH Action
Influence of Patterns of Pulsatile GnRH
Feedback Regulation of LH and FSH Secretion
Estrogens
Progesterone
Androgens
Inhibins, Activins, and Follistatins
Molecular Biology of LH and FSH Subunit Genes
α-Subunit
LHβ Subunit
FSHβ Subunit
Diagnostic Tests
GnRH Stimulation Test
Clomiphene Test
Detection and Characterization of Gonadotrophin Pulse Patterns
Clinical Disorders Affecting the Gonadotroph
Hypogonadotrophic Disorders
Congenital Hypogonadotrophic Disorders
Heterogeneity of Pulsatile Gonadotrophin Secretion in Patients with Congenital HH
Nonreproductive Phenotypes Associated with Kallmann Syndrome and HH
Mutations in the Genes Encoding LHβ and FSHβ Subunits
Hypogonadism Associated with Mutations in the FSHβ Gene
Hypogonadism Associated with Mutations of the LHβ Gene
Inactivating Mutations of LH and FSH Receptor Genes
Inactivating Mutations of the LH Receptor Gene
Inactivating Mutations of FSH Receptor Gene
Other Congenital Hypogonadotrophic Syndromes
Prader–Willi Syndrome
Laurence–Moon–Biedl Syndrome
Miscellaneous Congenital Hypogonadotrophic Syndromes
Transcription Factor Mutations
Acquired Hypogonadotrophic Disorders
Hypothalamic Amenorrhea
Chronic Renal Failure and Gonadal Dysfunction
Hemochromatosis
Hyperprolactinemia and Hypogonadotropism
Space-Occupying Lesions
Hypothalamic Syndromes
Hypergonadotrophic Disorders: Excessive or Nonphysiologic Secretion of Gonadotrophins
Ectopic Gonadotrophin Secretion
Central or Gonadotrophin-Dependent Precocious Puberty
Activating Mutations of the LH Receptor
Activating Mutations of the FSH Receptor
Polycystic Ovarian Syndrome
Treatment of Hypogonadotrophic Disorders
Gonadotrophin Treatment of HH
Pulsatile GnRH Therapy
GnRH Analogues
GnRH Agonists
GnRH Antagonists
Acknowledgments
References
8 The Posterior Pituitary
Introduction
Structure of the Neurohypophysis: Anatomy and Electrophysiology of Vasopressin-Producing Cells
The Vasopressin and Oxytocin Genes
Gene Structure
Expression of the Vasopressin Gene in Diabetes Insipidus Rats (Brattleboro Rats)
Expression of the Vasopressin Gene in Autosomal Dominant and Autosomal Recessive Diabetes Insipidus in Humans
Chemistry, Processing, and Metabolism of AVP
Control of AVP Secretion
Osmotic Stimulation
Osmoreceptors in the Brain and the Periphery
Osmotic Threshold: Sensitivity or Gain of the Osmoreceptor/AVP-Releasing Unit
Baroregulation
Hormonal Influences on the Secretion of Vasopressin
Vasopressin Receptors and Antagonists
Cellular Actions of Vasopressin
Quantitating Renal Water Excretion
Clinical Characteristics of Diabetes Insipidus Disorders
Neurogenic Diabetes Insipidus
Common Forms
Rare Forms
Autosomal Dominant and Recessive Neurogenic Diabetes Insipidus
Wolfram Syndrome
Syndrome of Hypernatremia and Hypodipsia
Nephrogenic Diabetes Insipidus
Loss-of-Function Mutations of the AVPR2
Rareness and Diversity of AVPR2 Mutations
Most Mutant V2 Receptors are not Transported to the Cell Membrane and are Retained in the Intracellular Compartments
Nonpeptide Vasopressin Receptor Antagonists Act as Pharmacological Chaperones to Functionally Rescue Misfolded Mutant V2 Re...
Gain-of-Function of the Vasopressin V2 Receptor: Nephrogenic Syndrome of Inappropriate Antidiuresis
Loss-of-Function Mutations of AQP2
Complex Polyuropolydipsic Syndrome
Acquired NDI (Table 8.3)
Primary Polydipsia
Diabetes Insipidus and Pregnancy
Pregnancy in a Patient Known to Have Diabetes Insipidus
Syndromes of Diabetes Insipidus that Begin during Gestation and Remit after Delivery
Investigation of a Patient With Polyuria
Indirect Tests for Diabetes Insipidus
Direct Tests of Diabetes Insipidus
Therapeutic Trial of dDAVP
Carrier Detection, Perinatal Testing, and Early Treatment
Radioimmunoassay of AVP, Copeptin, and Other Laboratory Determinations
Radioimmunoassay of AVP
AQP2 Measurements
Plasma Sodium, Plasma, and Urine Osmolality
Magnetic Resonance Imaging in Patients With Diabetes Insipidus
Treatment
Syndrome of Inappropriate Secretion of the Antidiuretic Hormone (SIADH)
Signs, Symptoms, and Treatment of Hyponatremia
Acknowledgments
References
II: Hypothalamic–Pituitary Disorders
9 The Hypothalamus
Introduction
Anatomy
Hypothalamic Physiology
Hypothalamic Endocrine and Metabolic Functions
Control of Anterior Pituitary Function
Appetite Control
Water Metabolism
Hypothalamic Nonendocrine Functions
Temperature Regulation
Sleep–Wake Cycle and Circadian Rhythm Control
Regulation of Visceral (Autonomic) Functions
Emotional and Cognitive Functions
Pathophysiology of Hypothalamic syndromes
Clinical Features of Hypothalamic Syndromes
Endocrine and Metabolic
Anterior Pituitary Dysfunction
Activating Lesions
Central Precocious Puberty
Acromegaly
Cushing Disease
Lesions with Hypothalamic Loss of Function
Hyperprolactinemia
Hypothalamic Hypogonadism
Growth Hormone Deficiency
Acquired GH Deficiency
Congenital GH Deficiency
Hypothalamic Hypoadrenalism
Hypothalamic Hypothyroidism
Disorders of Water Metabolism
Central Diabetes Insipidus
Adipsic or Essential Hypernatremia (Cerebral Salt Retention Syndrome)
Syndrome of Inappropriate Secretion of Antidiuretic Hormone
Disorders of Caloric Balance
Hypothalamic Obesity
Diencephalic Syndrome of Infancy
Anorexia Nervosa
Diencephalic Glycosuria
Nonendocrine
Deranged Control of Body Temperature
Hyperthermia
Hypothermia
Poikilothermia
Sleep–Wake Cycle and Circadian Abnormalities
Behavioral and Emotional Abnormalities
Diencephalic Epilepsy
Specific Hypothalamic Disorders
Prader–Willi Syndrome
Ciliopathies
Optic Nerve Hypoplasia
Environmental Deprivation Syndrome (Psychosocial Short Stature)
Hypothalamic Hamartoma
Germ Cell Tumors
Optic Chiasm and Hypothalamic Gliomas
Craniopharyngioma
Suprasellar Meningiomas
Suprasellar Arachnoid Cyst
Colloid Cyst of the Third Ventricle
Hematologic malignancies
Leukemia
Lymphoma
Inflammatory lesions
Infiltrative Disorders
Hypothalamic–Pituitary Sarcoidosis
Langerhans’ Cell Histiocytosis
Brain Irradiation
Traumatic brain injury
Acknowledgments
References
10 Anterior Pituitary Failure
Introduction
Mortality
Etiology
Structural Causes of Pituitary Failure
Mass Lesions
Pituitary Tumors
Pituitary Surgery
Nonpituitary Neoplasms
Cystic Lesions
Aneurysms
Infiltrative Lesions
Hypophysitis
Sarcoidosis and Other Granulomatous Diseases
Hemochromatosis
Pituitary Irradiation
Infectious Etiologies
Pituitary Hemorrhage
Apoplexy
Sheehan Syndrome
Congenital and Inherited Pituitary Insufficiency
Developmental Pituitary Dysfunction
Genetic Factors
Traumatic Brain Injury
Empty Sella Syndrome
Functional Causes of Pituitary Failure
Functional Central Adrenal Insufficiency
Functional Central Hypothyroidism
Functional Hypogonadotrophic Hypogonadism
Functional Growth Hormone Deficiency
Clinical Manifestations
Manifestations of Secondary Adrenal Insufficiency
Adrenal Androgen Insufficiency
Manifestations of Thyrotrophin Deficiency
Manifestations of Hypogonadotrophic Hypogonadism
Adult Males
Prepubertal Males
Neonatal Males
Female Hypogonadotrophic Hypogonadism
Adult Females (Secondary Amenorrhea)
Primary Amenorrhea/Pubertal Delay
Growth Hormone Deficiency
Adult-Onset GHD
Cardiovascular Risk
Childhood-onset GHD
Prolactin Deficiency
Diagnostic Testing
Assay Variability
Assessment of Pituitary Function
Corticotroph Assessment
Basal Testing
Dynamic Testing
Insulin Tolerance Testing
Overnight Metyrapone Test
ACTH Stimulation Testing
CRH Stimulation
Comparison of Tests
Testing After Pituitary Surgery
Diagnosis of Adrenal Androgen Deficiency in Women
Thyrotroph Assessment
TRH Testing
Gonadotroph Assessment
Adult Males
Adolescent Males
GnRH Stimulation Testing
Clomiphene Stimulation Test
hCG Stimulation Test
Adult Females
Primary Amenorrhea
Somatotroph Assessment
Adults
The ITT
GHRH–Arginine Stimulation Test
Glucagon Stimulation Test
Recommendations
Somatotroph Assessment in Children
GH Stimulation Testing in Children
Agents Used for GH Stimulation Testing in Children
Lactotroph Assessment
Special Considerations
Evaluation of Patients After TBI
Evaluation of Pituitary Function in Critical Illness
Treatment of Hypopituitarism
Recovery of Pituitary Function After Neurosurgical Treatment
Hormonal Replacement
Glucocorticoid Replacement
Androgen Replacement in Women
Thyroid Hormone Replacement
Gonadal Steroid Replacement
Female Hypogonadotrophic Hypogonadism
Estrogen Replacement in Adult Women
Inducing Puberty in Females
Inducing Fertility in Females
Male Hypogonadotrophic Hypogonadism
Testosterone Replacement in Men
Inducing Puberty in Males
Inducing Fertility in Males
Growth Hormone Replacement
Adult-Onset GHD
Clinical Benefits of GH Replacement
Body Composition
Bone Density
Cardiovascular Markers
Quality of Life
Monitoring Therapy
Children and Adolescents
References
11 Pituitary Dysfunction in Systemic Disorders
Introduction
Systemic Disorders Directly Affecting the Pituitary Gland
Pituitary Granulomas
Sarcoidosis
Granulomatosis With Polyangiitis (Formerly Wegener’s Granulomatosis)
Granulomatous Hypophysitis
Necrotizing Hypophysitis
Langerhans Cell Histiocytosis
Erdheim–Chester Disease
Autoimmune
Lymphocytic Hypophysitis
Immunoglobulin G4-related Hypophysitis
Polyglandular Autoimmune States
Amyloidosis
Infectious Diseases
Acquired Immune Deficiency Syndrome
Other Infectious Diseases
Pituitary Abscess
Iron Overload
Snakebite
Metastatic Cancer (see also chapter: Nonpituitary Sellar Masses)
Genetic Multiglandular Tumoral Syndromes (see also chapter: Genetics of Pituitary Tumor Syndromes)
Multiple Endocrine Neoplasia
Carney Complex (see also chapter: Acromegaly)
McCune–Albright syndrome (see also chapter: Acromegaly)
Other Stalk and Pituitary Lesions (see also chapter: Nonpituitary Sellar Masses)
Stalk Hemangioblastoma
Chordoid Glioma
Hemangiopericytoma
Fabry Disease
Changes in Pituitary Morphology and Function with Aging
General Effects of Systemic Illness on Pituitary Function
Pituitary Alterations Associated With Specific Systemic Disorders
Obesity
Malnutrition
Anorexia Nervosa
Diabetes Mellitus
Chronic Kidney Disease
Liver Disease
Other Endocrine Disorders
Primary Adrenal Insufficiency
Primary Hypothyroidism
Hyperthyroidism
Acknowledgments
References
12 Drugs and Pituitary Function
Introduction
Opiates and Opiate Antagonists
Opiates
Opiate Antagonists
Amphetamines and Methylphenidate
Caffeine
Benzodiazepines
Antidepressants
Lithium
Antipsychotic Drugs
Other Dopamine Antagonists
Dopamine Agonists
Cholinergic Agonists and Antagonists
Antihypertensives
Antihistamines
H1-Antihistamines
H2-Antihistamines
Cancer Therapies
Antineoplastic Chemotherapy
Immunotherapies
Estrogens
Androgens
Antiandrogens
Glucocorticoids
Endocrine-Disrupting Chemicals
Miscellaneous Drugs
Alcohol
Smoking
Cigarettes and Nicotine
Marijuana
Cocaine
Acknowledgments
References
13 The Pituitary Gland in Pregnancy
Introduction
Normal Pituitary during Pregnancy
Prolactin
Gonadotrophins
Growth Hormone
Thyrotrophin
Corticotrophin
Posterior Pituitary
Pituitary Tumors and Pregnancy
Prolactinomas
Acromegaly
Cushing Disease
Thyrotrophinomas
Clinically Nonfunctioning Pituitary Adenomas
Nontumoral Pituitary Disturbances Related to Pregnancy
Lymphocytic Hypophysitis
Hypopituitarism
Conclusions
References
14 Psychiatric Disease in Hypothalamic–Pituitary Disorders
Introduction
Historical Perspective
Psychiatric Diseases in Hypothalamic–Pituitary Disorders
Hypopituitarism
Corticotroph Insufficiency
Thyrotrophin Insufficiency
Gonadotrophin Insufficiency
Somatotrophin Insufficiency
Hormone Excess Syndrome and Psychiatric Disorders
Acromegaly
Cushing’s Syndrome
Prolactinoma
Rare Diseases of the Hypothalamus/Pituitary Gland and Psychiatric Disorders
Diagnosing Psychiatric Disorders in Hypothalamlic–Pituitary Diseases
Therapy of Psychiatric Disorders in Hypothalamic–Pituitary Disease
Acknowledgments
References
III: Pituitary Tumors
15 Acromegaly
Introduction
Epidemiology
Animal Models of Hypersomatotrophism
Pathogenesis
Pituitary Acromegaly
Pathogenesis of Somatotroph Cell Adenomas
Extrapituitary Acromegaly
Criteria for Diagnosis of Ectopic Acromegaly
GHRH Hypersecretion
GH Hypersecretion
Acromegaloidism
Genetic Syndromes
McCune–Albright Syndrome
Multiple Endocrine Neoplasia
Carney Complex
Familial Acromegaly
Gigantism
Clinical Features of Acromegaly
GH Action in Acromegaly
Effects of Excessive GH Secretion
Skeletal Changes
Skin Changes
Cardiovascular Complications
Respiratory Complications
Neuromuscular Changes
Psychologic Changes
Development of Neoplasms
Endocrine Complications
Effects on Morbidity and Mortality
Diagnosis
Differential Diagnosis
Treatment of Acromegaly
Aims
Goals of Therapy
Surgical Management
Side Effects of Surgery
Radiation Treatment
Stereotactic Radiosurgery
Side Effects of Radiotherapy
Medical Treatment
Dopamine Agonists
Somatostatin Receptor Ligands
Oral Octreotide Capsules
GH Receptor Antagonist
Selective Estrogen Receptor Modulators
Choice of Therapy
References
16 Prolactinoma
Introduction
Classification
Epidemiology and Natural History of Prolactinomas
Pathogenesis
Familial Prolactinomas
Altered Chromatin Remodeling
Cell Cycle Dysregulation
Growth Factor and Hormone Signaling
Aberrant Expression of Developmental Factors
Other Mechanisms
Clinical Manifestations
Endocrine Symptoms
Women
Men
Children and Adolescents
Local Mass Effects
Diagnosis
Hyperprolactinemia
Prevalence of Hyperprolactinemia
Causes of Hyperprolactinemia Other Than Prolactinomas
Pregnancy
Medications (Table 16.2)
Antipsychotics
Antidepressants
Opiates
Antihypertensive Drugs
Other Medications
Stress
Renal Disease
Cirrhosis
Hypothyroidism
Adrenal Insufficiency
Neurogenic
Ectopic Prolactin Secretion
Hypothalamic/Pituitary Stalk Disease
“Idiopathic” Hyperprolactinemia
Hyperprolactinemia Due to Genetic Resistance to PRL
Imaging
Treatment
Observation
Medical Therapy
Efficacy of Dopamine Agonists
Bromocriptine
Cabergoline
Pergolide
Quinagolide
Side Effects of Dopamine Agonists
Cabergoline: Possible Association with Cardiac Valve Disease
Discontinuation of Medical Therapy
Dopamine Agonist Resistance
Definition of Dopamine Agonist Resistance
Mechanisms of Dopamine Agonist Resistance
Resistance to Prolactin-Lowering and Antitumoral Effects of Dopamine Agonists
Medical Treatment of Prolactinomas in Children and Adolescents
Medical Therapy: Conclusions
Surgery
Surgical Indications and Approaches
Surgical Success Rates
Recurrence and Long-Term Cure
Predictors of Remission and Cure
Complications of Surgery
Radiotherapy
Pitfalls in Analysis of Radiotherapy Studies
Efficacy of Fractionated Radiotherapy
Efficacy of Stereotactic Radiosurgery
Selecting the Mode of Radiotherapy
Complications of Fractionated Radiotherapy
Complications of Stereotactic Radiosurgery
Which Therapeutic Strategy?
Microadenomas
Macroadenomas
Special Situations
Giant Prolactinomas
Malignant Prolactinomas
Prolactinomas in Multiple Endocrine Neoplasia
Acknowledgment
References
17 Cushing Disease
Introduction
Pathophysiology
Epidemiology
Chronic ACTH and Proopiomelanocortin (POMC) Peptide Oversecretion by the Pituitary
ACTH Synthesis and Secretion
Mechanisms of ACTH Biosynthesis
Regulation of ACTH Secretion
Oversecretion of ACTH in Cushing Disease
Cushing’s Hypothesis
Demonstrating ACTH Oversecretion
ACTH Secretion is Dysregulated, Not Autonomous
The Source and Mechanism of ACTH Oversecretion in Cushing Disease
Anterior Pituitary Corticotroph Adenoma
POMC Gene Expression is Qualitatively Unaltered
POMC Gene Expression is Relatively Resistant to Glucocorticoid Feedback
Variants of the Anterior Pituitary Corticotroph Adenoma
Familial Pituitary Adenomas and Cushing Disease
Intermediate Lobe Pituitary Adenoma
Hypothalamus-Dependent Cushing Disease
Corticotroph Cell Hyperplasia and Adenoma Formation
Effects of Chronic ACTH and POMC Peptide Oversecretion
Effects of ACTH on Corticosteroid Secretion and the Adrenal Gland
Extra-Adrenal Effects of ACTH and POMC Peptides
Pathology of the Adrenal in Cushing Disease
Simple Diffuse Hyperplasia
Multinodular Hyperplasia
Adrenal Rests
Other Causes of Cushing’s Syndrome
ACTH-Dependent Spontaneous Cushing’s Syndromes
CRH-Secreting Tumors
Ectopic ACTH Syndrome
ACTH-Independent Spontaneous Cushing’s Syndrome
Primary Adrenocortical Tumors
Other Adrenocortical Disorders
Gonadal Tumors
Iatrogenic Cushing’s Syndromes
Exogenous Glucocorticoids
Exogenous Cosyntropin
Clinical Features
Diagnosis of Cushing Disease
Routine Laboratory Tests
Clues to Clinical Diagnosis of Chronic Hypercortisolism
Challenges in Diagnosis
Cushing Disease in Children
Cushing Disease in Pregnant Women
Long-Term Outcome of Cushing Disease
Diagnostic Approach
Question 1: “Does This Patient Have Cushing’s Syndrome?”
Baseline Measurements
Suppression Tests
Question 2: “What is the Cause of Cushing’s Syndrome in this Patient?”
Plasma ACTH
Plasma Non-ACTH POMC Peptides
Plasma Adrenocortical Androgens
Establishing the Pituitary Origin of the ACTH-Driven Hypercortisolemic State
Noninvasive Baseline Tests
Dynamic Noninvasive Testing
Invasive Testing: Bilateral Inferior Petrosal Sinus Sampling
Imaging Techniques
Pituitary
MRI
CT Scanning
Skull X-rays
Adrenal Glands
Pitfalls in Diagnosis
Drug Interactions
Inducers of High CBG Plasma Levels
Liver Enzyme Inducers
Antiglucocorticoids (Mifepristone)
Glucocorticoids
Glycyrrhetinic Acid
Intercurrent Illness
Hypercortisolemic States Without Cushing’s Syndrome
Depression
Anorexia Nervosa
Alcohol
Stress
Strenuous Exercise
Pregnancy
Familial Resistance to Glucocorticoids
Tests to Distinguish Between “Pseudo-Cushing” and Cushing’s Syndrome
Pitfalls in Differential Diagnosis
Cushing Disease Mimicking an Autonomous Adrenocortical Tumor
Severe Cushing Disease Mimicking Classic Ectopic ACTH Syndrome
Mild Ectopic ACTH Syndrome Mimicking Classic Cushing Disease
Strategy for Diagnosis and Differential Diagnosis
Whom to Screen for Cushing’s Syndrome
When to Screen for Cushing’s Syndrome
A Stepwise Strategy
The Hypercortisolemic State
Causes of the Hypercortisolemic State
Treatment
Pituitary Surgery and Radiation
Surgery
Radiation
Conventional Radiotherapy
Stereotactic Radiosurgery with the Gamma Knife
Heavy-Particle Radiotherapy
Medical Treatments
Reversible Adrenal Steroidogenesis Inhibitors (Table 17.5)
Metyrapone
Imidazole Derivatives
Irreversible Adrenal Steroidogenesis Inhibitors
Therapy to Lower ACTH from the Corticotroph Tumor
Blockade of the Glucocorticoid Receptor
Adrenal Surgery
Total Bilateral Adrenalectomy
Nelson Syndrome
Overall Approach for Treatment
Pituitary Surgery as the First-Line Treatment
Failure of Pituitary Surgery
Pituitary Surgery for Failed Remission or Relapse
When Other Options May be Better as First-Line Treatment
Other Options for Persistent/Recurrent Disease
Future Directions
Acknowledgments
References
18 Thyrotrophin-Secreting Pituitary Tumors
Introduction
Pathogenesis
Hormone-Regulatory Pathways
Pituitary Hyperplasia in Long-Standing Hypothyroidism
Impaired Thyroid Hormone Negative Feedback
Altered Hypothalamic Signaling
Thyrotrophin-Releasing Hormone (TRH)
Dopamine
Somatostatin
Alterations in Pituitary Transcription Factors
Oncogenes, Tumor Suppressor Genes, and Growth Factors
Familial/Genetic Syndromes
Pathology
Clinical Features
Hyperthyroidism
Goiter
Pituitary Tumor Mass Effect
Hormone Cosecretion
Diagnosis
Laboratory Studies
Dynamic Tests
TRH Test
T3 Suppression Test
Octreotide Test
Circadian Secretion of TSH
Pituitary Imaging
Differential Diagnosis
Impaired Sensitivity to Thyroid Hormones
Resistance to Thyroid Hormone β (RTHβ)
Resistance to Thyroid Hormone α (TRHα)
Monocarboxylate Transporter 8 (MCT8) Defect
Selenocysteine Insertion Sequence-Binding Protein 2 (SBP2) Gene Defect
Euthyroid Hyperthyroxinemia
Treatment
Surgery
Medical Treatment
Somatostatin Analogues
Dopamine Agonists
Radiotherapy
Criteria of Cure and Follow-Up
Conclusions
References
19 Nonfunctioning and Gonadotrophin-Secreting Adenomas
Introduction
Epidemiology
Pathology
Gonadotroph Adenoma Hormone Secretion
Gonadotrophin Subunits: α, FSHβ, and LHβ
Other Secretory Products
Stimulated Secretion (Figs. 19.1 and 19.2)
Gross Pathology
Etiology
Clinical Features
Visual Field Defects
Headaches
Hypopituitarism
Assessment
Imaging
Neuro-Ophthalmological Evaluation
Hormonal Evaluation
Differential Diagnosis
Treatment
Surgery
Radiotherapy
Medical Therapy
Predictors of Regrowth
Follow-Up
Gonadotrophinomas
Clinical Features
Hormonal Evaluation
Men
Women
Distinguishing a Gonadotroph Adenoma From Primary Hypogonadism
References
20 Atypical Pituitary Adenomas
Introduction
Current WHO Definition of Atypical Pituitary Adenomas
Controversies Regarding the Nature and Diagnosis of Atypical Pituitary Adenomas
Genetics and Molecular Basis of Atypical Pituitary Adenomas
Genetic and Chromosomal Factors
Clinical Characteristics and the Functional Status of Atypical Pituitary Adenomas
Prognostic Factors in Atypical Pituitary Adenomas
Surgical Management of Atypical Pituitary Adenomas
Medical Management of Atypical Pituitary Adenomas
Temozolamide and Other Systemic Chemotherapies
Direct Chemotherapy
Radiation Therapy/Stereotactic Radiosurgery
Future Avenues
MicroRNA
Chemokines
Galectin-3
Novel Peptide Receptor Radionuclide Therapies
Conclusions
References
21 Genetics of Pituitary Tumor Syndromes
Introduction
Syndromic Conditions Associated With Pituitary Adenomas
Multiple Endocrine Neoplasia Type 1 (MEN1)
Multiple Endocrine Neoplasia Type 4 (MEN4)
Carney Complex
McCune–Albright Syndrome
Genetic Forms of Isolated Pituitary Adenomas
Familial Isolated Pituitary Adenomas
Aryl Hydrocarbon-Receptor Interacting Protein (AIP) Mutations
X-Linked Acrogigantism (X-LAG) Syndrome
Genetic Testing Overview
References
22 Nonpituitary Sellar Masses
Introduction
Clinical Diagnosis
Imaging Diagnosis
Morphological Diagnosis
Specific Lesions
Craniopharyngioma
Meningioma
TTF-1-Expressing Sellar Region Tumors
Metastatic Tumors [37,70–73]
Germinomas
Cysts
Inflammatory Lesions
Vascular Lesions
Conclusion
References
IV: Pituitary Procedures
23 Pituitary Imaging
Introduction
History of Pituitary Imaging
Plain Films and Tomograms
Size and Shape of Sella
Angiography
Computed Tomography
CT: Technical Factors
CT: Normal Anatomy
MR: Technique and Anatomy
Pituitary Size and Shape
Microadenomas
Gadolinium Enhancement
Macroadenomas
Posterior Pituitary
Other Intrasellar/Suprasellar Masses
Rathke’s Cleft Cysts
Craniopharyngiomas
Hamartomas
Aneurysms
Meningiomas
Hypothalamic–Chiasmatic Gliomas
Lymphocytic Hypophysitis
Other
Empty Sella
Imaging of the Empty Sella
References
24 Pituitary Surgery
Historical Overview
Diagnostic Evaluation
Surgical and FunctionalAnatomy
Surgical Techniques
Transsphenoidal Approach
Transcranial Pterional Approach
Complications
Further Treatment
Radiotherapy
Recurrences
Medical Treatment
Pituitary Tumors
Pituitary Adenomas
Nonfunctioning Pituitary Adenomas
Prolactinomas
Growth-Hormone-Producing Pituitary Adenomas
Cushing Disease
Nelson Syndrome
TSH-Producing Adenomas
Gonadotrophin-Producing Adenomas
Craniopharyngiomas
Supra- and Parasellar Meningiomas
Miscellaneous Cystic Lesions
Rathke’s Cleft Cysts
Intra- and Suprasellar Colloid Cysts
Arachnoid Cysts
Rare Pituitary Tumors
Optico–Hypothalamic Gliomas
Metastatic Tumors
Chordomas
Inflammatory Lesions
Hypophysitis
Pituitary Abscess
Hypothalamic Hamartomas
Germ Cell Tumors
Epidermoid Cysts
Evolving Technologies
References
Index

Citation preview

THE PITUITARY FOURTH EDITION

THE PITUITARY FOURTH EDITION

Edited by

SHLOMO MELMED, MB ChB, MACP Cedars Sinai Medical Center, Los Angeles, CA, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2017 Elsevier Inc. All rights reserved. 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). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability 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. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-804169-7 For Information on all Academic Press publications visit our website at https://www.elsevier.com

Publisher: Mica Haley Acquisition Editor: Tari Broderick Editorial Project Manager: Joslyn Chaiprasert-Paguio Production Project Manager: Julia Haynes Designer: Matt Limbert Typeset by MPS Limited, Chennai, India

Dedication

Dedicated to my wife Ilana, children, and grandchildren, in appreciation of their devoted support and continued inspiration. In appreciation of the superb expertise and professionalism of my scholarly colleagues who contributed to this text.

Contents

List of Contributors Preface

SECTION II

ix XI

HYPOTHALAMIC-PITU ITARY DISORDERS

SECTION I HYPOTHALAMIC-PITU ITARY FUNCTION 1. Pituitary Development

9. T he Hypothalamus

291

ANDREA GIUSTINA, STEFANO FRARA, ALFIO SPINA AND PIETRO MORTINI

3 10. Anterior Pituitary Failure

JACQUES DROUIN

329

JOHN D. CARMICHAEL

2. Hypothalamic Regulation of Anterior Pituitary Function

23

11. Pituitary Dysfunction in Systemic Disorders

ANAT BEN-SHLOMO AND SHLOMO MELMED

3. Adrenocorticotrophin

12. Drugs and Pituitary Function

47

13. The Pituitary Gland in Pregnancy

85

397

ANDREA GLEZER AND MARCELLO D. BRONSTEIN

VIVIEN S. BONERT AND SHLOMO MELv!ED

5. Prolactin

383

MARIA FLESERJU

CARMEN L. SOTO-RI VERA AND JOSEPH A. MAJZOUB

4. Growth Hormone

365

MARIA FLESERIU

14. Psychiatric Disease in HypothalamicPituitary Disorders

129

NADINE BINART

413

CAROLINE SIEVERS AND GUNTER K. STALLA

6. Thyroid-Stimulating Hormone

SECTION III

163

VIRGINIA D. SARAPURA AND MARY H. SAMUEL

7. Gonadotrophin Hormones

PITUITARY TUMORS 203

15. Acromegaly SHLOMO MELMED

URSULA B KAISER

8. The Posterior Pituitary

423

16. Prolactinoma

251

PHILIPPE CHANSON AND DOMINIQUE MAITER

DANIEL G. BICHET

vii

467

viii

17. Cushing Disease

CONTENTS

515

JOHN D.C NEWELL-PRJCE

18. Thyrotrophin-Secreting Pituitary Tumors

22. Nonpituitary Sellar Masses

631

LUIS V. SYRO, FABIO ROTONDO, OLGA MOSHKIN AND KALMAN KOVACS

573

SECTION IV

YONA GREENMAN

PITUITARY PROCEDURES 19. Nonfunctioning and GonadotrophinSecreting Adenomas

589

JOHN A.H. WASS AND NIKI KARAVITAKI

20. Atypical Pituitary Adenomas

21 . Genetics of Pituitary Tumor Syndromes

645

MARCEL MAYA AND BARRY D. PRESSMAN

605

DANIEL A. DONOHO AND GABRIEL ZADA

ADRIAN F. DALY AND ALBERT BECKERS

23. Pituitary Imaging

24. Pituitary Surgery

671

RUDOLF FAHLBUSCH AND MICHAEL BUCHFELDER

619

Index

689

List of Contributors

Albert Beckers, MD, PhD Belgium

Niki Karavitaki, MD, MSc, PhD University Birmingham, Birmingham, United Kingdom

University of Lie`ge, Lie`ge,

of

Anat Ben-Shlomo, MD Medicine/Pituitary Center Cedars Sinai Medical Center, Los Angeles, CA, United States

Kalman Kovacs, MD, PhD University of Toronto, Toronto, Ontario

Daniel G. Bichet, MD Universite´ de Montre´al, Hoˆpital du Sacre´-Coeur de Montre´al, Montre´al, QC, Canada

Dominique Maiter, MD, PhD Universite´ Catholique de Louvain, Brussels, Belgium Joseph A. Majzoub, MD Harvard Medical School Boston, Boston, MA, United States

Nadine Binart, PhD University Paris-Saclay, Le KremlinBiceˆtre Cedex, France

Marcel Maya, MD Cedars Sinai Medical Center, Los Angeles, CA, United States

Vivien S. Bonert, MD Cedars Sinai Medical Center, Los Angeles, CA, United States

Shlomo Melmed, MB ChB, MACP Cedars Sinai Medical Center, Los Angeles, CA, United States

Marcello D. Bronstein, MD, PhD University of Sa˜o Paulo Medical School, Sau Paulo, Brazil

Olga Moshkin, MD, PhD, FRCPC Peterborough Regional Health Centre, Peterborough, Canada

Michael Buchfelder, MD, PhD University of ErlangenNuremberg, Erlangen, Germany

Pietro Mortini, MD

John D. Carmichael, MD Keck School of Medicine at USC, Los Angeles, CA, United States

John D.C. Newell-Price, MA PhD FRCP University of Sheffield, Sheffield, United Kingdom

Philippe Chanson, MD Hoˆpital Biceˆtre and Universite´ Paris-Sud, Le Kremlin-Biceˆtre, France

Barry D. Pressman, MD, FACR Cedars-Sinai, Los Angeles, CA, United States

Adrian F. Daly, MB BCh, PhD University of Lie`ge, Lie`ge, Belgium

Fabio Rotondo, BSc University of Toronto, Toronto, Ontario Mary H. Samuels, MD Oregon Health and Science University, Portland, OR, United States

Daniel A. Donoho, MD University of Southern California, Los Angeles, CA, United States

Virginia D. Sarapura, MD University of Colorado Denver, Aurora, CO, United States

Jacques Drouin, PhD, FRSC Clinical Research Institute of Montreal (IRCM), Montre´al, QC, Canada Rudolf Fahlbusch, MD International Institute Hannover, Hannover, Germany

Caroline Sievers, PD, MD, MSc Max-Planck-Institute of Psychiatry, Munich, Germany

Neuroscience

Carmen L. Soto-Rivera, MD Harvard Medical School Boston, Boston, MA, United States

Maria Fleseriu, MD Oregon Health & Science University, Portland, OR, United States

Alfio Spina, MD Vita-Salute University, Milan, Italy

Stefano Frara, MD University of Brescia, Brescia, Italy Andrea Giustina, MD

Vita-Salute University, Milan, Italy

Gunter K. Stalla, MD Max-Planck-Institute of Psychiatry, Munich, Germany

University of Brescia, Brescia, Italy

Andrea Glezer, MD, PhD University of Sa˜o Paulo Medical School, Sau Paulo, Brazil

Luis V. Syro, MD Hospital Pablo Tobon Uribe and Clinica Medellin Carrera, Medelin, Colombia

Yona Greenman, MD Tel Aviv-Sourasky Medical Center, Tel Aviv, Israel Tel Aviv University, Tel Aviv, Israel

John A.H. Wass, MD, FRCP United Kingdom

Ursula B. Kaiser, MD MA, United States

Gabriel Zada, MD, MS University of Southern California, Los Angeles, CA, United States

Harvard Medical School, Boston,

ix

Churchill Hospital, Oxford,

Preface

The fourth edition of The Pituitary follows three widely read prior volumes published in 1995, 2002, and 2011. This textbook continues the tradition of a cogent blend of basic science and clinical medicine which was the successful hallmark of prior editions. The comprehensive text written by expert pituitary scholars is devoted to the pathogenesis, diagnosis, and treatment of pituitary disorders. The new fourth edition is extensively revised to reflect new knowledge derived from advances in genomics, molecular and cell biology, biochemistry, diagnostics, and therapeutics as they apply to the pituitary gland. Notably, new chapters devoted to genetics of pituitary tumor syndromes, atypical adenomas, and psychiatric dysfunction related to pituitary disease have been added to complement a comprehensive updated description of pituitary physiology, as well as management options for patients harboring pituitary tumors or exhibiting features of pituitary failure. The wide spectrum of clinical disorders emanating from dysfunction of the “master gland” is described in detail, as is the fundamental science underlying

pituitary dysfunction. Descriptions of mechanisms for disease pathogenesis provide the reader with an indepth understanding of both subcellular and extrinsic mechanisms subserving normal and disordered pituitary hormone secretion and action. I am especially indebted to my erudite expert colleagues for their creative scholarly contributions and dedicated efforts in compiling this extensive body of knowledge for students, trainees, physicians, and scientists geared to understanding pituitary function and caring for patients with pituitary disorders. Our desire is to continue to provide medical and doctoral students, clinical and basic endocrinology trainees, endocrinologists, internists, pediatricians, gynecologists, and neurosurgeons with a comprehensive yet integrated text devoted to the science and art of pituitary medicine.

Shlomo Melmed Cedars-Sinai Medical Center, Los Angeles, CA, United States

xi

C H A P T E R

1 Pituitary Development Jacques Drouin

INTRODUCTION

the seat of the soul. It was at the beginning of the 20th century that its endocrine functions became recognized [1] and thereafter the various hormones produced by the pituitary were characterized, isolated and their structure determined. The major role of the hypothalamus in the control of pituitary function was recognized by Harris in the mid-20th century and that marked the beginning of the new discipline of neuroendocrinology [2]. The adult pituitary is linked to the hypothalamus through the pituitary stalk that harbors a specialized portal system through which hypophysiotrophic hypothalamic hormones directly reach their pituitary cell targets [3,4]. The adult pituitary is composed of three lobes, the anterior and intermediate lobes that have a common developmental origin from the ectoderm, and the posterior lobe that is an extension of the ventral diencephalon or hypothalamus. Whereas the intermediate pituitary is a relatively homogeneous tissue containing only melanotroph cells that produce α-melanotrophin (αMSH), the anterior lobe contains five different hormonesecreting lineages, including the corticotrophs that produce adrenocorticotrophin (ACTH), the gonadotrophs that produce the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), the somatotrophs that produce growth hormone (GH), the lactotrophs that produce prolactin (PRL) and, finally, the thyrotrophs that produce thyroid-stimulating hormone (TSH). In addition, these tissues contain support cells, known as pituicytes or folliculostellate cells. The neural or posterior lobe of the pituitary is largely constituted of axonal projections from the hypothalamus that secrete arginine vasopressin and oxytocin (OT) as well as support cells. The intermediate lobe is present in many species, in particular in rodents, mice and rats, that have been used extensively to study pituitary development and function, but it regresses in humans at about the 15th week of gestation: it is thus

The pituitary gland has a relatively simple organization despite its central role as chef d’orchestre of the endocrine system. Indeed, the glandular portion of the pituitary, comprised of the anterior and intermediate lobes, contains six secretory cell types, each dedicated to the production of a different hormone. Long thought to be a random patchwork of cells, we are just now discovering that pituitary cells are organized in threedimensional structures and that the tissue develops following a precise stepwise plan. As for most tissues and organs, numerous signaling pathways are involved in pituitary organogenesis but it is mostly the discovery of regulatory transcription factors that has provided insight into mechanisms of pituitary development. Genetic analyses of the genes encoding these transcription factors have defined mechanisms for the formation of Rathke’s pouch, the pituitary anlage, and for expansion and differentiation of this simple epithelium into a complex network of endocrine cells that produce hormones while integrating complex inputs from the hypothalamus and bloodstream. The understanding of normal developmental processes provides a novel insight into mechanisms of pathogenesis: e.g., critical regulators of pituitary cell differentiation become the cause of hormone deficiencies when their genes carry mutations. This chapter surveys current notions of pituitary development highlighting the impact of this knowledge on understanding pituitary pathologies as well as identifying the challenges and gaps for the future.

THE PITUITARY GLAND The pituitary gland was ascribed various roles by anatomists over the centuries, including the source of phlegm that drained from the brain to the nose or

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00001-5

3

© 2017 Elsevier Inc. All rights reserved.

4

1. PITUITARY DEVELOPMENT

absent from the adult human pituitary gland. In view of the critical importance of the intermediate lobe in embryonic development, it is possible that the tissue is maintained in the developing human embryo for this very reason. Most of our recent insight into the mechanisms of pituitary development has come from studies in mice: the review of our current knowledge presented in this chapter will therefore primarily focus on mouse development with references to other species (including humans) when significant differences are known or in cases of direct clinical relevance.

FORMATION OF RATHKE’S POUCH The glandular or endocrine part of the pituitary gland derives from the most anterior segment of the surface ectoderm. It ultimately comprises the anterior and intermediate lobes of the pituitary. This was shown using chick-quail chimeras [5,6]. It is thus the most anterior portion of the midline surface ectoderm, the anterior neural ridge, which harbors the presumptive pituitary. Interestingly, fate-mapping studies also indicated that the adjoining neural territory will form the ventral diencephalon and hypothalamus. As head development is initiated and the neuroepithelium expands to form the brain, the anterior neural ridge is displaced ventrally and eventually occupies the lower facial and oral area. It is thus the midline portion of the oral ectoderm that invaginates to become the pituitary anlage, Rathke’s pouch. This invagination does not form through an active process but it rather appears to result from sustained contact between neuroepithelium and oral ectoderm at the time when derivatives of prechordal mesoderm and neural crest invade the space between neuroepithelium and surface ectoderm and thus separate these tissue layers everywhere except in the midline at the pouch level. Rathke’s pouch is thus a simple epithelium that is a few cells thick extending at the back of the oral cavity towards the developing diencephalon, with which it maintains intimate contact. This contact is essential for proper pouch and pituitary development since its rupture either physically [7 10] or through genetic manipulations [11,12] leads to aborted pituitary development. Indeed, a number of transcription factors expressed in diencephalon and infundibulum, but not in the pituitary itself, such as Nkx2.1 [11,13], Sox3 [14], and Lhx2 [12], are required for proper diencephalon development and secondarily affect pituitary formation. In humans, SOX3 mutations have been associated with hypopituitarism [14]. Collectively, these data have supported the importance of signal exchange between diencephalon and forming pituitary [15] for proper development of both tissues.

Rathke’s pouch rapidly forms a closed gland through disruption of its link with the oral ectoderm. This occurs through apoptosis of the intermediate epithelial tissue [16]. The oral ectoderm and Rathke’s pouch are marked by expression of transcription factors that are essential for early pouch development (Fig. 1.1). The earliest factors are the pituitary homeobox (Pitx, Ptx) factors, Pitx1 and Pitx2 [17,18]. Indeed, these two related transcription factors are coexpressed throughout the oral ectoderm and their combined inactivation results in blockade of development at the early pouch stage [16]. The double mouse mutant Pitx12/2Pitx22/2 exhibits delayed and incomplete disruption of tissues between developing pituitary and oral ectoderm, and pituitary development does not appear to be able to progress beyond this stage. The single Pitx22/2 mutant is somewhat less affected, reaching the late pouch stage [19 21]. The Pitx12/2 mutant has relatively normal pituitary organogenesis, except for underrepresentation of the gonadotroph and thyrotroph lineages [22] that express higher levels of Pitx1 protein in the adult [23]. The two Pitx factors thus have partly redundant roles in early pituitary development with Pitx2 having predominant and unique functions in organogenesis. Another pair of homeodomain transcription factors, the Lim-homeo factors Lhx3 and Lhx4, are also expressed in Rathke’s pouch after Pitx1 and Pitx2. The expression of Lhx3 and Lhx4 is in fact dependent on Pitx factors, and thus, the Pitx pair of factors may be considered to be at the top of a regulatory cascade for pituitary development. Interestingly, the double Lhx32/2Lhx42/2 mutant mice pituitary exhibits blocked development at the early pouch stage; it is thus a phenocopy of the double Pitx1/2 mutant [24]. The single Lhx3 and Lhx4 mutants have less-pronounced phenotypes, indicating that the actions of the two Lhx factors are also partly redundant with each other [25]. The phenocopy of the Pitx12/2Pitx22/2, and Lhx32/2/Lhx42/2 pituitary phenotypes clearly suggests that many of the actions of the Pitx factors are mediated through the Lhx3/4 factors. Consistent with these mouse studies, mutations in the LHX3 and LHX4 genes have been associated with combined pituitary hormone deficiency (CPHD), together with neck and/or skull malformations [26]. Rathke’s pouch is also marked by expression of the paired-like homeodomain factor Hesx1 (also known as Rpx). This factor has a complex pattern of expression in the early prechordal area, but its expression becomes restricted to the ventral diencephalon and Rathke’s pouch by e9.5 [27,28]. It thus marks the two sides of the developing neuroendocrine hypothalamo-pituitary system [28,29]. Pituitary Hesx1 expression is transient and is extinguished by about

I. HYPOTHALAMIC PITUITARY FUNCTION

FORMATION OF RATHKE’S POUCH

5

FIGURE 1.1 Development of the pituitary gland in mouse. Critical steps, signaling molecules, and transcription factors for pituitary development are highlighted on drawings representing the developing pituitary or Rathke’s pouch (red) from e9.5 to e17.5 of mouse embryonic development. At e9.5 and e10.5, the ventral diencephalon sequentially expresses Bmp4 and Fgf8 that are critical for Rathke’s pouch development; also, sonic hedgehog (Shh) expressed throughout the oral ectoderm, but excluded from Rathke’s pouch, is important for pituitary formation. The expression of critical transcription factors for either pituitary organogenesis or cell differentiation is listed in the middle column whereas the consequence of their gene inactivation is listed on the left. The position of the various mouse genotypes along the developmental time sequence indicates the stage at which pituitary development is interrupted by these mutations.

e12.5 following a pattern that is complementary to the appearance of Prop1 which antagonizes Hesx1 [15,30,31]. Inactivation of the mouse Hesx1 gene results in complex brain, optic and olfactory developmental defects; pituitary development is also perturbed, ranging from complete absence to multiple invaginations and

nascent glands [32]. Hesx1 thus appears to be involved in restriction of the neuroepithelium ectoderm contact at the midline where Rathke’s pouch is normally induced. This restriction/induction may be mediated through FGF10 since its expression is extended rostrally in Hesx12/2 mutants [31].

I. HYPOTHALAMIC PITUITARY FUNCTION

6

1. PITUITARY DEVELOPMENT

Hesx1 is a transcriptional repressor that recruits the Groucho-related corepressor Tle1 [31]. Other Tle-related proteins are expressed during pituitary development and interestingly, inactivation of Aes that is transiently expressed in the pituitary results in bifurcated pouches and dysplastic pituitaries [33]. Consistent with mouse studies, mutations of human HESX1 have been associated with septo-optic dysplasias that cause brain and optic nerve defects, together with hypopituitarism ranging from GH deficiency to CPHD [26]. A striking demonstration of the importance of tissue interactions for pituitary development was provided by the reproduction in tissue culture of self-forming pituitary pouches in association with aggregates of neural cells [34]. Upon addition of appropriate signals mimicking the normal developmental sequence, these pouches sequentially express Pitx1 followed by Lhx3; terminal differentiation toward the corticotroph lineage as marked by Tpit and ACTH expression, is achieved upon Notch signaling inhibition. This culture system thus reproduces the normal developmental scheme, highlighting the critical role of interactions between neural and surface ectoderms.

GLANDULAR OR ENDOCRINE GLAND DEVELOPMENT The early pituitary gland is constituted of an epithelial layer that is a few cells thick and encloses a lumen that will become the pituitary cleft between intermediate and anterior lobes. The portion of this pouch that is in close contact with the infundibulum will differentiate into the intermediate lobe. The first sign of glandular development is observed at the ventrorostral tip of the early gland where cells appear to leave the epithelial layer to take a more disorganized mesenchymal appearance. This period of transition is accompanied by intense cell proliferation and differentiated cells appear at the same time, as discussed below. This process is similar to epithelium mesenchyme transition (EMT) and it appears to be dependent on the homeodomain transcription factor, Prophet-of-Pit (Prop1). Prop1 is transiently expressed in the e10.5 e14.5 developing pituitary [35] and all adult pituitary cells derive from cells that have expressed Prop1 [36]. The Prop1 mutation prevents EMT and exhibits extensive expansion of the epithelial pituitary that becomes convoluted with an extended lumen [37,38]. At early stages, this mutant gland appears to be larger than normal but it then decreases in size through cell loss by apoptosis [38,39]. The Prop12/2 mutant is not entirely deficient in EMT and anterior lobe

development eventually proceeds. However, Prop1 is also required for activation of the Pit1 transcription factor gene, as indicated by its name [35,40], which is itself required for differentiation of the somatotroph, lactotroph, and thyrotroph lineages. Hence, Prop1 mutants are deficient in these lineages [35,40,41] but the mutation does not prevent corticotroph, melanotroph, or gonadotroph differentiation. The Prop1 mutant mice are thus dwarfed because of their deficit in GH and, indeed, PROP1 mutations have been associated with dwarfism and CPHD in humans [42]. With age, patients with PROP1 mutations often develop more extensive pituitary hormone deficiencies [43,44].

SIGNALS CONTROLLING PITUITARY DEVELOPMENT One of the early evidences for asymmetry and signaling at the onset of pituitary-hypothalamic development is the expression of BMP4 in the region of the ventral diencephalon that is overlying the area of stomodeal oral ectoderm where Rathke’s pouch will develop (Fig. 1.1). This expression is present at e8.5, and by around e10.5 it is replaced by Fgf8. Although inactivation of the BMP4 gene is early-lethal, analysis of a few surviving embryos at e9.5 suggested a failure of ectoderm thickening and initiation of Rathke’s pouch development [13]. The early phases of pituitary development are accompanied by complex and dynamic patterns of expression for many signaling molecules involved in development and organogenesis [45,46]. The BMPs are actually a good illustration of this complex interplay. As noted above, early expression of BMP4 in the ventral diencephalon appears to be important for induction of the ectodermal pituitary anlage and experiments designed to further test this role have used transgenic overexpression of the BMP antagonist Noggin in the oral ectoderm, including Rathke’s pouch, driven by the Pitx1 promoter [45]. This blockade of BMP signaling led to arrested pituitary development at the pouch stage, without much cell differentiation except for a few corticotrophs. This phenotype is similar to that of Pitx22/2 and Lhx32/2 mice [25]. BMP4 signaling may thus regulate Lhx3 expression or even the upstream Pitx factors, but this experiment tested the importance of continued BMP signaling more than its initial action as assessed in BMP42/2 embryos. Inactivation of the Noggin gene itself supported the critical role of BMPs in pituitary induction [47]. The early expression of BMP4 in ventral diencephalon is thus on the dorsal side of the developing pouch; in parallel with its extinction, the related BMP2 is expressed on the ventral side of

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the developing pituitary and in the surrounding mesenchyme (around e10.5). It has been proposed that ventral BMP2 may promote differentiation of so-called “ventral” lineages such as corticotrophs and this has been supported through transgenic gain-of-function experiments [45]. However, the use of organ culture systems to test the role of BMP2/4 in differentiation rather led to the conclusion that BMP signaling is repressing the corticotroph fate [46]. This latter finding is actually in agreement with a repressor effect of BMP signaling on proopiomelanocortin (POMC) gene transcription [48]. Whereas the highly dynamic pattern of expression of these two related BMPs, BMP4, and BMP2, and the consequences of their manipulation are highly suggestive of important roles in pituitary development and cell differentiation, the same rapid changes in expression and seemingly contradictory experimental results also hint that BMPs have multiple effects depending on the timing of action and target cells. We are thus still lacking a coherent and complete picture for the multiple actions of these signaling molecules. Another important signaling molecule for pituitary development is Sonic Hedgehog (Shh). Indeed, Shh is expressed in the ventral diencephalon and fairly widely in the oral ectoderm, but it is specifically excluded from the region of the oral ectoderm that becomes Rathke’s pouch [49]. In contrast, Shh target genes such as Patched1 are expressed in the developing pituitary, indicating that it is responsive to Shh signaling. These patterns are thus suggestive of an important role for the Shh pathway in pituitary induction. However, the Shh2/2 mutant mouse was not extremely informative in precisely defining this role since Shh is critical for formation of midline structures and the bulk of these structures are affected in the Shh mutants [50]. Nonetheless, the importance of Shh signaling for early pituitary development is also supported by mouse mutants for the Gli zinc finger transcription factors that mediate the effects of the Shh pathway. Indeed, the double mouse mutant Gli12/2; Gli22/2 fails to develop the pituitary whereas the single Gli22/2 mutant exhibits variable defects in pituitary formation [51]. Furthermore, overexpression of the Shh antagonist HIP blocked Rathke’s pouch development [49]. As indicated above, the early expression of BMP4 in the ventral diencephalon is replaced from about e10.5 by FGF8 and FGF10 and the expression of these growth factors is maintained throughout the active phase of pituitary expansion (e11.5 e14.5). The FGFs appear to be important for survival of early pituitary cells since mutant mice for FGF10 or for its receptor FGFR2IIIb initially form Rathke’s pouch and then it

regresses because of widespread apoptosis [52,53]. In agreement with this, transgenic overexpression of FGF8 led to pituitary hyperplasia [45]; further, these experiments suggested that FGF8 stimulates Lhx3 expression. This idea was also supported by analyses of the Nkx2.1 mutant mice that fail to express FGF8 in diencephalon and pituitary Lhx3 [13]. It is thus possible that the FGF effect on proliferation and/or maintenance of early pituitary cells is mediated through induction of Lhx3. The Wnt pathway also appears important for proliferation and/or survival of pituitary cells, but again the large number of Wnt molecules and their receptors expressed in and around the developing pituitary make it difficult to develop a coherent and complete picture of their role. Canonical Wnt signaling involves beta-catenin and targeted deletion of this gene using a Pitx1-Cre transgene resulted in a small pituitary, together with deficient Pit1 expression and Pit1-dependent lineages [30]. It was suggested that beta-catenin is acting directly on the Pit1 gene to regulate its expression through interaction with the upstream factor Prop1. Further, the canonical Wnt/beta-catenin pathway is acting through transcription factors related to Lef/TCF and targeted deletion of some members of this family altered pituitary development [30,32]. The involvement of these factors, such as TCF4, in both ventral diencephalon and Rathke’s pouch produces complex mutant phenotypes that result from intrinsic pituitary defects as well as from defective pituitary induction by overlying diencephalon [54]. Finally, the Notch pathway is also active in early pituitary development and recent work has suggested that its major involvement may be in pituitary progenitor cells; hence, this aspect is discussed below.

CELL DIFFERENTIATION Cell differentiation starts early during pituitary development, as assessed by expression of the hormone genes characteristic of each lineage [55]. The hormonecoding genes have also served as a starting point to identify cell-autonomous transcription factors that are involved in their own expression but also in lineagerestricted functions and differentiation. Hence, most of what we know about pituitary cell differentiation relates to the terminal stages of differentiation for each lineage and involves cell-restricted transcription factors that are responsible for terminal differentiation. The transcription factors that mark terminal differentiation are usually expressed 12 24 hours before the hormone gene itself and they have so far not been useful in directly identifying or studying multivalent progenitors of the

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developing or adult pituitary. However, the analysis of their loss-of-function mutations has provided considerable insight into the relationships between different lineages. Investigation of the Jackson and Snell dwarf mice that carry Pit1 mutations thus revealed the requirement for this Pou-homeo transcription factor for differentiation of three lineages, the somatotrophs, lactotrophs, and thyrotrophs [56,57]. Analyses of Pit1 mutants in both mice and humans thus supported the model of a common precursor for these three lineages [58].

Similarly, the Tpit2/2 mutant mice revealed an antagonistic relationship between corticotrophs/ melanotrophs and gonadotrophs, suggesting that these lineages share a common precursor [59]. Taken collectively, the data on these mutants have suggested a binary model of pituitary cell differentiation (Fig. 1.2). Although consistent with current data, this model has not been ascertained more directly, e.g., through characterization of the putative common progenitors. Nonetheless, it provides a useful framework for ongoing

FIGURE 1.2 Differentiation of pituitary cells. A scheme for sequential differentiation of cells in the developing pituitary was derived from studies of mutants for the critical cell-restricted regulators of differentiation. Putative pituitary stem and progenitor cells are marked by expression of Sox2. While critical regulators of terminal differentiation such as Tpit, SF1, Pit1, and Pax7 have been well characterized, regulators for the early commitment of putative precursors are still elusive. IL, intermediate lobe.

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PITUITARY CELL CYCLE CONTROL

investigation of the mechanisms of early commitment to each pituitary lineage. The salient features of this model and its regulatory molecules are discussed below in greater detail for each lineage.

TISSUE ARCHITECTURE AND CELL NETWORKS Until quite recently, the pituitary anterior lobe was considered to be a patchwork of intermingled cells of different lineages. This idea was challenged by the discovery that all somatotrophs are interconnected and form a homotypic network [60]. All cells of the gland may thus be part of the same (or very few) network(s) and the tridimensional (3D) organization of the GH cell network is unique compared to cell networks of other lineages or to the vasculature [61]. The exchange of signals between cells of homotypic networks may serve to mount a strong and coordinated secretory response and to adjust local blood flow accordingly. Thus, these 3D networks appear to increase the efficiency of hormone response and alter the patterns of response following endocrine resetting, such as occurs at sexual maturity [60] or as modified by gonadal steroids to create the sexual dimorphism of GH response [62]. Network plasticity is also implicated in adaptation of the prolactin response to lactation and this is mediated through changes in gap junctions between lactotrophs [63]. Somatotrophs and lactotrophs being the most abundant cells in the anterior pituitary, it is not difficult to imagine that these cells would have direct cell cell contacts but the cells of the less abundant lineages, such as corticotrophs and gonadotrophs, also form homotypic networks that extend throughout the gland. The corticotroph homotypic network has unique features: indeed, corticotrophs form columns or sheets of cells that extend from the ventral surface of the pituitary into the soma of the anterior lobe; many of these cell cell contacts could not be visualized previously before the availability of whole-tissue imaging since they are mediated by long cytoplasmic projections or cytonemes that extend between cells of other lineages to reach and establish contacts with other corticotrophs [64]. The relationship of the corticotroph network to the capillary bed is also striking as this network is often the furthest away from capillaries, whereas the gonadotroph network is closer to capillaries. Nonetheless, cells of both lineages have direct intimate contact with capillaries and these also rely on cytonemes [64,65]. The establishment of the cell networks during fetal development revealed an active process of cell cell interactions. Indeed, upon differentiation, newly differentiated cells migrate towards each other

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to establish homotypic contacts [65]. The first cells to reach terminal differentiation and to organize into a network are the corticotrophs that form streaks on the ventral surface of the developing pituitary; the streaks then infiltrate dorsally into the soma of the developing gland. It is presently not clear whether the initial establishment of the corticotroph network might serve a scaffolding role for organization of the other homotypic networks and, further, the molecular basis of these interactions remains undefined, although lineage-specific patterns of cadherin expression were described [66]. From the physiological perspective, the organization of cells into homotypic networks enhances the robustness, synchronicity, and amplitude of responses to secretagogues and regulation of network architecture can influence the nature of secreted hormone pulses as for the GH sexual dimorphism [62] and also provide a memory of recent stimulations as in lactation [63].

PITUITARY CELL CYCLE CONTROL There is extensive cell proliferation during fetal pituitary organogenesis; this expansion involves proliferation of Sox2-positive progenitors [67] and marker-negative putative precursors, both being negative for markers of differentiation [68]. These proliferating cells express cyclins that are involved in cell cycle progression such as cyclin A and cyclins D1, D2, and D3 (Fig. 1.3) and do not express significant levels of cell cycle inhibitors such as the inhibitors of the Cip/Kip family, p21Cip1, p27Kip1, and p57Kip2, or of the INK4 family [69]. These cells exit the cell cycle upon expression of the cell cycle inhibitor p57Kip2 and the same cells coexpress detectable levels of cyclin E [69]. These double-positive cells do not express any markers of hormone-producing cells and thus appear to be progenitors or precursors that have recently exited the cell cycle. They appear to represent a transient cell population from which differentiated cells arise: this interpretation is supported by the temporal sequence of appearance of appropriate markers and by their physical distribution going from the periluminal area of the developing anterior lobe that contains the proliferating progenitors towards the midgland that contains most of the noncycling p57Kip2 and cyclin E double-positive progenitors, and finally, more ventrally, the first differentiated cells (Fig. 1.1, e13.5). The first differentiated cells appear to express Tpit and POMC and they are followed by αGSU-expressing cells. These differentiated cells switch off p57Kip2 expression and switch on the related p27Kip1 in its place. Expression of p27Kip1 is maintained throughout adulthood in normal differentiated pituitary cells,

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FIGURE 1.3 Cell cycle exit of pituitary progenitors. The expression of various cyclins and cell cycle inhibitors of the Cip/Kip family is shown below a diagram representing different stages of pituitary cell differentiation, starting from cycling progenitors to differentiated adult hormone-producing cells. Although the scheme of differentiation highlights the Tpit-dependent differentiation into POMC lineages, expression of the various cell cycle regulators is similar during the course of differentiation of the other pituitary lineages. Adapted from Bilodeau S, Roussel-Gervais A, Drouin J. Distinct developmental roles of cell cycle inhibitors p57Kip2 and p27Kip1 distinguish pituitary progenitor cell cycle exit from cell cycle re-entry of differentiated cells. Mol Cell Biol 2009; 29(7): 1895 908.

whereas p57Kip2 is undetectable in differentiated pituitary cells. Both loss-of-function mutations for p57Kip2 and gain-of-function transgenic experiments have supported the model that p57Kip2 is responsible for driving pituitary progenitors out of the cell cycle [69,70]. Expression of p27Kip1 in differentiated cells is required to restrain cell cycling of these cells as supported by the presence of cycling differentiated cells in p27Kip12/2 pituitaries [69]. Furthermore, the loss of p27Kip1 expression in the adult pituitary leads to the formation of pituitary tumors, particularly in the intermediate lobe [71 73] and the double mutant p57Kip22/2; p27Kip12/2 presents fetal pituitaries in which all cells are proliferating, both progenitors and differentiated cells [69]. These later observations clearly indicate that mechanisms of cell differentiation are independent of cell cycle exit. Conversely, at least one model of blocked pituitary

differentiation, the Tpit2/2 intermediate lobe, indicated that expression of p27Kip1 is not dependent on differentiation, although switch-off of p57Kip2 expression appears to be partly dependent on this process [69]. However, p27Kip1 participates in silencing the pluripotency state by directly repressing the pluripotency Sox2 gene [74]. Nonetheless, the mechanisms for control of cell cycle and differentiation during pituitary development appear to be largely independent but the exact nature of the specific signals that are involved remains to be identified. While differentiated adult pituitary cells have been thought to be essentially nondividing, it appears that this is not the case but rather that they divide extremely slowly. Indeed, genetic ablation of dividing corticotrophs indicated that maintenance of these cells in the adult pituitary relies significantly on self-duplication; thus, adult corticotrophs are not postmitotic [75].

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PROGENITORS AND STEM CELLS

It is noteworthy that the third member of the Cip/Kip family p21Cip1 that is expressed at low levels in adult differentiated pituitary cells does not appear to play a major role in control of cell cycle progression or in pituitary tumorigenesis compared to p27Kip1 [76]. Rather, it was involved in control of cellular senescence [77]. Cellular senescence controlled by p21Cip1 may thus play a watchdog role by counterbalancing the effect of oncogenes associated with pituitary tumor development [78,79]. Interestingly, the impairment of progenitor state resulting from Hes1 inactivation in developing pituitaries increased p21Cip1 expression and led to apoptosis [70], consistent with a purported watchdog role for abnormal pituitary cell proliferation.

PROGENITORS AND STEM CELLS Expansion of the early anterior pituitary between e12 and e15 of mouse development is due to the rapid proliferation of cells that do not express any marker of terminal cell differentiation, such as hormones or cell-restricted transcription factors. These cells have thus been considered to be progenitors, but their level of commitment or partial differentiation cannot be evaluated because of a lack of appropriate markers. These cells contribute to significant growth of the gland during this transient period of expansion. Otherwise, it is the characterization of adult pituitary stem cells that has provided clues to the origin and early differentiation steps of pituitary lineages. Putative pituitary stem cells were first identified through a cell sphere assay and using cell markers developed in other tissues [80]. These pituisphereforming cells have the potential to differentiate into most hormone-producing lineages. It is, however, the realization that these putative adult pituitary stem cells express the embryonic stem cell marker, Sox2, that allowed their better characterization [67]. The Sox2positive pituitary stem cells are predominantly found along the cleft of the adult gland, and they are similarly positioned but far more abundant in the developing gland. After the initial cell expansion phase, a subset of Sox2-positive progenitors coexpress the related Sox9 transcription factor and it was suggested that Sox2; Sox9 double-positive cells may represent a shift to a quiescent adult progenitor state [81]. These cells also appear to express Prop1 and the Ret coreceptor GFRa2 [82]. Progenitors expressing either or both Sox2 and Sox9 can self-renew and differentiate into all lineages [83 85]. Undifferentiated cells of Rathke’s pouch and of the early pituitary express a subset of Notch pathway genes and their expression is lost upon differentiation [86].

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Notch signaling may be required to maintain the proliferative progenitor state [87] as suggested by gene inactivation of the Notch direct target transcription factor Rbp-J or its downstream target Hes1 [88 90]. In these knockout models, differentiation into corticotrophs that occurs early in organogenesis is accelerated, whereas differentiation into the later Pit1-dependent lineages is impaired. The premature differentiation observed in these models is correlated with decreased progenitor proliferation and increased expression of cell cycle inhibitors of the Cip/Kip family [70] that have been implicated in progenitor cell cycle exit [69]. Conditional inactivation of the Notch2 gene in progenitors supported this interpretation and also indicated that postnatal Notch22/2 progenitors may have altered identity as they show abnormal localization [91]. Collectively, these data support the idea that Notch signaling maintains the pituitary progenitor state and that during development it is essential for the sequential action of differentiation cues and the emergence of distinct lineages. The presence of putative stem cells in the adult pituitary suggested that cell renewal takes place in the adult gland. Although the normal adult pituitary has very low cell proliferation, most cells positive for proliferation markers in the adult gland do not express markers of the hormone-producing lineages but genetic marking studies showed very little differentiation from progenitors [83,84]. Further, organ ablation models, such as adrenalectomy and gonadectomy, showed expansion of mostly hormone-negative cells before the appearance of hormone-positive cells [92]. Interestingly, the combination of these two end-organ ablation paradigms [92] suggested expansion of a common pool of undifferentiated cells, in agreement with the existence of a common precursor for corticotrophs and gonadotrophs [59] and with tracing of the progeny of Sox2-positive cells [83]. These data argue in favor of a model in which stem cells expand before differentiation for tissue adaptation to stress, such as is caused by a major loss of feedback signals from end-organs. Nonetheless, various studies showed proliferation of differentiated cells [92,93]. The use of lineage markers [94] or of a conditional system to kill proliferating differentiated cells [75] has rather suggested that normal adult tissue maintenance primarily relies on division of differentiated cells in the anterior pituitary. Thus, both stem and differentiated cells of the adult pituitary divide in response to different physiological or pathophysiological signals. The nature of those signals remains elusive but they appear powerful: indeed, work aimed at testing the tumorigenic potential of pituitary stem cells using constitutive activation of Wnt signaling in those cells resulted in tumors that do not derive from stem cells themselves but rather surprisingly from adjacent cells [84].

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CORTICOTROPHS The corticotrophs are the first cells to reach terminal differentiation, and in the mouse they are first detected around e12.5 in the nascent anterior gland [55]. In fact, they appear to be the only cells to differentiate in mutant pituitaries that exhibit blocked organogenesis at the early pouch stage, such as in Pitx1/2 or Lhx3/4 mutants [16,25]. A highly cell-restricted transcription factor, the Tbox factor Tpit, was identified on the basis of its action on cell-specific regulatory elements of the POMC promoter [95]. Inactivation of the mouse Tpit gene showed that Tpit is critical for corticotroph differentiation; in addition, it is required for corticotroph expansion and/or maintenance [59]. In accordance with its late expression during differentiation (a half-day before POMC), Tpit deficiency does not appear to impair commitment of a subset of pituitary progenitors to the corticotroph lineage but rather blocks their terminal differentiation. Tpit is thus a positive regulator for differentiation of corticotrophs (Fig. 1.2). Tpit is also a negative regulator of the gonadotroph fate, and as a result, Tpit2/2 anterior pituitaries have an increased number of gonadotrophs [59]. At least part of this antagonism is exerted between Tpit and the gonadotroph-specific transcription factor SF1 on their respective gene targets through a mechanism of trans-repression [59]. This reciprocal mechanism results in blockade of SF1 target genes by Tpit, and vice versa; it is thus an excellent mechanism to implement a molecular switch between two cell fates. For this mechanism to be relevant, common precursors of corticotrophs and gonadotrophs would need to express both Tpit and SF1 and the balance between the two only needs to be tipped one way in order to ensure selection of one cell fate at the exclusion of the other. Such double-positive cells were indeed observed (albeit at very low frequency) in the fetal pituitary. As was predicted from its highly restricted cell distribution, mutations in the human TPIT gene result in isolated ACTH deficiency (IAD), a condition that was barely recognized before the discovery of Tpit [95,96]. IAD is a recessive inherited condition caused by the deficiency of pituitary ACTH, resulting in secondary adrenal glucocorticoid deficiency; it can be lethal for newborns and neonates because of abrupt and severe hypoglycemia [97]. IAD patients have no detectable pituitary ACTH and hypoplastic adrenal glands. The hormonal deficit is corrected by glucocorticoid therapy resulting in normal development. Many different TPIT gene mutations have been identified including premature stops, splice defects,

genomic deletions, and point mutations [95 98]. Many point mutations affect DNA binding and transcriptional activity and one particularly interesting mutation, Tpit M86R, is specifically deficient in protein protein interactions but not in DNA binding per se [99]. As a highly restricted marker of corticotroph cells, Tpit is a very convenient marker of corticotroph adenoma cells, particularly since its expression is not affected by glucocorticoids in these glucocorticoid-resistant tumors [100]. NeuroD1, a basic helix-loop-helix (bHLH) transcription factor, is another factor identified on the basis of its action on cell-specific transcription of the POMC promoter [101 103]. During fetal pituitary development, NeuroD1 is expressed transiently at high levels in corticotrophs but it is excluded from melanotrophs [103]. Consistent with this pattern, inactivation of the NeuroD1 gene results in a delay of POMC expression in anterior pituitary corticotrophs [102]. However, this delay is fully recovered by e15.5, a time when normal NeuroD1 expression has decreased in corticotrophs. This is suggestive of a transient requirement on NeuroD1 but not necessarily on its target sequence, the Eboxneuro, within the POMC promoter and, indeed, mutagenesis of this target sequence in a transgenic mouse assay indicated sustained dependence on the Eboxneuro for POMC expression throughout development and adulthood [104]. It was suggested that another bHLH factor takes over the role of NeuroD1 at mid-development and throughout adult corticotroph function. This factor is likely Mash1 (Ascl1) that occupies both enhancers of the POMC gene [105]. Mash1 is also highly expressed in adult melanotrophs and gonadotrophs. Corticotroph function is highly dependent on activation by hypothalamic signals and feedback repression by glucocorticoids. Activation of corticotroph function, POMC transcription, and ACTH release occurs primarily through the action of corticotrophin-releasing hormone (CRH) and its membrane receptor [106,107]. Expression of the CRH-R1 receptor appears upon corticotroph differentiation and corticotroph sensitivity to CRH action becomes active at midfetal development when the portal system between hypothalamus and pituitary becomes functional [108]. Similarly, the onset of glucocorticoid feedback repression on corticotrophs and ACTH secretion occurs at the same time [108,109]. POMC expression is also stimulated by the growth factors epidermal growth factor (EGF) [110] and leukemia inhibitory factor, the latter acting through the Stat3 transcription factor [111]; Stat3 action is antagonized by glucocorticoids [112]. CRH stimulates POMC gene transcription through activation of the orphan nuclear receptor NGFI-B

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MELANOTROPHS

(NR4A1, Nur77) and the related NR4A2 (Nurr1) and NR4A3 (NOR1) [113,114]; these MAPkinasedependent transcription factors form dimers that act on a palindromic DNA target site, the NurRE that was first described in the POMC promoter [113,115]. Glucocorticoids acting through their receptor GR exert their negative-feedback repression on POMC transcription by, at least in part, antagonizing the activity of the Nur factors through direct protein interactions, a mechanism known as trans-repression [116,117]. GR trans-repression of POMC transcription requires the chromatin remodeling proteins Brg1 and HDAC2 [118]. The action of signal-dependent transcription factors mediating the inputs of extracellular signals on POMC regulatory sequences is tightly associated with the activity of developmental regulators that define the corticotroph gene expression identity [119]. These regulatory mechanisms are maintained throughout adult life at apparently constitutive levels. However, they can be perturbed in pathological conditions. Notably, feedback repression of corticotroph POMC becomes desensitized in chronic stress and depressive states, but the mechanism of this misregulation is complex and not well understood [120 126]. Pituitary corticotroph adenomas that cause Cushing disease are also characterized by relative resistance to glucocorticoid feedback. In rare cases, these adenomas express a mutant GR [127]. Recent studies have identified more frequent deficiencies in two proteins that are required for glucocorticoid feedback and that may account for hormone resistance in corticotrophinomas. Indeed, about 50% of corticotrophinomas are deficient in nuclear expression of either Brg1, the ATPase subunit of the chromatin remodeling Swi/snf complex, or in the histone deacetylase HDAC2 [118]. The loss of these proteins provides a molecular explanation for resistance to glucocorticoid feedback. Exome sequencing of Cushing adenoma samples identified frequent (about 50%) mutations at a hotspot within the USP8 gene that encodes a deubiquitinase [128,129]. The mutations activate USP8 activity by altering a 14-3-3 regulatory binding site and leading to USP8 cleavage that releases the active enzyme. This activation is correlated with persistent EGF signaling and stimulation of POMC expression.

MELANOTROPHS All hormone-producing cells of the intermediate pituitary are melanotrophs and they express the same single-copy POMC gene as anterior lobe corticotrophs.

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However, regulation of melanotroph function is quite different compared to corticotrophs. During fetal development, POMC expression starts around e15.5 in melanotrophs (Fig. 1.2) and it is preceded by expression of Tpit [95]. Tpit is as essential for melanotroph POMC expression as it is for corticotrophs and Tpit2/2 pituitaries maintain POMC expression in only a few percent of melanotrophs [59,69]. In the absence of Tpit, about 10 15% intermediate lobe cells switch fate and become bona fide gonadotrophs. Cells that switch fate in this model do not express markers of melanotrophs and thus it appears that melanotroph and gonadotroph markers are mutually exclusive, in agreement with the observed antagonism between Tpit and SF1 [59]. Interestingly, a significant portion of Tpit2/2 intermediate pituitary cells that do not differentiate retain expression of the fetal cell cycle inhibitor p57Kip2. Nonetheless, these p57Kip2-positive putative precursors as well as all cells in the mutant intermediate lobe switch on the related p27Kip1 [69]. This model has suggested that differentiation, whether driven by Tpit, SF1, or by default, results in switching off p57Kip2; in this context, p57Kip2 may represent the last (temporally) marker of the precursor state. Whereas Tpit exerts a similar role for terminal differentiation of both melanotrophs and corticotrophs, its action cannot explain the unique identity of each lineage. The Pax7 transcription factor is largely responsible for specifying the melanotroph identity. Pax7 expression is restricted to pituitary melanotrophs where its expression precedes expression of Tpit and partly overlaps with extinction of Sox2 in intermediate lobe progenitors. Consistent with the idea that Pax7 specifies intermediate lobe melanotroph identity, inactivation of the Pax7 gene does not prevent Tpitdependent differentiation of intermediate lobe cells but, in this mutant, the cells adopt a corticotroph-like fate [130]. Pax7 has unique properties by comparison to Tpit or other terminal differentiation factors such as Pit1. Indeed, Pax7 is a pioneer transcription factor that can access its target DNA sequence in compacted heterochromatin and initiate chromatin remodeling, thus providing access to a new repertoire of enhancers (Fig. 1.4). These newly accessible enhancers can then be targeted by Tpit and other transcription factors for activation of melanotroph-specific genes such as the PC2 (PCSK2) gene that encodes the protein convertase 2 that cleaves ACTH into αMSH [131]. In contrast, Tpit does not have pioneer activity and cannot access its target sequences at melanotroph-specific enhancers before Pax7-dependent chromatin remodeling. The establishment of a specific epigenome through chromatin remodeling is thus a critical mechanism during specification of cell fate.

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FIGURE 1.4 Epigenetic remodeling of melanotroph-specific PC2 gene enhancer located at 146 Kb from the PC2 transcription start site. The repressive chromatin mark H3K9me3 is the only feature of this genomic region in corticotroph AtT-20 cells; after Pax7 expression, this mark is decreased and marks of active enhancers, H3K4me1, p300, H3K27Ac, are present together with Tpit. The ATACseq signal indicates accessible DNA within enhancer sequences. Diagrams on the left illustrate these changes that occur at about 2500 melanotroph-specific enhancers genome-wide.

GONADOTROPHS Gonadotrophs appear to be specified relatively early during pituitary organogenesis despite the fact that their marker hormones, LH and FSH, are the last to be expressed at e16.5 of mouse development [68]. Indeed, gonadotrophs are first marked by the restricted expression of the nuclear receptor transcription factor SF1 and this expression starts at around e13.5 [132]. Different transcription factors were shown to be important for gonadotroph function, including Pitx1 that is expressed at higher protein levels in gonadotrophs than in other lineages [23]. The level of Pitx1 protein appears to play a role in regulating the abundance of gonadotrophs relative to other cells, since the Pitx1 knockout has fewer gonadotrophs [133]. Sites of direct Pitx1 action have been identified in the LHβ and FSHβ promoters [134,135]. Also, the related paired homeodomain transcription factor of bicoid specificity Otx1 is expressed in the pituitary and Otx12/2 mice have transient deficiencies of LH, FSH, and GH, resulting in hypogonadism and dwarfism at prepubertal

stages [136]. Furthermore, mutations in the related OTX2 were found to cause CPHD [137]. GATA-2 also contributes to gonadotroph differentiation since GATA-2 gene inactivation led to reduced gonadotrophin expression [138]. GATA-2 is expressed earlier that SFI [139] and its action may be partially redundant with the related GATA-3 [138] and/or GATA-4 [140]. In addition to its expression in pituitary gonadotrophs, SF1 marks every tissue of the hypothalamo pituitary gonadal axis as well as another steroidogenic tissue, the adrenals [141,142]. And accordingly, inactivation of the SF1 gene results in gonadal and adrenal agenesis, hypothalamic defects, and deficient LHβ, FSHβ, and GnRH receptor expression in the pituitary [132,143 145]. Although these studies supported the idea that SF1 is important for function of gonadotrophs and gonadotrophin gene expression, expression of LHβ and FSHβ is restored in SF12/2 mice by treatment with GnRH [146] suggesting that SF1 is not essential for gonadotroph cell fate. Hence, it may be a relatively late regulator of differentiation.

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LACTOTROPH DIFFERENTIATION

The action of GnRH on gonadotroph function is in part mediated by the zinc finger transcription factor Egr1 which acts in synergism with SF1 on the LHβ promoter [147 150]. The compensation of SF1 deficiency may thus be exerted through Egr1 in GnRH-treated SF12/2 mice. In the context of this promoter, both SF1 and Egr1 activate transcription by synergism with Pitx1 [148], thus suggesting a mechanism by which they may partially replace each other. FSHβ gene transcription selectively requires the Foxl2 factor. Foxl2 mutant mice are deficient in FSH and infertile but they are not depleted of gonadotrophs [151,152]. Foxl2 is thus the first factor to selectively control FSH gene expression independently of LH.

SOMATOTROPHS Somatotrophs represent one of the three lineages, together with lactotrophs and thyrotrophs, that are marked by and require the Pou homeodomain transcription factor Pit1 for terminal differentiation (Fig. 1.2). Expression of Pit1 starts at about e13.5 of mouse development in the medial region of the developing anterior lobe (Fig. 1.1); its expression is maintained throughout adult life in somatotrophs, lactotrophs, and thyrotrophs. The consequences of Pit1 loss-of-function were first established when the Jackson and Snell dwarf mice were studied and found to carry mutations of the Pit1 gene. These Pit1 mutant mice are deficient in the three Pit1-expressing lineages: this factor is thus critical for their terminal differentiation. In addition, Pit1 is required for transcription of the GH, PRL, TSHβ, and GHRH receptor genes [153]; the factor was in fact first identified on the basis of this transcriptional activity [154,155]. A similar requirement on Pit1 was also shown recently in zebrafish [156], and human mutations of the PIT1 gene cause CPHD [58]. Initial expression of Pit1 requires Prop1 [35] as suggested by the name of this factor (prophet-of-Pit) but other factors, such as AtbF1, contribute to high-level Pit1 expression [157]. Maintenance of Pit1 expression is also dependent on a positive regulatory feedback exerted on a distal enhancer of the Pit1 gene [158]. The importance of this autoregulatory feedback was well supported by studies of the Snell mutant in which initial activation of the Pit1 gene occurs but where Pit1 expression then fails to be maintained [159]. PROP1 mutations also cause CPHD [42,58,159]. The maintenance of GH expression, as well as expression of the GHRH receptor, requires the bHLH factor NeuroD4 (Math3), whose expression is itself

15

dependent on Pit1 [88]. NeuroD4 selectively acts on GH, but not PRL or TSH expression, thus providing differential control between the three Pit1-dependent genes and lineages.

LACTOTROPH DIFFERENTIATION Although lactotrophs appear to be specified early in part through the critical action of Pit1 [57], Prl expression is mostly upregulated postnatally. Analysis of Prl gene expression has led to identification of transcription factors that are required for Prl expression but these studies have not yet defined the basis for lactotroph-specific mechanisms of differentiation. A critical signal for lactotroph function and Prl expression is provided by estrogens, and their receptor ER was shown to act synergistically with Pit1 on a Prl gene enhancer [160 162]. The importance of ER and estrogen action on Prl expression was supported in mice inactivated for the ERα gene that exhibit fewer lactotrophs and marked reduction of Prl expression [163]. However, specification of the lactotroph lineages is not affected in the ERα2/2 mice. Similarly, Ets transcription factors were found to be important for Prl expression [164] and the action of Ets factors is synergistic with Pit1 [165,166]. Ets factors integrate Ras-MAPK signaling through phosphorylation of Ets1 [167] and synergism with Pit1 [168]. Different Ets transcription factors have been involved in Prl expression and target sites of the Prl promoter appear to show preference for Ets factors GABPα and GABPβ [169]. In addition, Prl expression depends on the Pitx factors [133,170 172] and on c/EBPα [173]. In addition to the strong activation of lactotroph function by estrogens, their function and Prl expression are under sustained negative action of hypothalamic dopamine. On the Prl promoter, dopamine repression may be exerted in part by the Ets repressor factor ERF [174]. The repressive action of dopamine is mediated through the dopamine D2 receptor (D2R) and the importance of this constitutive negative control was best exemplified in D2R mutant mice. These mice exhibit lactotroph hyperplasia and excessive prolactin production leading to formation of lactotroph adenomas in old mice [175,176]. Since derepressed growth of lactotrophs appears to predispose to lactotroph adenoma development, the balance between the inhibitory action of dopamine and the stimulatory action of estrogens on proliferation of these cells may serve in part to control the size of the lactotroph population but also to control lactotroph adenoma development.

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THYROTROPHS The third Pit1-dependent lineage is thyrotrophs and they are also deficient as a result of mouse and human Pit1 mutations [57]. The thyrotrophs are an intriguing and interesting lineage compared to the others since they share properties and regulatory transcription factors of both branches of the pituitary cell differentiation tree (Fig. 1.2). Indeed, these cells are dependent on Pit1 and they are thus related to the somatotroph and lactotroph lineages. But also, they express and are dependent on GATA-2, a factor that is shared with gonadotrophs [139]. The importance of GATA-2 for thyrotroph differentiation was directly assessed by conditional knockout of its gene in gonadotrophs and thyrotrophs using an αGSU-cre transgene for GATA-2 inactivation [138]. These mutant pituitaries have fewer thyrotrophs and gonadotrophs in agreement with the importance of this factor for both lineages. Notwithstanding the possibility that GATA-2 function is partially compensated by GATA-3, these studies do not as yet define the basis for specificity of the thyrotroph program relative to the somato-lactotrophs or to gonadotrophs. Transcription of the TSHβ gene was shown to depend on Lhx3 as well as GATA-2 [139,177,178]. Thyrotroph function and TSHβ gene transcription are stimulated by the hypothalamic hormone TRH and subject to feedback inhibition by thyroid hormones. TRH stimulation of THSβ transcription requires Lhx3, activated CBP, and Pit1 [178,179] and is, at least in part, mediated by the orphan nuclear receptor NR4A1 (NGFI-B, Nur77) [180]. Thyroid hormone repression of TSHβ transcription requires the thyroid receptor β (TRβ) that appears to be acting downstream of transcription initiation within the TSHβ gene [181,182]. The thyrotroph lineage thus appears to represent an interesting case since its specification may respond to signals that positively control both gonadotroph and somato-lactotroph lineages as exemplified by their expression of GATA-2 and Pit1. It will be interesting to determine whether an active mechanism is responsible for maintenance of these two signals/ transcription factors or whether it is the default maintenance of these factors that otherwise marks other lineages that are responsible for specification of the thyrotroph fate.

PERSPECTIVES The last few decades have been rich in teachings about regulators and mechanisms of pituitary cell differentiation with the discovery of the transcription

factors Pit1, Prop1, SF1, Tpit, and Pax7 that control pituitary cell differentiation. Mutations in the genes encoding these factors are important causes of pituitary hormone deficiencies. Many other factors involved in development of either the pituitary itself and/or of surrounding tissues have provided candidates and culprits for various pituitary malformations that result in multiple hormone deficiencies. There is still much that we do not understand: mechanisms and regulators for many cell fate choices remain to be identified. For example, what controls the difference between somatotrophs, lactotrophs, and the third Pit1dependent lineage, the thyrotrophs? Answers to these questions will no doubt provide tools for diagnosis of pituitary hormone deficiencies in addition to information on the underlying mechanisms of cell differentiation. This knowledge will be of even greater importance, now that we have identified putative pituitary stem cells and that we are developing the means to manipulate stem cells. As for stem cells of any other tissue, we now realize that the greatest challenge ahead will be the control of differentiation if we are to realize the promise offered by these multipotent cells for treatment of various deficits. It is thus critical that we identify the missing regulators of pituitary cell differentiation. Furthermore, the sequential differentiation of cells starting from progenitors towards terminally differentiated cells involves multiple steps and epigenetic reprogramming of precursors as cell fate is determined and differentiation choices are made. These epigenetic choices involve reprogramming of pituitary stem cells in order for these cells to lose their “stemness” character and its underlying active proliferation state, towards a differentiated program and tightly controlled cell growth. It is quite likely that growth control mechanisms that shift during development and differentiation of pituitary cells are also relevant in adenoma tissues that may contain pituitary (cancer) stem cells together with hormoneproducing differentiated cells. The developmental mechanisms for control of growth are thus very likely to be informative about processes that are deregulated when pituitary adenomas or tumors develop. The investigation of developmental processes is thus likely to have a major impact not only on our understanding of inherited forms of hormone deficiencies, but also on mechanisms of pituitary tumorigenesis and the hormone excesses that accompany some pituitary adenomas. Finally, we are just realizing the nature and importance of tridimensional cell network organization in the pituitary: the direct contacts between cells of the same lineage, their organization within unique 3D networks and the interactions between networks of the different

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REFERENCES

lineages are likely critical for the efficient delivery of synchronous and rapid hormone responses, and hence for appropriate integrated function of the gland. The molecular bases for establishment of these homotypic cell networks as well as for heterotypic cell contacts remain unknown, but are likely critical for optimal function. Conversely, misregulation of these mechanisms may be associated with pituitary dysfunction, in particular partial or progressive loss-of-function that we may still need to recognize at the clinical level. Establishment of these networks during fetal development, but also during the postfetal period, is likely critical to transform fetal hormone-producing pituitary cells into the hormone factories that these cells become in the adult gland. Conversely, impaired reorganization of these networks during critical phases of life, such as during puberty, pregnancy, or lactation, may have serious clinical implications that it is now our challenge to understand. Whereas developmental biologists have so far focused most of their interest on embryonic and fetal development, it now appears that postnatal development also includes critical events for the formation of a functional pituitary and hence our focus should in the future also include this period of development.

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Acknowledgments We are grateful to many colleagues and lab members who have contributed over the years to decipher the regulatory mechanisms responsible for pituitary development. The help of Lionel Budry for figure preparation and the secretarial assistance of Lise Laroche are gratefully acknowledged. Work in the author’s laboratory has been supported by grants from the Canadian Institutes of Health Research.

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[105] Zhang F, Tanasa B, Merkurjev D, et al. Enhancer-bound LDB1 regulates a corticotrope promoter-pausing repression program. Proc Natl Acad Sci USA 2015;112(5):1380 5. [106] Smith GW, Aubry JM, Dellu F, et al. Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 1998;20(6):1093 102. [107] Bale TL, Picetti R, Contarino A, Koob GF, Vale WW, Lee KF. Mice deficient for both corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 have an impaired stress response and display sexually dichotomous anxiety-like behavior. J Neurosci 2002;22(1):193 9. [108] Lugo DI, Pintar JE. Ontogeny of basal and regulated secretion from POMC cells of the developing anterior lobe of the rat pituitary gland. Dev Biol 1996;173(1):95 109. [109] Lugo DI, Pintar JE. Ontogeny of basal and regulated proopiomelanocortin-derived peptide secretion from fetal and neonatal pituitary intermediate lobe cells: melanotrophs exhibit transient glucocorticoid responses during development. Dev Biol 1996;173(1):110 18. [110] Fukuoka H, Cooper O, Ben-Shlomo A, et al. EGFR as a therapeutic target for human, canine, and mouse ACTHsecreting pituitary adenomas. J Clin Invest 2011; 121(12):4712 21. [111] Bousquet C, Melmed S. Critical role for STAT3 in murine pituitary adrenocorticotropin hormone leukemia inhibitory factor signaling. J Biol Chem 1999;274(16):10723 30. [112] Langlais D, Couture C, Drouin J. The Stat3/GR interaction code: predictive value of direct/indirect DNA recruitment for transcription outcome. Mol Cell 2012;47(1):38 49. [113] Philips A, Lesage S, Gingras R, et al. Novel dimeric Nur77 signaling mechanisms in endocrine and lymphoid cells. Mol Cell Biol 1997;17(10):5946 51. [114] Maira MH, Martens C, Philips A, Drouin J. Heterodimerization between members of the Nur subfamily of orphan nuclear receptors as a novel mechanism for gene activation. Mol Cell Biol 1999;19(11):7549 57. [115] Maira MH, Martens C, Batsche E, Gauthier Y, Drouin J. Dimer-specific potentiation of NGFI-B (Nur77) transcriptional activity by the protein kinase A pathway and AF-1-dependent coactivator recruitment. Mol Cell Biol 2003;23(3):763 76. [116] Philips A, Maira MH, Mullick A, et al. Antagonism between Nur77 and glucocorticoid receptor for control of transcription. Mol Cell Biol 1997;17(10):5952 9. [117] Martens C, Bilodeau S, Maira M, Gauthier Y, Drouin J. Protein:protein interactions and transcriptional antagonism between the subfamily of NGFI-B/Nur77 orphan nuclear receptors and glucocorticoid receptor. Mol Endocrinol 2005; 19(4):885 97. [118] Bilodeau S, Vallette-Kasic S, Gauthier Y, et al. Role of Brg1 and HDAC2 in GR trans-repression of pituitary POMC gene and misexpression in Cushing disease. Genes Dev 2006;20(20): 2871 86. [119] Drouin J. Transcriptional and epigenetic regulation of POMC gene expression. J Mol Endocrinol 2016;56(4):T99 112. [120] Heuser I. Anna-Monika-Prize paper. The hypothalamicpituitary-adrenal system in depression. Pharmacopsychiatry 1998;31(1):10 13. [121] Holsboer F, Barden N. Antidepressants and hypothalamicpituitary-adrenocortical regulation. Endocr Rev 1996;17(2): 187 205. [122] Holsboer F, Liebl R, Hofschuster E. Repeated dexamethasone suppression test during depressive illness. Normalisation of test result compared with clinical improvement. J Affect Disord 1982;4(2):93 101.

[123] Nemeroff CB, Widerlov E, Bissette G, et al. Elevated concentrations of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science 1984;226 (4680):1342 4. [124] Pace TW, Hu F, Miller AH. Cytokine-effects on glucocorticoid receptor function: relevance to glucocorticoid resistance and the pathophysiology and treatment of major depression. Brain Behav Immun 2007;21(1):9 19. [125] Watson S, Gallagher P, Ferrier IN, Young AH. Postdexamethasone arginine vasopressin levels in patients with severe mood disorders. J Psychiatr Res 2006;40(4):353 9. [126] Schule C, Baghai TC, Eser D, Rupprecht R. Hypothalamicpituitary-adrenocortical system dysregulation and new treatment strategies in depression. Expert Rev Neurother 2009;9(7):1005 19. [127] Lamberts SWJ. Glucocorticoid receptors and Cushing’s disease. Mol Cell Endocrinol 2002;197(1 2):69 72. [128] Reincke M, Sbiera S, Hayakawa A, et al. Mutations in the deubiquitinase gene USP8 cause Cushing’s disease. Nat Genet 2015;47(1):31 8. [129] Perez-Rivas LG, Theodoropoulou M, Ferrau F, et al. The gene of the ubiquitin-specific protease 8 is frequently mutated in adenomas causing Cushing’s disease. J Clin Endocrinol Metab 2015;jc20151453. [130] Budry L, Balsalobre A, Gauthier Y, et al. The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling. Genes Dev 2012;26(20): 2299 310. [131] Drouin J. Minireview: pioneer transcription factors in cell fate specification. Mol Endocrinol 2014;28(7):989 98. [132] Ingraham HA, Lala DS, Ikeda Y, et al. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 1994;8:2302 12. [133] Szeto DP, Ryan AK, O’Connell SM, Rosenfeld MG. P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development. Proc Natl Acad Sci USA 1996;93(15):7706 10. [134] Quirk CC, Lozada KL, Keri RA, Nilson JH. A single Pitx1 binding site is essential for activity of the LHbeta promoter in transgenic mice. Mol Endocrinol 2001;15(5):734 46. [135] Zakaria MM, Jeong KH, Lacza C, Kaiser UB. Pituitary homeobox 1 activates the rat FSHbeta (rFSHbeta) gene through both direct and indirect interactions with the rFSHbeta gene promoter. Mol Endocrinol 2002;16(8):1840 52. [136] Acampora D, Mazan S, Tuorto F, et al. Transient dwarfism and hypogonadism in mice lacking Otx1 reveal prepubescent stage-specific control of pituitary levels of GH, FSH and LH. Development 1998;125(7):1229 39. [137] Tajima T, Ohtake A, Hoshino M, et al. OTX2 loss of function mutation causes anophthalmia and combined pituitary hormone deficiency with a small anterior and ectopic posterior pituitary. J Clin Endocrinol Metab 2009;94(1):314 19. [138] Charles MA, Saunders TL, Wood WM, et al. Pituitary-specific Gata2 knockout: effects on gonadotrope and thyrotrope function. Mol Endocrinol 2006;20(6):1366 77. [139] Dasen JS, O’Connell SM, Flynn SE, et al. Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell 1999;97(5):587 98. [140] Lo A, Zheng W, Gong Y, Crochet JR, Halvorson LM. GATA transcription factors regulate LHbeta gene expression. J Mol Endocrinol 2011;47(1):45 58. [141] Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL. Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 1993;7(7):852 60.

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[142] Ikeda Y, Shen WH, Ingraham HA, Parker KL. Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 1994;8(5):654 62. [143] Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994;77(4):481 90. [144] Shinoda K, Lei H, Yoshii H, et al. Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dyn 1995; 204(1):22 9. [145] Zhao L, Bakke M, Krimkevich Y, et al. Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function. Development 2001;128(2):147 54. [146] Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL. The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 1995;9(4):478 86. [147] Topilko P, Schneider-Maunoury S, Levi G, et al. Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr- )-targeted mice. Mol Endocrinol 1998;12(1):107 22. [148] Tremblay JJ, Drouin J. Egr-1 is a downstream effector of GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance luteinizing hormone β gene transcription. Mol Cell Biol 1999;19(4):2567 76. [149] Dorn C, Ou Q, Svaren J, Crawford PA, Sadovsky Y. Activation of luteinizing hormone beta gene by gonadotropin-releasing hormone requires the synergy of early growth response-1 and steroidogenic factor-1. J Biol Chem 1999;274(20):13870 6. [150] Wolfe MW, Call GB. Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression. Mol Endocrinol 1999;13(5):752 63. [151] Justice NJ, Blount AL, Pelosi E, Schlessinger D, Vale W, Bilezikjian LM. Impaired FSHbeta expression in the pituitaries of Foxl2 mutant animals. Mol Endocrinol 2011;25(8):1404 15. [152] Tran S, Zhou X, Lafleur C, et al. Impaired fertility and FSH synthesis in gonadotrope-specific Foxl2 knockout mice. Mol Endocrinol 2013;27(3):407 21. [153] Andersen B, Rosenfeld MG. POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease. Endocr Rev 2001;22(1):2 35. [154] Bodner M, Castrillo JL, Theill LE, Deerinck T, Ellisman M, Karin M. The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 1988;55(3):505 18. [155] Ingraham HA, Chen R, Mangalam HJ, et al. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 1988;55(3):519 29. [156] Nica G, Herzog W, Sonntag C, Hammerschmidt M. Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism. Mol Endocrinol 2004;18(5):1196 209. [157] Qi Y, Ranish JA, Zhu X, et al. Atbf1 is required for the Pit1 gene early activation. Proc Natl Acad Sci USA 2008;105(7):2481 6. [158] Rhodes SJ, Chen R, DiMattia GE, et al. A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene. Genes Dev 1993;7:913 32. [159] Mullis PE. Genetic control of growth. Eur J Endocrinol 2005;152(1):11 31. [160] Day RN, Koike S, Sakai M, Muramatsu M, Maurer RA. Both pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol Endocrinol 1990;4(12):1964 71, 2. [161] Crenshaw III EB, Kalla K, Simmons DM, Swanson LW, Rosenfeld MG. Cell-specific expression of the prolactin gene in transgenic mice is controlled by synergistic interactions between promoter and enhancer elements. Genes Dev 1989;3:959 72.

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[162] Nowakowski BE, Maurer RA. Multiple Pit-1-binding sites facilitate estrogen responsiveness of the prolactin gene. Mol Endocrinol 1994;8(12):1742 9. [163] Scully KM, Gleiberman AS, Lindzey J, Lubahn DB, Korach KS, Rosenfeld MG. Role of estrogen receptor-alpha in the anterior pituitary gland. Mol Endocrinol 1997;11(6):674 81. [164] Gutierrez-Hartmann A, Duval DL, Bradford AP. ETS transcription factors in endocrine systems. Trends Endocrinol Metab 2007;18(4):150 8. [165] Bradford AP, Wasylyk C, Wasylyk B, Gutierrez-Hartmann A. Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression. Mol Cell Biol 1997;17(3):1065 74. [166] Bradford AP, Conrad KE, Wasylyk C, Wasylyk B, GutierrezHartmann A. Functional interaction of c-Ets-1 and GHF-1/ Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain. Mol Cell Biol 1995;15(5):2849 57. [167] Wasylyk B, Hagman J, Gutierrez-Hartmann A. Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signaling pathway. Trends Biochem Sci 1998;23(6):213 16. [168] Bradford AP, Conrad KE, Tran PH, Ostrowski MC, GutierrezHartmann A. GHF-1/Pit-1 functions as a cell-specific integrator of Ras signaling by targeting the Ras pathway to a composite Ets-1/GHF-1 response element. J Biol Chem 1996;271(40):24639 48. [169] Schweppe RE, Melton AA, Brodsky KS, et al. Purification and mass spectrometric identification of GA-binding protein (GABP) as the functional pituitary Ets factor binding to the basal transcription element of the prolactin promoter. J Biol Chem 2003;278(19):16863 72. [170] Tremblay JJ, Lanctoˆt C, Drouin J. The pan-pituitary activator of transcription, Ptx-1 (pituitary homeobox1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Limhomeodomain gene Lim3/Lhx3. Mol Endocrinol 1998;12(3): 428 41. [171] Quentien MH, Manfroid I, Moncet D, et al. Pitx factors are involved in basal and hormone-regulated activity of the human prolactin promoter. J Biol Chem 2002;277(46): 44408 16. [172] Tremblay JJ, Goodyer CG, Drouin J. Transcriptional properties of Ptx1 and Ptx2 isoforms. Neuroendocrinol 2000;71:277 86. [173] Jacob KK, Stanley FM. CCAAT/enhancer-binding protein alpha is a physiological regulator of prolactin gene expression. Endocrinology 1999;140(10):4542 50. [174] Liu JC, Baker RE, Sun C, Sundmark VC, Elsholtz HP. Activation of Go-coupled dopamine D2 receptors inhibits ERK1/ERK2 in pituitary cells. A key step in the transcriptional suppression of the prolactin gene. J Biol Chem 2002;277(39): 35819 25. [175] Saiardi A, Bozzi Y, Baik JH, Borrelli E. Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia. Neuron 1997; 19(1):115 26. [176] Asa SL, Kelly MA, Grandy DK, Low MJ. Pituitary lactotroph adenomas develop after prolonged lactotroph hyperplasia in dopamine D2 receptor-deficient mice. Endocrinology 1999;140(11):5348 55. [177] Gordon DF, Lewis SR, Haugen BR, et al. Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin β-subunit promoter. J Biol Chem 1997;272(39):24339 47. [178] Hashimoto K, Yamada M, Monden T, Satoh T, Wondisford FE, Mori M. Thyrotropin-releasing hormone (TRH) specific interaction between amino terminus of P-Lim and CREB binding protein (CBP). Mol Cell Endocrinol 2005;229(1 2):11 20.

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[179] Hashimoto K, Zanger K, Hollenberg AN, Cohen LE, Radovick S, Wondisford FE. cAMP response element-binding proteinbinding protein mediates thyrotropin-releasing hormone signaling on thyrotropin subunit genes. J Biol Chem 2000; 275(43):33365 72. [180] Nakajima Y, Yamada M, Taguchi R, et al. NR4A1 (Nur77) mediates thyrotropin-releasing hormone-induced stimulation of transcription of the thyrotropin beta gene: analysis of TRH knockout mice. PloS One 2012;7(7):e40437.

[181] Sasaki S, Lesoon-Wood LA, Dey A, et al. Ligand-induced recruitment of a histone deacetylase in the negative-feedback regulation of the thyrotropin beta gene. EMBO J 1999; 18(19):5389 98. [182] Abel ED, Moura EG, Ahima RS, et al. Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone. Mol Endocrinol 2003; 17(9):1767 76.

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C H A P T E R

2 Hypothalamic Regulation of Anterior Pituitary Function Anat Ben-Shlomo and Shlomo Melmed

INTRODUCTION

was subsequently observed by Popa and Fielding [7]. The idea that the hypothalamus could neuroregulate the anterior pituitary was formulated by Berta and Scharrer in the 1930s, and supported by the work of Geoffrey Harris, who used conscious female rabbits to show that although an intact pituitary gland was required for ovulation, only direct and highly selective electrical stimulation of the hypothalamus induced ovulation. This argued against neural pathways passing from the hypothalamus to the anterior pituitary, and supported a humoral mechanism, whereby hormones pass from the hypothalamus and through the hypothalamic pituitary portal vein system to the anterior pituitary [8]. Crystallization of this concept marked the beginning of a new era in neuroendocrinology and the search for hypothalamic neurohormones that regulate the pituitary [2]. Ovine luteinizing hormone (LH) was isolated by Evans in 1940, followed by isolation of ovine adrenocorticotropic hormone (ACTH) in 1943, growth hormone (GH) in 1945, and follicle-stimulating hormone (FSH) in 1949. Evans’ student Li isolated a nonhydrolyzed (α) ovine ACTH and determined its amino acid sequence in 1955. The discovery of pituitary hormones enabled the discovery of their hypothalamic regulators, their synthesis and release mechanisms, and led to Nobel Prizes for three investigators: Du Vigneaud characterized and synthesized oxytocin and ADH (vasopressin), Guillemin discovered thyrotrophin-releasing hormone (TRH), and somatostatin [9], and both Guillemin and Schally discovered gonadotrophinreleasing hormone (GnRH) [10]. Over the past decades, key milestones have included cloning and recombinant production of all known pituitary and hypothalamic hormones, cloning

Morphological and functional relationships between the nervous and endocrine systems have established the concept that the pituitary is centrally regulated by the hypothalamus [1]. In 1928, Scharrer suggested that the pituitary is regulated by the hypothalamus, proposing that hypothalamic neurons secreted hormones and speculating that these neurons control pituitary function [2]. Cajal identified the connection between the hypothalamus and the neurohypophysis (posterior pituitary), and also showed unmyelinated nerve fibers crossing from the murine hypothalamus to the neurohypophysis, later localized by Pines to the supraoptic and paraventricular hypothalamic nuclei [3]. The functional significance of this finding was developed in the 1930s by Fisher and colleagues, who demonstrated that bilateral interruption of the feline supraopticohypophyseal tract dramatically increased urine output and caused atrophy of the neurohypophysis. Urine output was normalized again after injection of fresh, but not atrophic posterior pituitary extracts. Transection of the monkey median eminence, but not the infundibular stem, resulted in severe polyuria [4]. Across species, the posterior pituitary was later shown to be directly regulated by hypothalamic antidiuretic hormone (ADH) and oxytocin produced by hormonesecreting neurons in the supraoptic nucleus, traveling through axons in the pituitary stalk, and stored and secreted by the posterior pituitary gland [3,5]. A physical connection between the hypothalamus and the anterior pituitary had been established at the end of the 19th century by Berkeley [6] and blood flow communication via complex hypophysio-portal vessels

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00002-7

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and characterization of their receptors, and discovery of additional hypothalamic peptides regulating the pituitary. These have enabled an in-depth understanding of hypothalamic pituitary physiology and pathophysiology, and, ultimately, development of an extensive pharmacotherapeutic armamentarium to treat hypothalamic pituitary disorders.

ANATOMY AND HISTOLOGY OF THE HYPOTHALAMIC PITUITARY UNIT The Hypothalamus The hypothalamus comprises less than 1% of brain volume and weighs approximately 5 grams [11,12]. The hypothalamic sulcus defines the upper border and extends from the interventricular foramen to the cerebral aqueduct, above which lies the thalamus. The anterior border is roughly defined as a line through the anterior commissure, lamina terminalis, and optic chiasm. The posterior border is adjacent to the midbrain tegmentum superiorly and the mammillary bodies inferiorly. Lateral borders are defined by the substantia innominata, the internal capsule, the subthalamic nucleus, and the cerebral peduncle. Hypothalamic neuronal bodies producing factors controlling the pituitary are clustered in different nuclei. Each hypothalamic hormone may be produced in more than one nucleus, and a single nucleus may express several hormones. Nuclei predominantly involved in pituitary regulation are mostly located in the medial hypothalamus. The median eminence is the major functional link between the hypothalamus and the pituitary and lies outside the blood brain barrier [13]. Its blood supply is separate from the rest of the hypothalamus and is largely shared with the pituitary [14 16]. The median eminence is composed of ependymal, internal, and external zones [17]. The innermost ependymal zone resides on the floor of the third ventricle; tight junctions and tanycytes prevent exchange of large molecules between the cerebrospinal fluid (CSF) and the extracellular median eminence spaces, as well as backtrafficking of releasing factors into the hypothalamus [18]. The internal zone consists of axons arising from the supraoptic and paraventricular nuclei to the posterior pituitary, and axons from the hypophysiotropic neurons to the external zone of the median eminence. The external zone of the median eminence contains axons from periventricular hypophysiotropic neurons, including the periventricular hypothalamic nucleus, and paraventricular and arcuate nuclei. This zone is the primary source for transfer of releasing factors into the hypophyseal portal circulation, eventually reaching

anterior pituitary trophic hormone-secreting cells [17]. Axons from peptidergic neurons release peptides including TRH, GnRH, corticotrophin-releasing hormone (CRH), growth hormone releasing hormone (GHRH), and somatostatin. Axons from monoaminesecreting neurons release dopamine and serotonin. Some releasing factors reaching the median eminence do not enter the hypophyseal portal circulation, but are released locally to regulate secretion by other nerve terminals [19]. The hypophyseal portal circulation derives from the superior hypophyseal artery, a branch of the internal carotid artery, forming a capillary loop network that penetrates and surrounds the internal and external median eminence zones. Arterial blood in this network receives releasing factors secreted upon depolarization of hypothalamic neurons, and transports these peptides to a large network of sinusoids surrounding the pituitary stalk, supplying the entire anterior pituitary [17,20]. Utilizing plasmalemmal vesicle-associated protein 1 (PV1) staining, extensive fenestrations were demonstrated at the median eminence, at the arcuate nucleus, and proximal to the pituitary stalk [21]. Estrous cycledependent PV1 expression and glycosylation levels in this area suggest that time-dependent enhanced permeability controls exposure to feedback regulators between the mediobasal hypothalamus, the pituitary, and the periphery [21]. The large surface area of this vascular network [22,23] and its fenestrations [21] facilitate efficient diffusion of hypothalamic releasing factors to pituitary cells. Hypophyseal portal circulation blood likely flows predominantly, if not exclusively, from the hypothalamus to the pituitary [20], but humoral feedback regulation on hypothalamic neurons controlling the pituitary likely also occurs. Potential routes for peripheral humoral factors regulating the hypothalamus include transcytosis through glial and endothelial cells in the blood brain barrier, permeable capillaries that allow access of peripheral factors to the CSF, and bidirectional fenestrated capillaries. Non-neuroendocrine supporting cell types including pituicytes and tanycytes also contribute to hypothalamic pituitary regulation. Pituicytes are gliallike cells that engulf axon terminals of vasopressin neurons when the hormone is not required, but retract when vasopressin secretion is increased, such as during dehydration [24]. Tanycytes, activated by growth factors and adhesion molecules, operate similarly on axon terminals of GnRH neurons [18,25].

Pituitary Anatomy The human pituitary gland (hypophysis) is subdivided into the anterior pituitary (adenohypophysis)

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and the posterior pituitary (neurohypophysis or pars nervosa) (Fig. 2.1). The adult human pituitary weighs about 0.6 g and comprises a space of about 0.13 cm (longest transverse dimension) by 0.06 0.09 cm (vertical dimension) by 0.09 cm (anteroposterior dimension). Releasing and inhibiting hormones Oxytocin

Hypothalamus

(ADH) Axons

Median eminence

Superior hypophyseal artery

Portal veins Hypophyseal vein (ADH,OCT)

Anterior pituitary

Posterior pituitary

The gland is lined by dura mater and lies within the hypophyseal fossa, the sella turcica, a bony convection in the upper surface of the sphenoid bone. The sella turcica protects the lower, anterior, and posterior pituitary margins, and a thin bony plate separates the pituitary from the sphenoid sinus. The tuberculum sella is a bony ridge located anterior to the pituitary, while the middle and anterior clinoid processes are anterolateral bony protrusions in the sphenoid bone. The posterior edge of the sella turcica is marked by the dorsum sella and the posterior clinoid processes on two sides. On its upper end, the pituitary is protected from CSF pressure by the diaphragma sella, which is an extension of the dura mater with a small central opening traversed by the pituitary stalk. The optic chiasm is located just anterior to the pituitary stalk. On its two lateral ends the pituitary is adjacent to the cavernous sinuses, large networks of thin-walled veins bordered by the temporal bone, the sphenoid bone, and the dura lateral to the sella turcica (Fig. 2.2). Cranial nerves III (oculomotor), IV (trochlear), V1 (ophthalmic, a branch of the trigeminal nerve), V2 (maxillary, also a branch of the trigeminal nerve), and VI (abducens) pass through this space arranged from superior to inferior within the lateral wall of the cavernous sinus. Cranial nerve VI (abducens) runs medial to the other nerves and lateral and below the internal carotid artery. The internal carotid

Inferior hypophyseal artery GH ACTH PRL TSH LH FSH Hypophyseal veins

FIGURE 2.1 Structural functional humoral, endocrine, and neuroendocrine relationships within the hypothalamic pituitary unit emphasize the unique and intimate interdependence of neural structures and hormone secretion with the circulation. Oxytocin (OCT) and vasopressin (ADH) neuron bodies located in the hypothalamus send axons through the pituitary stalk that terminate in the posterior pituitary, where they release OCT and ADH into blood vessels within the posterior pituitary. Hypothalamic neurons that produce growth hormone releasing hormone (GHRH), corticotrophinreleasing hormone (CRH), thyrotrophin-releasing hormone (TRH), and gonadotrophin releasing hormone (GnRH) send their axons through the median eminence to terminate and release their hormones into the hypophyseal portal circulation. This network of blood vessels is located at the median eminence, which surrounds the pituitary stalk and penetrates into the anterior lobe of the pituitary. These hypothalamic neurohormones stimulate responsive anterior pituitary cells to secrete growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroxin-stimulating hormone (TSH), luteinizing hormone (LH), and follicule-stimulating hormone (FSH), respectively. Dopamine neurons reaching the median eminence are responsible for tonic inhibition of prolactin secretion from the anterior pituitary, while somatostatin released from somatostatinergic neurons inhibit GH and TRH release. Source: Adapted from Melmed S. Pituitary. In: Dale DC, Federman DD, editors. ACP medicine, vol. 1; 2006. pp. 571 86.

Carotid artery Cavernous sinus Temporal lobe Intracavernous carotid artery

Optic chiasm

Pituitary gland

III IV VI V1 V2

Sphenoid sinus

FIGURE 2.2 Coronal view of the pituitary gland within the human skull. The borders of the pituitary are composed of the sella turcica below, the optic chiasm above, and the cavernous sinuses on both sides. The sella turcica is situated immediately above the sphenoid sinus. The cavernous sinuses constitute a thin-walled venous network that receive blood from the superior and inferior ophthalmic veins, the sphenoparietal sinus, and the superficial middle cerebral veins. Blood from the cavernous sinuses drains into the superior and inferior petrosal sinuses, the emissary veins, and the pterygoid plexus. Structures crossing the cavernous sinuses lateral to the pituitary gland include the carotid artery, cranial nerve III (oculomotor), cranial nerve IV (trochlear), cranial nerve VI (abducens) nerve, two branches of cranial nerve V (trigeminal nerve branch 1 and 2), the ophthalmic branch V1, and the maxillary branch V2. Source: Adapted from Stiver SI, Sharpe JA. Neuro-ophthalmologic evaluation of pituitary tumors. In: Thapar K, Kovacs K, Scheithauer BW, Lloyd RV, editors. Diagnosis and management of pituitary tumors. Totowa, NJ: Humana Press; 2001. pp. 173 200.

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artery ascends from the neck, enters the cavernous sinus medial to the abducens nerve, then traverses the roof of the cavernous sinus medial to the anterior clinoid process to enter the supracavernous portion. This vascular path constitutes the carotid siphon. Using T1-weighted magnetic resonance imaging, the posterior pituitary is visually distinguishable from the anterior lobe as a bright small area in the posterior part of the gland. The posterior pituitary is the distal component of the hypothalamo-neurohypophyseal tract, which also includes the infundibular stalk, the hypothalamic median eminence, and the tuber cinereum, a part of the base of the hypothalamus. The posterior pituitary comprises a collection of axon terminals originating from the magnocellular secretory neuron bodies located in the hypothalamic paraventricular and supraoptic nuclei. These axons traverse the infundibular stalk, terminating at the posterior pituitary, and secrete vasopressin and oxytocin into the systemic circulation. Pituicytes are scattered between axon terminals. The anterior lobe of the pituitary can be divided into the pars distalis (pars glandularis), which constitutes B80% of the gland, the pars intermedia, and the pars tuberalis, which engulfs the posterior pituitary stalk. The pars intermedia is rudimentary in the human, although in other species it is more developed.

CELLULAR COMPOSITION OF THE ANTERIOR PITUITARY The anterior pituitary is derived from oral ectoderm and, typical of an endocrine gland, has cells grouped in cords and follicles. Approximately 50% of the cells are somatotrophs that produce GH (Fig. 2.3). These cells are primarily located in the lateral wings of the anterior lobe, but can also be scattered in the median wedge [26]. Prolactin (PRL)-secreting lactotrophs represent B15% of cells in the anterior pituitary, and are randomly distributed throughout the lobe, but are most numerous in the posteromedial and posterolateral portions [26]. Corticotrophs express proopiomelanocortin (POMC), which is the precursor of adrenocorticotropic hormone (ACTH), melanocortin hormone (MSH), lipotropic hormone, and endorphins. Corticotrophs comprise about 15 20% of anterior pituitary cells. They mostly cluster in the central mucoid wedge in the center of the gland, but are also scattered in the lateral wings, and are the predominant cell type in the poorly developed human intermediate lobe. Gonadotroph cells produce LH and FSH and represent up to 10% of the human anterior pituitary cell population. (In the rat, gonadotroph cell number varies with age, sex, and hormonal status [27].) Gonadotrophs are scattered

FIGURE 2.3 Distribution and percentage of anterior pituitary cell subtypes, horizontal view. Gonadotroph cells are scattered throughout the anterior pituitary and constitute B10% of cells. PRL, prolactin-secreting cells; GH, growth hormone-secreting cells; ACTH, adrenocorticotropic hormone; TSH, thyrotrophin-secreting cells. Source: Adapted from Scheithauer BW, Horvath E, Lloyd RV, Kovacs K. Pathology of pituitary adenoma and pituitary hyperplasia. In: Thapar K, Kovacs K, Scheithauer BW, Lloyd RV, editors. Diagnosis and management of pituitary tumors. Totowa, NJ: Humana Press; 2001. pp. 91 154.

throughout the pars distalis and are the major constituent of the pars tuberalis [28]. Thyrotrophs are the least abundant cell type in the anterior pituitary, comprising approximately 5% of the total cell population, and are mostly found in the anteromedial portion of the gland [26], but are also found in the pars tuberalis [28]. Supporting and/or non-neuroendocrine cells are scattered throughout the anterior pituitary, including follicular cells surrounding follicles [26,29], agranular folliculostellate cells with long-branched cytoplasmic processes [30,31], incompletely differentiated null cells that do not secrete specific hormones [32], and mitochondria-rich oncocytes [26].

HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION The hypothalamic pituitary unit is a neuroendocrine transducer that mediates signals between the brain and the peripheral hormone-secreting target glands by positive and negative feedback regulators. In response to circadian, pulsatile, or acute brain stimuli, relevant hypothalamic neurons secrete hypothalamic releasing or inhibiting neurohormones into the hypophyseal portal circulation to regulate trophic hormone synthesis and release. Pituitary peptidic hormones are secreted to the systemic circulation to reach their distal target endocrine glands and regulate peripheral hormone release (Fig. 2.4). Negative feedback regulation of hormone secretion exists at all levels of the hypothalamic pituitary unit, but mostly by target gland hormones suppressing the pituitary and

I. HYPOTHALAMIC PITUITARY FUNCTION

27

HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION

CNS

Hypothalamus

Pituitary

Tier 1

Tier 2

Tier 3 Target

FIGURE 2.5 Three tiers regulating pituitary function. denotes stimulation; denotes inhibition. Source: Adapted from Ray D, Melmed S. Endocr Rev 1997;18:206 28.

FIGURE 2.4 The three control levels of hypothalamic pituitary target organ regulation. The first level is composed of hypothalamic neurons that secrete releasing and inhibiting hormones into the hypophyseal portal circulation that allows communication between the hypothalamus and the pituitary. The second control level involves the release of pituitary hormones to the circulation, reaching target glands and organs. The third control level constitutes the distal target organs secreting hormones that elicit the required effect on peripheral tissues.

hypothalamus. Local factors including cytokines, growth factors, nutrients, neuropeptides, and neurotransmitters, add further paracrine/autocrine complexity and can alter the delicate hormonal balance along each respective axis. Pituitary hormonal secretion is regulated at three tiers (Fig. 2.5, Table 2.1). The first tier comprises hypothalamic releasing and inhibiting hormones impinging directly upon the pituitary. The second tier involves secretion/inhibition of paracrine and autocrine intrapituitary hormone, cytokine, and growth factor signals. The third tier involves feedback regulation by hormones secreted from target glands and organs regulating pituitary or hypothalamic control.

Thyrotrophin-Releasing Hormone TRH is the major endogenous stimulator of the hypothalamic pituitary thyroid axis (Fig. 2.6). TRH is a phylogenetically ancient tripeptide, synthesized and released from TRH neurons with cell bodies in the

paraventricular nucleus [33]. High concentrations of TRH-positive terminals are found in the median eminence. The human TRH preprohormone gene encodes six copies of TRH [34]. Translation produces a peptide precursor that is subsequently enzymatically cleaved by prohormone convertases (PC1/3, PC2), carboxypeptidase E, pyroglutamyl cyclase, and peptidylglycine α-hydroxylating monooxygenase to create the active TRH pyroGlu-His-Pro-NH2 [35,36]. TRH secretion is regulated by membrane depolarization through Ca21dependent exocytosis. TRH is rapidly inactivated by proline endopeptidase, pyroglutamyl peptidase I (PPI), and the membrane-bound PPII, which hydrolyze active TRH in the hypothalamus and pituitary [36]. TRH receptors (TRHRs) are seven transmembrane domain GTP (G)-protein coupled receptors (GPCR) that are members of group A7 of the rhodopsin-like receptor family (class 1). Two subtype TRHRs have been reported in rodents, TRHR1 (the dominant receptor subtype [37]) and TRHR2; in humans, the single TRHR type is similar to TRH-R1 [38,39]. TRHR1 is highly expressed in the anterior pituitary, neuroendocrine brain regions, the autonomic nervous system, and the brainstem, and signaling is mediated primarily by Gq/11 proteins. Upon TRH binding, activation of phosphoinositide stimulates phospholipase C, which, in turn, induces hydrolysis of phosphatidylinositol 4,5,-P2 (PIP2) to form inositol 1,4,5-triphosphate (InsP3) and 1,2-diacylglycerol. This leads to increased intracellular Ca21 concentrations ([Ca21]i) and phosphokinase C (PKC) activation [40]. TRHR1 also increases [Ca21]i by coupling to Gi2 or Gi3 and activates calcium/calmodulin-dependent protein kinase and mitogenactivated protein kinase (MAPK) [39]. Pituitary TRHR1 expression is regulated by thyroid hormone but not by TRH [41].

I. HYPOTHALAMIC PITUITARY FUNCTION

TABLE 2.1 Hypothalamic Pituitary Axis Target Overview Stimulatory Hypothalamus Neuron

Pituitary

Target

Effects

TRH

Inhibitory CRH

GHRH

GnRH

Somatostatin

Dopamine Arcuate periventricular

Hypothalamic nucleus

Paraventricular Paraventricular

Arcuate ventromedial

Medio-basal, infundibular, periventricular regions

Periventricular paraventricular arcuate ventromedial

Gene

TRH

CRH

GHRH

GNRH-1

SST

Precursor

Prepro-TRH

Prepro-CRH

Prepro-GHRH

Prepro-GnRH

Preprosomatostatin Tyrosine L-DOPA

Human chromosome

3q13.3-q21

8q13

20q11.2

8p21-p11.2

3q28

Amino acid sequence

pGlu-His-ProNH2

Ser-Glu-Glu-ProPro-Ile-Ser-LeuAsp-Leu-ThrPhe-His-Leu-LeuArg-Glu-Val-LeuGlu-Met-AlaArg-Ala-Glu-GlnLeu-Ala-Gln-GlnAla-His-Ser-AsnArg-Lys-LeuMet-Glu-Ile-IleNH2

Tyr-Ala-Asp-AlaIle-Phe-Thr-AsnSer-Tyr-Arg-LysVal-Leu-Gly-GlnLeu-Ser-Ala-ArgLys-Leu-Leu-GlnAsp-Ile-Met-SerArg-Gln-Gln-GlyGlu-Ser-Asn-GlnGlu-Arg-Gly-Ala or [-Arg-Ala-ArgLeu-NH2]

pGlu-His-TrpSer-Tyr-GlyLeu-Arg-ProGly-NH2

Ala-Gly- Cys-LysAsn-Phe-Phe-TrpLys-Thr-Phe-ThrSer-Cys

Neurohormone

TRH

CRH

GHRH(1 40) GHRH(1 44)

GnRH

Somatostatin

Dopamine

Dominant GPCR

TRH-R1

CRH-R1CRH-R2

GHRHR

GnRHR

SSTR1, SSTR2, SSTR3, SSTR5

D2R

Gα protein subunits

Gαq/11 (Gαi2, Gαi3)

Gαs (Gαq, Gαi)

Gαs

Gαq/11 (Gαs, Gαi/o)

Gαi/o

Gαi/o

Signaling pathways shown to be involved in signaling

-PLCCalciumMAPK

-Adenylate cyclase-cAMPPKA-MAPKPLC-PKCCalcium

-Adenylate -PLC-Calciumcyclase-cAMPMAPK PKA-MAPK-PLCCalcium

-Adenylate cyclase-cAMPPKA-MAPKCalcium-PTP

-Adenylate cyclase-cAMPPKA-PLCCalcium-PTP

Pituitary cell

Thyrotroph, lactotroph

Corticotroph

Somatotroph

Gonadotroph

Somatotroph, thyrotroph, corticotroph

Lactotroph, thyrotroph, melanotroph

Pituitary hormone affected

TSH, PRL

ACTH

GH

LH, FSH

GH, TSH, ACTH

PRL, TSH, MSH

Pituitary hormone GPCR

TSHR, PRLR

Mc2R

GHR

LHR, FSHR

Target gland

Thyroid

Adrenal cortex

Liver

Gonads

Peripheral hormone

T4, T3

Cortisol androgens

IGF-1

Estrogen, testosterone progesterone

Receptors

TRα1, TRβ2

GCR type I, type II

IGF-1R

ERα, ERβ, AR, PR

Metabolic homeostasis

Metabolic homeostasis stress response

Tissue growth

Sexual development fertility

TRH, thyrotrophin-releasing hormone; CRH, corticotrophin-releasing hormone; GHRH, growth hormone releasing hormone; GnRH, gonadotrophin-releasing hormone; L-DOPA, 3,4-dihydroxyphenylalanine; PKA, phosphokinase A; PKC, phosphokinase C; PLC, phospholipase C; MAPK, mitogen-activating phosphokinase; PTP, phosphotyrosine kinase; TSH, thyroxin-stimulating hormone; ACTH, adrenocorticotropic hormone; GH, growth hormone; LH, luteinizing hormone; FSH, follicule-stimulating hormone; PRL, prolactin; TSHR, TSH receptor; PRLR, prolactin receptor; Mc2R, melanocortin receptor type 2; GHR, GH receptor; LHR, LH receptor; FSHR, FSH receptor; T4, thyroxine; T3, triiodothyroxine; GCR, glucocorticoid receptor; IGF-1, insulin-like growth factor type 1; IGF-1R, IGF-1 receptor; ER, estrogen receptor; AR, androgen receptor; PR, progesterone receptor.

HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION

TRH induces secretion and synthesis of pituitary thyroid-stimulating hormone (TSH, thyrotrophin). TRH injection in humans elicits up to a 22-fold increase in TSH within 30 minutes [42]. Sustained infusion of TRH over 4 hours results in a biphasic TSH increase. The first peak represents the early release of preformed TSH, and the second peak represents release of newly synthesized TSH [43]. TRH affects TSH bioactivity by altering its glycosylation pattern [44]. TSH secretion increases thyroid T4 secretion, which is converted to the more active triiodothyronine (T3) [45].

SRIF

TRH –

–

+ –

Thyrotroph TSH + Thyroid T4/T3

FIGURE 2.6 Specific hypothalamic neurons release thyrotrophinreleasing hormone (TRH), which stimulates pituitary thyrotrophs to release thyroxine-stimulating hormone (TSH). TSH then induces the thyroid to produce and release thyroxine (T4 and T3). T4 and T3 negatively regulate further release of both TSH and TRH, thus constituting a negative feedback loop. Somatostatin (SRIF) inhibits both TRH and TSH release. Source: Reproduced from Melmed S. J Clin Invest 2003;112:1603 18.

29

TRH also stimulates pituitary prolactin secretion [46] and may induce hyperprolactinemia in some hypothyroid patients. PRL levels are reduced by 40% in TRH-null mice. Although TRH is not essential for pregnancy and lactation, it is required for lactotroph function, particularly during murine lactation [47]. TRH may stimulate GH release in acromegaly, renal or liver failure, anorexia nervosa, and psychotic depression, as well as in some hypothyroid children [46]. Multiple factors, either directly or indirectly, regulate TRH neuron activity (Fig. 2.7). Thyroid hormones are the most potent negative regulators of TRH. T4, efficiently taken up by epithelial cells of the choroid system in the lateral ventricle, binds to transthyretin (T4-binding prealbumin) and is secreted across the blood brain barrier into the CSF. In the paraventricular nucleus, type II deiodinase converts T4 to T3, which interacts with thyroid hormone receptors on TRH neurons to reduce TRH synthesis and secretion. Approximately 80% of T3 at the paraventricular nucleus originates from peripheral T4; only 20% of hypothalamic T3 crosses the blood brain barrier directly from the periphery [48,49]. Type II deiodinase, mainly expressed in third ventricle tanycytes, is an important regulator of TRH neuron activity and plays a major role in T3 availability in the paraventricular nucleus. Fasting and infection upregulate tanycyte type II deiodinase expression, resulting in local increases of hypothalamic T3, which may partially explain the reactive decrease in peripheral TSH observed during fasting or inflammatory diseases [49]. T3 inhibits TRH gene transcription [33,50] and TSH synthesis and release both in vitro [51] and in thyrotroph xenografts [52]. T3 also influences processing of proTRH by altering paraventricular nucleus

FIGURE 2.7 Stimulators (up arrow) and inhibitors (down arrow) of the thyrotrophin-releasing hormone (TRH) neuron in the hypothalamic paraventricular nucleus. thyroid hormone receptor, glucocorticoid receptor, leptin receptor. CART, cocaine- and amphetamineresponsive transcript; αMSH, α-melanocortin stimulating hormone; NPY, neuropeptide Y.

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prohormone convertase (PC) levels [44]. T4 reduces TRH levels measured in the hypophyseal portal circulation [53]. Other factors regulate the pituitary TSH response to TRH, but it is difficult to distinguish between hypothalamic and pituitary effects. Somatostatin inhibits TRHinduced pituitary TSH and PRL release in cultured rat anterior pituitary cells and TRH-induced TSH release in hypothyroid rats [54]. Injection of somatostatinblocking antibodies into rat cerebral ventricles increases both TSH and GH levels, suggesting that TRH increases when somatostatin release is inhibited [55]. Dopamine inhibits TRH stimulation of TSH in normal and hyperprolactinemic females, while the dopamine synthesis inhibitor alpha-methyl-p-tyrosine enhances the TSH response to TRH [56]. The medulla oblongata mediates temperature regulation of TRH neurons. Cold exposure mediates adrenergic input from the medulla and stimulates TRH release, mainly through α1 adrenoreceptors, and reverses T3 suppression of TRH transcription [44]. Cocaine- and amphetamine-regulated transcript and neuropeptide Y (NPY) are both released by adrenergic medullary neurons; the former stimulates TRH release and potentiates epinephrine action on TRH neurons, while the latter inhibits TRH transcription [44]. TRH plays a role in appetite control. The arcuate nucleus contains leptin neurons that regulate appetite, and leptin upregulates TRH expression. However, leptin also indirectly modulates TRH through POMC and NPY-agouti-related peptide (AgRP) neurons in the arcuate nucleus [57]. Leptin reduction during fasting is associated with reduced prepro-TRH transcription, and hypothyroidism in leptin null mice can be corrected with exogenous leptin [58]. TRH neurons express glucocorticoid receptors and TRH gene contains glucocorticoid response elements. A dual effect of glucocorticoid on TRH expression has been observed: Mice acutely injected with corticosterone show TRH stimulation; but prolonged exposure to corticosterone reduces TRH production. Many TRH neurons at the median eminence do not supply the pituitary but rather communicate with neurons in other brain centers.

Corticotrophin-Releasing Hormone Mammalian CRH belongs to a family of stress response-regulating proteins including CRH, urocortin I, II (stresscopin-related peptide), and III (stresscopin) [59 61]. CRH is the dominant stimulator of the hypothalamic pituitary adrenal (HPA) axis [60] (Fig. 2.8). CRH is highly conserved among human, mouse, and rat species, but the ovine peptide differs

CRH

–

+ Corticotroph

–

ACTH + + Adrenal Cortisol

FIGURE 2.8 Hypothalamic corticotrophin-releasing hormone (CRH) stimulates the corticotroph to release adrenocorticotropic hormone (ACTH), which, in turn, stimulates the adrenal glands to produce and release cortisol and androgens. Cortisol inhibits further release of both CRH and ACTH. Source: Reproduced from Melmed S. J Clin Invest 2003;112:1603 18.

by seven amino acids, increasing its potency. The human CRH gene is located on the long arm of chromosome 8, 8q13 [62] and contains two exons. The amidated 41-amino-acid CRH peptide is cleaved from its carboxyl-terminus by PC1,3 and PC2 [63]. CRH preprohormone precursor is produced in the parvocellular neurons of the hypothalamic paraventricular nucleus along with vasopressin, encephalin, and neurotensin [59,64]. CRH receptor subtype 1 (CRHR1) is the predominant receptor subtype in pituitary corticotrophs [59]; it is also highly expressed in the adrenal medulla and to a lesser extent in the adrenal cortex, specifically in the zona fasciculata and reticularis [65]. CRHR2 has several splice variants. CRHR2α mRNA is localized to the hypothalamus, hippocampus, lateral brain septum, and adrenals, while CRHR2β is expressed in the brainstem, vasculature, heart, lung, skeletal muscle, adrenals, and gastrointestinal tract [65]. Stress results in significant upregulation of CRHR1 in the CRH neurons in the paraventricular nucleus in rodents [59], suggesting the existence of a CRH autocrine feedback. Expression of CRHR2 variants in the adrenal glands suggest an intraadrenal CRH-dependent circuit distinct from the hypothalamic pituitary CRH unit [65]. CRH and urocortin I bind both CRHR1 and CRHR2, while urocortin II and III bind only CRHR2 [66]. Both receptors are GPCRs coupled to the Gαs subunit and stimulate adenylyl cyclase to increase cAMP levels and activate phosphokinase-A (PKA). Both receptor subtypes also mediate increased calcium levels in vitro, mainly via Gαs but also to a lesser degree through Gαq [67], an effect that is mediated through stimulation of phospholipase-C, -β, and -ε [67]. Upon stimulation with either CRH or urocortin CRHR1 is rapidly

I. HYPOTHALAMIC PITUITARY FUNCTION

HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION

phosphorylated, desensitized, and internalized via PKC pathway regulation [59]. CRH action on the pituitary is modified by CRHbinding protein (CRH-BP), a highly conserved 37-kDa glycoprotein that is abundantly expressed in the pituitary and hypothalamus, but minimally expressed in the paraventricular nucleus and the median eminence where CRH neurons reside. Cytoplasmic CRH-BP is localized in cells expressing CRH receptors, and is also found in the plasma [59,65]. CRH-BP rapidly but transiently binds with high affinity to CRH and urocortins, and thus competitively inhibits ligand action [65]. ACTH and cortisol follow a 24-hour circadian rhythm driven by CRH and essential to maintaining normal adrenal function [68,69]. Human ACTH and cortisol begin rising between 1 and 4 a.m. and peak during the early morning hours; a smaller peak occurs in the early afternoon, and levels then fall over the remainder of the day to reach a nadir at around midnight. CRH administration results in a rapid release of pituitary ACTH (peak at 30 minutes) followed by release of adrenal cortisol (peak at 60 minutes) [70] and androgens. Urocortin I administration also stimulates release of ACTH (peak at 60 minutes) and cortisol (peak at 90 minutes) [71]. Continuous CRH infusion produces marked diurnal changes in corticosterone levels, indicating that CRH is requiring for maintaining a normal ACTH circadian rhythm [72]. Adult CRH null mice have atrophic adrenal glands and do not exhibit a normal circadian ACTH corticosterone rhythm [72]. CRH neurons are tightly regulated by a wide array of stimulators and inhibitors that originate in the periphery, as well as by an extensive network of afferent neurons from the hypothalamus, limbic forebrain, and brainstem (Fig. 2.9). Systemic or physiologic stressors including cytokines, salt loading, hemorrhage,

31

adrenalectomy, hypoglycemia, and fasting activate CRH release [73]. Neurogenic, emotional, or psychological stressors can activate CRH release, but these responses are likely indirect as the paraventricular nucleus is not known to receive direct input from the cerebral cortex or the thalamus [73,74]. Glucocorticoids (GC) freely cross the blood brain barrier, bind glucocorticoid nuclear receptors [GR, GCR, NR3C1 (nuclear receptor subfamily 3, group C, member 1)] subtype I and II, and inhibit CRH expression [75] and secretion [76] from CRH neurons in the PVN. GC also affect CRH translation and mRNA stability [77]. As the CRH gene promoter does not express GRE binding site, the effect of GC on CRH gene transcription suppression is moderate, and likely indirect [64,78]. Indirect actions may be mediated through afferents that make synaptic contact with CRH neurons in the arcuate nucleus and catecholaminergic neurons, as well as via afferents that regulate neuronal input from other brain areas. This extensive network allows for a wide range of neural influences on CRH function and stress response [64]. Rapid GC inhibition of PVN neuronal secretion via release of postsynaptic endocannabinoid (anandamide and 2-arachidonoylglycerol) acting as a retrograde messenger to activate presynaptic CB1 receptors has been described [79], as has somatostatin inhibition of hypothalamic CRH release due to the close proximity of paraventricular somatostatin neurons to CRH neurons [80 82]. Glutamatergic and GABAergic neurotransmission have been implicated as critical regulators of the stress response. Glutamate stimulates while GABA inhibits CRH release, and the two neurotransmitters also influence each other [83 85]. Other potent regulators of CRH include inflammatory cytokines and neurotransmitters. During inflammation,

FIGURE 2.9 Stimulators (up arrow) and inhibitors (down arrow) of the corticotrophin-releasing hormone (CRH) neuron in the hypothalamic paraventricular nucleus. cytokine receptors, glucocorticoid receptor, leptin receptor. GABA, γ aminobutyric acid; CRHR1, CRH receptor subtype 1; IL, interleukin; TNF, tumor necrosis factor; GLP-1, glucagon-like peptide-1.

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cytokines such as IL-1, TNFα, and IL-6 increase CRH release and mediate CNS responses [86 88] to stimulate ACTH and adrenal secretion. Increased GC levels, in turn, limit inflammation by inhibiting lymphocyte proliferation and production of immunoglobulins and cytokines. Leptin stimulates CRH release from CRH neurons, but this effect is likely indirect as leptin receptor mRNA is mostly concentrated in the arcuate nucleus and has not been detected in the PVN [89,90]. Catecholamines, including dopamine, increase CRH expression directly and indirectly [91,92]. Ghrelin may also stimulate CRH directly via efferents terminating on CRH neurons, but these actions are mostly indirect, acting through GABAergic NPY/AgRP neurons [93]. Glucagon-like protein 1 (GLP-1)-secreting neurons originating in the nucleus of the solitary tract project onto PVN CRH neurons [94]. Centrally administered GLP-1 activates Ca21 signaling in CRH neurons and increases ACTH and corticosterone [95]. In rats, peripheral or central GLP-1 administration has been shown to abruptly increase ACTH and corticosterone levels, an effect completely abrogated by astressin, a CRH antagonist [96]. Although CRH regulation of ACTH and cortisol secretion and synthesis is well established, comprehensive understanding of central and peripheral stimuli regulating CRH transcription and their crosstalk is still unclear. Further elucidation of mechanisms of CRH production is crucial to our understanding of extrapituitary functions of CRH and urocortin associated with HPA regulation, including sympathetic activity, behavioral changes such as arousal, locomotion, reward and feeding, and immune, cardiac, gastrointestinal, and reproductive functions.

Growth Hormone Releasing Hormone Hypothalamic control of pituitary GH secretion was predicted from animal studies involving hypothalamic lesions [97] as well as pituitary cell treatment with hypothalamic extracts [98]. Further support was derived from the observed GH depletion after stalk resection [99], the circadian nature of GH release [100], acute GH response to stress [101] and GH-inhibiting somatostatin [102]. GHRH was isolated in 1982 in two patients with ectopic GHRH secreted from a pancreatic adenoma [103 105] and from human hypothalami [106,107]. The GHRH gene includes five exons and is located on chromosome 20 in humans [108], and on chromosome 2 in mice. GHRH preprotein precursor contains 108 amino acids, and is subsequently processed into GHRH(1 40)-OH and GHRH(1 44)-NH2, which comprise 40 and 44 amino acids, respectively. While the

NH2-terminal is essential for GHRH bioactivity, the COOH-terminal is not, and fragments as short as GHRH(1 29)-NH2 are active [109]. GHRH(1 44)-NH2 is the major form of the peptide [107]. The circulating enzyme dipeptidylpeptidase type IV inactivates GHRH to a stable metabolite, GHRH (3 44)-NH2 [109]. The COOH-terminal of GHRH accounts for most interspecies sequence diversity [110]. GHRH neurons are located in the arcuate nucleus and around the ventromedial nucleus, projecting into the median eminence and terminating on capillaries of the hypophyseal portal circulation before releasing GHRH [111]. GHRH is detected in the human hypothalamus between 18 and 29 weeks gestation, and coincides with the appearance of fetal pituitary somatotrophs. In vitro, fetal pituitary cells respond to GHRH and somatostatin in mid-gestation. During mid-puberty, GHRH levels increase significantly, in girls more than in boys, but then decline with age, in part accounting for the age-related somatopause [112]. Sex steroids activate GHRH neurons and inhibit somatostatin neurons, accounting, at least in part, for sexual dimorphic GH secretion patterns [113]. Adult males have more GHRH neurons and higher hypothalamic GHRH and somatostatin mRNA expression as compared to females, corresponding to higher peaks and lower troughs in GH secretion. The synaptic organization in the arcuate nucleus also differs between genders. Females exhibit more synapses, and synapse number varies with the estrous cycle [113]. GHRH receptor (GHRHR), mostly expressed on pituitary somatotroph cells, is a GPCR coupled to the Gαs subunit. Activation of GHRHR increases intracellular calcium levels, stimulates adenylyl cyclase, increases intracellular cAMP [114], and activates pituitary PI3K and MAPK pathways [115]. Pituitary GHRH actions include increasing GH synthesis and release and somatotroph cell proliferation (Fig. 2.10). A single intravenous GHRH bolus dosedependently increases serum GH levels, peaking at 15 45 minutes, and returning to baseline within 90 120 minutes [116]. Repeated or constant administration of GHRH maintains elevated GH and insulinlike growth factor-1 (IGF-1) levels without axis downregulation [117]. GHRH also directly induces somatotroph proliferation, hyperplasia, and even neoplastic transformation [118], such that transgenic mice overexpressing GHRH exhibit somatotroph hyperplasia in enlarged pituitaries and ultimately develop multifocal somatotroph adenomas. Clinical examples of GHRH or GHRH-like trophic effects include ectopic GHRH release by neuroendocrine tumors, increased GHRH action in up to 40% of acromegaly patients as a consequence of activating somatic mutations in the Gαs subunit gene, and loss-of-function PRKAR1A gene

I. HYPOTHALAMIC PITUITARY FUNCTION

HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION

FIGURE 2.10 Hypothalamic growth hormone releasing hormone (GHRH) stimulates the somatotroph to release growth hormone (GH) which, in turn, stimulates the liver to produce and release insulin-like growth factor 1 (IGF-1). Both GH and IGF-1 negatively regulate further release of both GHRH and GH. Somatostatin (SRIF) inhibits both GHRH and GH release. Source: Reproduced from Melmed S. J Clin Invest 2003;112:1603 18.

33

mutations, which encode type 1A regulatory subunit of PKA in some families with Carney complex. These clinical conditions result in somatotroph hyperplasia and, sometimes, adenomatous transformation [118]. Negative feedback control of GHRH release is achieved mainly by direct GH stimulatory effects on somatostatin neurons rather than on GHRH neurons (Fig. 2.11), such that increased serum GH stimulates somatostatin release into the median eminence, which subsequently inhibits pituitary GH secretion. Neuroanatomic and neurofunctional bidirectional interactions occur between somatostatinergic neurons in the periventricular and paraventricular nuclei and GHRH neurons in the arcuate nucleus [119,120]. GHRH neurons express somatostatin receptor subtype 2 (SST2) and subtype 1 (SST1) but not GH receptor (GHR), while somatostatin neurons express both GHR and GHRHR [121,122]. SST2 null mice are refractory to GH-negative feedback regulation in the arcuate nucleus [123], and GH or IGF-1 administered to hypophysectomized rats increase somatostatin but do not change GHRH levels [124,125]. These observations suggest that

FIGURE 2.11 Stimulators (up arrow) and inhibitors (down arrow) of the growth hormone releasing hormone (GHRH) neuron and somatostatin (SRIF) neuron in the hypothalamic nuclei. somatostatin receptors, GH receptor, GHRH receptor, GH secretagogue receptor. GABA, γ-aminobutyric acid; SRIF, somatostatin; GHRH, growth hormone releasing hormone; CRH, corticotrophin-releasing hormone; NMDA, N-methyl-D-aspartate; NPY, neuropeptide Y; GH, growth hormone; IGF-1, insulin-like growth factor 1; E2, estradiol; T, testosterone.

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GH and IGF-1 indirectly inhibit GHRH secretion by increasing hypothalamic somatostatin secretion and SST2 receptor subtype. Direct somatostatinergic inhibition of GHRH neurons is also suggested by the abundance of somatostatinergic neurons contacting the GHRH perikarya in the arcuate nucleus, as GHRH axonal varicosities rarely contact somatostatinergic perikarya [126]. Somatostatin also reaches to the pituitary through the hypophyseal portal circulation to directly inhibit somatotroph GH secretion. Both GHRH and somatostatin neurons express receptors for several neurotransmitters and peptides, and multiple extrahypothalamic brain regions project to GHRH and somatostatin neurons. Catecholaminergic neurons intimately associate with GHRH and dopaminergic neurons in the median eminence, suggesting a direct effect of catecholamines on GHRH production and secretion [127]. Other regulators of somatostatinergic neurons indirectly affect GHRH neurons. For example, activation of β2 adrenoreceptor inhibits GH secretion through stimulation of somatostatin neurons, while activation of α2 adrenoreceptors stimulates GH secretion in part by stimulation of GHRH and inhibition of somatostatin. Acetylcholine also enhances GHRHinduced GH secretion, mostly by activation of muscarinic receptor subtype 1 (M1) on somatostatin neurons and inhibition of somatostatin release. Acetylcholine also activates M3 muscarinic receptors on hypothalamic GHRH neurons. M3 null mice exhibit pronounced anterior pituitary gland hypoplasia and a marked decrease in GH and PRL levels, which can be corrected by exogenous GHRH treatment [128]. Neuropeptides that regulate rodent GHRH-GH secretion include galanin, ghrelin, and melatonin, which stimulate GH secretion, as well as calcitonin, NPY, and CRH, which inhibit GH secretion. Galanin, a 29-amino-acid peptide expressed in the paraventricular and arcuate nuclei, is coexpressed with GHRH. Both central and peripheral galanin administration induce GH secretion and potentiate GHRH-stimulated GH secretion [129]. In humans, galanin causes a significant increase in plasma GH through direct GHRH stimulation [130] and somatostatin inhibition [129]. As galanin mRNA expression in GHRH neurons is considerably higher in males than females [129], sexually dimorphic GH pulsatility may be related to galanin expression. Ghrelin is a gut-derived 28-amino-acid GH secretagogue that acts on the ghrelin receptor (GHS-R) to increase intracellular calcium concentration via inositol phosphate to stimulate GH release. GHS-R mRNA is expressed in the arcuate and ventromedial hypothalamic nuclei and in the pituitary. Intravenous ghrelin injection dose-dependently stimulates GH release in both rats and humans [131]. Direct ghrelin action on hypothalamic GHRH to stimulate GH secretion is

likely, as ghrelin does not stimulate GH release in patients with hypothalamic lesions, but does increase GHRH release from hypothalamic tissue in vitro. In addition, in rats, ghrelin induction of GH in primary pituitary cell cultures is significantly lower than that observed in vivo. Of note, ghrelin and GHRH cotreatment has a synergistic effect on GH secretion, suggesting that GHRH is important for ghrelin-induced GH release [131]. In vitro analysis suggests GHRH allosterically interacts with ghrelin receptor GHS-R1α, modifying the ghrelin-associated intracellular signaling pathway [132]. Centrally administered ghrelin to mice initiates a GH pulse by stimulating GHRH and inhibiting somatostatin release. NPY is a 36-amino-acid peptide widely distributed in the brain and in sympathetic neurons; NPYcontaining neurons are located in the arcuate nucleus, and to a lesser extent, in paraventricular and periventricular nuclei. In the rat, intracerebral NPY injection or chronic infusion inhibits GH secretion, most likely through somatostatin activation [133 135]. As somatostatinergic and GHRH neurons possess multiple receptors for different peptides and neurotransmitters, it is often difficult to differentiate between direct and indirect effects of different factors on GHRH production and secretion. Nevertheless, it appears that regulation of somatostatin secretion is the most direct determinant of GHRH neuron activity.

Somatostatin Somatotrophin release inhibiting hormone or somatostatin 14 was inadvertently discovered in 1973 during the search for a hypothalamic growth hormone releasing factor [82]. Somatostatin 28, a 28-amino-acid somatostatin peptide that is a longer form of somatostatin 14, was discovered immediately thereafter [82]. The human somatostatin gene is located on the long arm of chromosome 3 and comprises two exons and one intron. The two exons encode preprosomatostatin, and exon 2 encodes somatostatin 14 and somatostatin 28. Both peptides are derived from posttranslational cleavage of prosomatostatin by PC1 and PC2 and carboxypeptidase E [82]. Somatostatins are cyclic peptides with a single covalent bond between 2 Cys residues and a bioactive core composed of a PheTrp-Lys-Thr amino acid sequence. Somatostatin 14 is predominant in the brain, while somatostatin 28 predominates in the gastrointestinal tract [82]. The name somatostatin was given as this peptide was initially thought to primarily inhibit pituitary GH release, however, it was later shown to also inhibit TSH, ACTH, and multiple central and peripheral hormones [136]. Moreover, in some tissues, somatostatin 14 exhibits both stimulatory and inhibitory acute actions [136].

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HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION

Members of the somatostatin family of peptides are derived from a common primordial gene that divided at about the time of the advent of chordates, to create two parallel genes, preprosomatostatin and preprocortistatin. Preprosomatostatin was further duplicated into preprosomatostatin I and preprosomatostatin II approximately 400 million years ago [82]. While fish and amphibians both have preprosomatostatin I and II, mammals have a single preprosomatostatin similar to preprosomatostatin I that gives rise to somatostatin 14 and somatostatin 28. Somatostatin 14 is identical in all vertebrates and exhibits a high degree of homology to somatostatin 14 in invertebrates and protozoa [82]. Somatostatin 14 is the main form in the brain including the hypothalamus, while cortistatin is mostly expressed in the hippocampus and cortex [82]. Hypothalamic somatostatin is produced predominantly in the anterior periventricular nucleus, and is also produced in the paraventricular, arcuate, and ventromedial nuclei. These neurons project into the median eminence, and their fibers terminate in the hypophyseal portal circulation, where they release somatostatin into the blood supplying the anterior pituitary cells [82]. Some neuronal axons course through the neural pituitary stalk and terminate directly in the posterior pituitary [137]. Additional minor routes for somatostatin to reach the pituitary include CSF leakage from the third ventricle to the portal system, peripheral somatostatin, and short portal blood vessels crossing from the posterior to the anterior pituitary [137]. Thus, somatostatin acts as an endocrine hormone on the anterior pituitary. The primary function of somatostatin is to inhibit pituitary hormone secretion, accomplished by inhibition of hypothalamic peptides responsible for pituitary hormone synthesis and secretion and by direct effects on pituitary cells. In the hypothalamus, somatostatin inhibits release of hypothalamic CRH, TRH, dopamine, norepinephrine [82], and GHRH [123,138]. Somatostatin also inhibits its own secretion from the periventricular nucleus [82]. In the pituitary, somatostatin inhibits GH, TSH, and ACTH secretion in the presence of low glucocorticoid levels, and also blocks estrogen-induced PRL [139]. Somatostatin signals through five somatostatin receptor subtypes, SST1 5, all of which are GPCR encoded by specific genes on different chromosomes. SST2 is alternatively spliced to SST2a and SST2b in rodents but not in humans; however, only the SST2a isoform is expressed in the pituitary [139]. Somatostatin 14 exhibits high binding affinities to all the receptor subtypes, but has greatest affinity to SST2, which is the subtype largely responsible for inhibiting pituitary hormone secretion. Most studies on somatostatin receptor action in pituitary cells have involved

35

SST2 and SST5. Upon ligand binding, the receptors bind the Gαi/o subunit of the Gαβγ tetramer, which, in turn, inhibits adenylyl cyclase and thus reduces cytoplasmic cAMP levels. Receptor activation also initiates opening of potassium channels and closure of calcium channels. These mechanisms result in suppression of pituitary hormone secretion [139]. Multiple factors regulate hypothalamic somatostatin 14 production, including ions and nutrients, neuropeptides, neurotransmitters, classical hormones, growth factors, and cytokines (Fig. 2.11). Intracellular stimulators of somatostatin include calcium, cAMP, cGMP, and nitric oxide, all participate in the induction of cell membrane depolarization. Stimulators of hypothalamic somatostatin include GHRH, CRH, bombesin, neurotensin, N-methyl-D-aspartate, GH, IGF-1, estrogen, testosterone, thyroxine, insulin, and glucagon, as well as a variety of cytokines. Inhibitors of somatostatin release include opiates, GABA, leptin, glucose, and TGFβ. Glucocorticoids have a dual effect on somatostatin release from the hypothalamus: at low doses, glucocorticoids induce somatostatin, but high doses inhibit somatostatin expression [82]. Somatostatin receptor expression is regulated by multiple factors. Pituitary SST2 is upregulated by prolonged exposure to somatostatin, GHRH, and testosterone, as well as by short exposure to glucocorticoids and TGFβ. In contrast, somatostatin receptor expression is downregulated by ghrelin and chronic glucocorticoid exposure. SST5 is upregulated by sustained exposure to somatostatin and thyroxine, and downregulated by GHRH, ghrelin, and estrogen [139].

Gonadotrophin-Releasing Hormone GnRH is a hypothalamic neuropeptide that regulates release of FSH and LH by pituitary gonadotroph cells (Fig. 2.12). Mammals express two GnRH genes; GnRH-I is located on chromosome 8, and GnRH-II is on chromosome 20 [140]. GnRH-I is the predominant peptide in the brain and hypothalamus and regulates gonadotroph function. GnRH-II is expressed mostly outside the brain. The GnRH-I gene encodes a precursor protein of 92 amino acids that is further processed by PC to create the 10-amino-acid GnRH, retaining a pyroGlu at the N-terminus and a Gly-amide at the C-terminus that are important for bioactivity [140]. In humans and nonhuman primates, small GnRH-I neuronal cell bodies are not localized to a specific nucleus, but are scattered mostly in the mediobasal hypothalamus, infundibular, and periventricular regions, and form a diffuse neural network that coordinates the GnRH pulse generator [141,142]. GnRH-I neurons project to the median

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GnRH –

– Gonadotroph

+ –

– LH +

FSH +

+

+

Testosterone { Estradiol Progesterone

FIGURE 2.12 Hypothalamic gonadotrophin-releasing hormone (GnRH) stimulates the gonadotroph to release follicule-stimulating hormone (FSH) and luteinizing hormone (LH). These two hormones, in turn, stimulate gonads to produce and release estradiol and testosterone and regulate germ cell function. Both estradiol and testosterone negatively regulate further release of GnRH, FSH, and LH. Source: Reproduced from Melmed S. J Clin Invest 2003;112:1603 18.

eminence and infundibular stalk, terminating in the hypophyseal portal circulation and in the neurohypophysis. GnRH-I mRNA has been demonstrated in both the normal human pituitary as well as in pituitary adenomas [143]. Unlike other hypothalamic and pituitary neurons, GnRH neurons originate outside the CNS and migrate to the hypothalamus, using neuronal and glial cell scaffolding as well as chemotactic and adhesion molecules. GnRH neurons likely arise from the olfactory placode, as GnRH deficiency in humans is accompanied by loss of sense of smell (Kallmann syndrome) and by studies showing that ablation of the olfactory placode in animals causes loss of GnRH cells. GnRH-I neurons have also been proposed to arise from the region giving rise to the anterior pituitary placode, as zebrafish mutants lacking a pituitary but with normal olfactory organs show loss of hypothalamic GnRH-I neurons [144]. The GnRH receptor (GnRHR), a small GPCR, lacks a typical intracellular C-terminal cytoplasmic domain [143]. It mostly binds Gαq/11 but may also dosedependently bind Gαs and Gαi. Upon binding, Gαq/11mediated phospholipase C is activated and stimulates the diacylglycerol PKC and PI3K pathways to increase intracellular calcium levels and stimulate FSH secretion [143,145]. These signaling pathways also activate MAPK cascades [143,145]. In the normal anterior pituitary, GnRHR immunopositivity colocalizes with cells producing α-subunit, FSHβ, LHβ, TSHβ, and GH, suggesting GnRHR expression in gonadotrophs, thyrotrophs, and somatotrophs. GnRH levels regulate

gonadotroph GnRHR expression levels [143]. When GnRH levels are low (e.g., during lactation and malnutrition), GnRHR expression declines substantially. Long-term deprivation of GnRH necessitates longer periods of pituitary priming with repeated (but not continuous) pulses of GnRH until optimal sensitivity to GnRH is achieved. By contrast, continuous GnRH exposure downregulates GnRHR by enhancing receptor internalization and degradation, rendering the gonadotroph insensitive to GnRH [146 148]. GnRH neurons exhibit a pulsatile pattern of coordinated, repetitive GnRH release into the hypophyseal portal circulation. This GnRH pulse generator is controlled by both intrinsic [149] and external regulators [142]. Pulse frequency determines the rate of pulsatile FSH/LH release from gonadotrophs, the ratio between FSH and LH, and the degree to which FSH and LH are glycosylated, which determines their stability and potency. Although GnRH pulses match LH surges, FSH release is only partially concordant with LH surges. Therefore, LH, and not FSH, is used as an indicator of GnRH pulsatility. Higher GnRH frequencies increase LH and decrease FSH, thus increasing the LH/FSH ratio, while lower frequencies result in decreased LH/FSH ratios. Precise mechanisms of how gonadotrophs decode pulsatile GnRH signals to preferentially produce FSH or LH remain unclear [150]. Glycosylation of FSH and LH, especially addition of terminal sialic acid, is important for physiological action, protecting these hormones from liver degradation and prolonging half-life while reducing potency. Slow GnRH pulse frequencies increase FSH glycosylation during the ovarian follicular phase and support follicle development, while increased GnRH frequencies prior to ovulation generate more robust FSH with a shorter half-life [151]. GnRH release decreases during the postnatal period and begins to increase again in late childhood. Increased GnRH during puberty results from a combination of increased stimulatory tone and simultaneously decreased inhibitory tone [152]. Despite significant advances in the field, mechanisms for GnRH regulation remain enigmatic, and may involve an extensive network of intermediate regulatory neurons [153]. Some of these known factors are summarized in Fig. 2.13. Hypothalamic kisspeptin is the most potent GnRH secretagogue involved in puberty and reproduction [154]. GnRH neurons receive kisspeptin afferent inputs, express the Gq/G11 protein coupled kisspeptin receptor (KISS1R previously known as GPR54) mRNA, and respond to kisspeptin with cFos expression and depolarization [155]. Kisspeptin enhances GnRH pulse amplitude and duration, while GnRH inhibits kisspeptin secretion [142,156]. Importantly, kisspeptin responses are sexually dimorphic. Men respond with abrupt LH release upon kisspeptin treatment, while the

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HYPOTHALAMIC FACTORS REGULATING PITUITARY FUNCTION

37

FIGURE 2.13 Stimulators (up arrow) and inhibitors (down arrow) of the gonadotrophin-releasing hormone (GnRH) neuron in the hypothalamic medio-basal, infundibulum, and periventricular hypothalamic regions.  Under certain physiological conditions, including mid-cycle LH-surge. ^Hypothalamic factors enhancing estradiol-induced GnRH release. Kisspeptin receptor, GABAA receptor, Y1R, NPY receptor, GRP147, gonadotrophin inhibitory hormone receptor. GnRH, gonadotrophin-releasing factor; GALP, galanin-like peptide; NPY, neuropeptide Y; GABA, γ-aminobutyric acid; CRH, corticotrophin-releasing hormone; GnIH, gonadotrophin-inhibiting hormone; LH, luteinizing hormone; FSH, follicule-stimulating hormone; GnRHR1, gonadotrophin-releasing hormone receptor subtype 1.

response in women varies depending on the menstrual cycle phase. Neurokinin B and dynorphin are coexpressed in most kisspeptin neurons in the human hypothalamic infundibular nucleus, which corresponds to the rodent arcuate nucleus. These three peptides, cosecreted from kisspeptin/neurokinin B/dynorphin (KNDy) neurons, are thought to be tightly involved in regulation of the GnRH pulse generator and may be the long- predicted intermediate neurons that regulate effects of a plethora of peptides on GnRH neurons [153,157 161]. In humans, KNDy neurons are found in the infundibular nucleus and extend a network of axons into the hypothalamic infundibular stalk, in close proximity to GnRH axons. While GnRH neurons express only kisspeptin receptors, KNDy neurons express kisspeptin, neurokinin B, and kappa opioid peptide receptors (the receptor for dynorphin), creating both direct and indirect control of GnRH release [154]. Neurokinin B, a member of the tachykinin family, exerts paracrine/autocrine stimulatory action predominantly via its cognate GPCR TAC3 receptor located on KNDy neurons. Dynorphin is an opioid that binds its cognate kappa-opioid receptor expressed on KNDy neurons to inhibit their activity, thereby indirectly inhibiting GnRH neuron activity [162]. Thus, kisspeptin directly, and neurokinin B indirectly, stimulate GnRH release and dynorphin indirectly inhibits GnRH release, the latter two by autocrine/paracrine kisspeptin secretion regulation [153]. Moreover, GnRHR and

GPR54 may heterodimerize, and this receptor complex may preferentially couple Gαi and thereby inhibit GnRH release. Sex steroid hormones have a dramatic effect on GnRH release patterns. Estrogen receptors α and β (ERα and ERβ) [163], progesterone receptor (PR) [164], and androgen receptors (AR) [165] exhibit considerable overlap, and are found in hypothalamic areas involved with reproduction and sexual behavior. It is unclear whether GnRH neurons express estrogen receptors and are directly regulated by estrogen [142,166]; however, it is clear that GnRH neurons are regulated indirectly by sex steroids via interacting neighboring neurons, including the KNDy neurons, that abundantly express sex steroid receptors [142,153]. In general, estradiol, progesterone, and testosterone reduce GnRH pulse frequency and GnRH release to the hypophyseal portal circulation [167]. In humans, estradiol inhibition of LH/FSH is dominant at the pituitary level, while progesterone and testosterone inhibition is mostly regulated by the hypothalamus and KNDy neurons [153,167]. Estrogen feedback on GnRH secretion changes from negative to positive in the late follicular phase of the menstrual cycle and prior to and during LH surge. Estradiol-positive feedback, which also involves kisspeptin release [153], increases GnRH production and expression of GnRHR on gonadotroph membranes, increasing GnRH sensitivity. Neural enhancers of the estradiol stimulatory effect on GnRH include glutamate and aspartate [168],

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catecholamines, and neurotensin [169]. Mechanisms mediating the dual inhibitory/stimulatory effect of estradiol on GnRH neurons and the switch between the two forms are unclear, but are likely due to afferents from other estradiol-responsive neurons that directly regulate GnRH neurons. Progesterone and testosterone inhibit GnRH neurons. As a small subset of GnRH neurons express progesterone or AR [153], it is assumed that, as with estrogen, intermediate neurons are responsible for inhibitory effects of progesterone and testosterone on GnRH neurons. There is evidence to suggest that progesterone mediates KNDy neurons expressing PR to secrete dynorphin, which, in turn, suppresses GnRH release [170]. KNDy neurons also express AR and may play a role in mediating the testosterone-related inhibitory effect on GnRH [171]. Importantly, sex steroids affect the hypothalamic pituitary gonads axis at all levels, and the effect is species-specific. It is therefore challenging to accurately dissect the extent to which sex steroids alter GnRH secretion and/or affect gonadotroph sensitivity to GnRH action. Moreover, a direct deduction from animal to human GnRH regulation may be misleading. The effect of metabolic changes on GnRH release is a well-established phenomenon. Food deprivation impairs GnRH secretion and adiposity is required to initiate puberty in humans [172]. While leptin, the satiety hormone, is essential for puberty and fertility, NPY, a potent appetite-stimulator, exerts sex-steroiddependent inhibition of the gonadotroph axis [173]. GnRH neurons do not exhibit leptin receptors; these effects are mediated via projecting interneurons. Kisspeptin neurons in the arcuate nucleus express leptin and NPY receptors, and may have a role in mediating the metabolic signals of leptin and NPY on GnRH neurons [153,173]. In addition, NPY Y1 receptor subtype is expressed on GnRH neurons in vivo and may partially mediate NPY action on these neurons [173]. Galanin-like peptide (GALP) is another food-intake regulator shown to increase GnRH release from GnRH neurons. GALP neurons are in close contact with GnRH neuron bodies and have a direct estradioldependent stimulatory action on GnRH secretion [174,175]. Both GALP and kisspeptin neurons express leptin receptors and are stimulated by leptin, thus linking GnRH neuron function and the reproductive system to nutrition and energy reserves [176]. Stress, whether physical or psychological, is associated with changes in hypothalamic GnRH secretion. Prolonged activation of the hypothalamic pituitary adrenal axis, as in Cushing’s syndrome, is also associated with hypogonadotrophic hypogonadism. It is as yet unclear whether cortisol regulates GnRH release at the hypothalamic or pituitary level,

although there are compelling data suggesting that, in ewes, cortisol-mediated inhibition of LH secretion is mediated via reduction of pituitary sensitivity to GnRH action and not via a direct effect on GnRH neurons. CRH, vasopressin, catecholamines, and opioids, all of which participate in the stress response, have been suggested to play a mediatory role on GnRH neurons [177]. GABA and opioids are neural inhibitors of the stimulatory effect of estradiol on GnRH, and a significant decrease in their tone prior to the LH surge is critical for surge generation [169]. GABA neurons express ERα and are sensitive to estradiol action. GABA likely acts directly on GnRH neurons, as GABAergic neuron synapses terminate on GnRH neurons that express functional GABAA receptors. Endogenous opioids contribute to inhibition of stimulatory estradiol effects on GnRH neurons through indirect neural circuits, as GnRH neurons do not express opioid receptors. Gonadotrophin inhibitory hormone (GnIH) inhibits gonadotrophin synthesis and/or release by decreasing GnRH and/or kisspeptin neuron activity or by inhibiting pituitary gonadotrophs [178]. GnIH is therefore important for reproductive development and maintenance. GnIH binds primarily GPR147, a Gαi protein coupled receptor, inhibiting adenylate cyclase and reducing intracellular cAMP. GPR147 is expressed in GnRH and kisspeptin neurons and also in pituitary gonadotrophs and gonads. GnIH neuronal cell bodies are found in the hypothalamus, mostly in the periventricular nucleus, and project to the diencephalon and midbrain. They may also regulate kisspeptin, NPY, CRH, dopamine, POMC, NPY, orexin, melaninconcentrating hormone, and oxytocin neurons, thus mediating stress responses, appetite, and energy balance. Although the discovery of KNDy and GnIH neurons has significantly enriched our knowledge of GnRH neuron function, interspecies variability, sexual dimorphism, an inability to dissect the effect on GnRH neurons from the changed sensitivity of pituitary gonadotroph cell to GnRH, as well as an extensive intermediate neuronal network that interacts with GnRH neurons, all contribute to the complexity and uncertainty challenging our understanding of GnRH neuron regulation.

HYPOTHALAMIC PROLACTIN REGULATORS Prolactin-Inhibitory Factors Pituitary prolactin secretion is under tonic inhibition by hypothalamic dopamine (Fig. 2.14), with a marked

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HYPOTHALAMIC PROLACTIN REGULATORS

Dopamine

–

Lactotroph

PRL + Breast

FIGURE 2.14 Hypothalamic dopamine neurons release dopamine that exerts tonic pituitary lactotroph inhibition, reducing prolactin (PRL) synthesis and secretion. Source: Reproduced from Melmed S. J Clin Invest 2003;112:1603 18.

increase in lactotroph production and PRL secretion in the absence of this inhibition, such as when the pituitary stalk is damaged, or when the gland is ectopically transplanted, and a marked increase in lactotroph production and secretion of PRL ensues [179]. Studies in rats demonstrated that hypothalamic dopamine is released from neurons located in the arcuate and anterior periventricular nuclei. Tuberoinfundibular dopaminergic (TIDA) neurons project into the external zone of the median eminence, and are considered the major source of dopamine to the anterior pituitary. Tuberohypophyseal dopamine (THDA) neurons also project into the posterior and intermediate pituitary, while periventricular hypophyseal dopaminergic (PHDA) neurons project to the intermediate pituitary lobe. The anterior pituitary also receives inhibitory dopaminergic input from the posterior and intermediate pituitary through interconnecting short portal veins [180]. Dopamine also inhibits MSH release from melanotrophs located in the intermediate lobe, which is rudimentary in humans. Increased dopamine release from rat THDA and PHDA is associated with decreased melanotroph POMC gene expression, proliferation [181], and serum MSH levels [182]. Dopamine is a member of the catecholamine neurotransmitter group that shares a common synthetic pathway. Tyrosine is metabolized to dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase; DOPA is then decarboxylated to dopamine by aromatic L-amino acid decarboxylase. In dopaminergic neurons, further metabolism of dopamine does not occur, but in noradrenergic neurons, dopamine is subsequently hydroxylated to norepinephrine by dopamine β hydroxylase, and then to epinephrine by phenylethanolamine N-methyl transferase.

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Dopamine binds dopamine receptor subtype 2 (D2R) on pituitary lactotrophs, inhibiting both prolactin synthesis and release and lactotroph proliferation. D2R null mice develop lactotroph hyperplasia, hyperprolactinemia, and lactotroph adenomas, indicating that, at least in mice, D2R is the predominant dopamine receptor mediating the dopamine effect on prolactin [183]. D2R is a GPCR that couples the Gαi/o subunit to decrease adenylyl cyclase and intracellular cAMP levels, increase potassium influx, inhibit inositol phosphate production, and decrease intracellular calcium concentration. These signaling pathways acutely inhibit PRL [184], and, to a lesser extent, TSH secretion. Multiple downstream signaling pathways involved in lactotroph proliferation inhibition include adenylyl cyclase-PKA, MAPK [185], and phosphotyrosine phosphatase [186], although exact mechanisms are unclear. Dopamine is considered the main prolactininhibitory factor (PIF), but frequent inconsistencies are seen between dopamine levels in the rat hypophyseal portal circulation and serum prolactin levels. For example, even though serum prolactin levels are similar in males and females, dopamine levels in the hypophyseal portal circulation are five to seven times lower in males. The apparent inconsistency between dopaminergic activity and prolactin secretion has been attributed to other hypothalamic factors that may alter prolactin release in vitro and in vivo, including GABA, somatostatin that inhibits, and calcitonin that stimulates prolactin [179].

Prolactin-Releasing Factors Unlike for other pituitary hormones, a physiological specific prolactin-releasing factor (PRF) has yet to be identified. A number of hypothalamic factors stimulate prolactin secretion, including TRH, oxytocin, vasopressin, vasoactive intestinal polypeptide (VIP), angiotensin II, NPY, galanin, substance P, bombesin-like peptides (gastrin-releasing peptide, neuromedin B and C), and neurotensin [179,187], but none is a prolactin-specific hypothalamic factor and their demonstrated action on prolactin is inconsistent. Lactotroph prolactin secretion increases as dopamine levels reaching the pituitary are decreased. Interestingly, at very low (pM) concentrations, dopamine may actually stimulate prolactin secretion, but the physiological relevance of these studies in humans is unclear [188 190]. Interestingly, transient dopamine antagonism or withdrawal enhances stimulatory effects of TRH on prolactin secretion [179]. Oxytocin and vasopressin reach anterior pituitary lactotrophs through both the long and short hypophyseal portal circulation and stimulate prolactin secretion.

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Oxytocin increases male rat prolactin levels; in female rats, inhibition of oxytocin reduces prolactin surges induced by suckling or by estrogen, and blocks endogenous stimulatory rhythms controlling prolactin secretion. In contrast, low-dose oxytocin injections reduce basal and stress-induced prolactin secretion in male rats, and blocking vasopressin activity with antiserum attenuates the suckling-induced rise of plasma prolactin in females [179]. VIP and peptide histidine-isoleucine, or its human homologue peptide histidine methionine, are synthesized from neurons located in the paraventricular nucleus and also influence pituitary prolactin secretion. Both stimulate prolactin release in rats and in vitro. pituitary adenylyl cyclase activating polypeptide, a VIPlike hypothalamic peptide, dose-dependently stimulates pituitary prolactin in both male and nonsuckled lactating female rats [179]. Prolactin receptors are expressed on all dopaminergic neurons that supply the pituitary, enabling a short negative feedback loop of prolactin on dopamine synthesis. Increasing prolactin levels increases tyrosine hydroxylase expression, which, in turn, increases dopamine expression and leads to decreased prolactin levels [191,192]. Factors that increase prolactin secretion, including estrogen [193] and placental lactogens [179], eventually increase dopamine production. Estrogen increases prolactin mainly by reducing expression of lactotroph dopamine receptors, and long-term estrogen treatment reduces levels of tyrosine hydroxylase and dopamine content in TIDA neurons. Placental lactogens also inhibit tyrosine hydroxylase and dopamine content [194]. Multiple neural systems regulate dopaminergic neurons and PIF and PRF neurons, and subsequently regulate prolactin secretion. TIDA neurons are stimulated by acetylcholine, glutamate, and opioids, and are inhibited by stress, high levels of glucocorticoids, and histamine. PRF neurons in the paraventricular nucleus are stimulated by serotonin. TIDA neurons are also controlled by light, constituting the major neuroendocrine mechanism underlying the prolactin circadian rhythm. Since both the pituitary and the hypothalamus express dopamine receptors, most neuroleptics that inhibit dopamine secretion also increase prolactin levels [195].

SUMMARY There has been considerable progress in our knowledge of the anatomical, physiological, and pathophysiological aspects of the hypothalamic pituitary unit since their interaction was recognized almost a century ago. Discovery of pituitary hormones was followed by isolation of hypothalamic neuropeptides that regulate

their synthesis, cognate receptors, and multiple signaling pathways that they activate. Extensive feedback mechanisms, essential for neuroendocrine fine-tuning, and complex brain networking widely regulate hypothalamic neuropeptides to alter pituitary and target gland responses. Study of human hypothalamic pituitary path regulation is hampered by tissue inaccessibility for biopsy. The generation of neuropeptidergic hypothalamic neurons from pluripotent human stem cells yielding hypothalamic neurons producing POMC, AgRP, oxytocin, arginine, vasopressin, CRH, and TRH [196,197], could serve as cellular models to expand our understanding of the hypothalamic pituitary interaction in health and disease.

References [1] Sawin CT. Berta and Ernst Scharrer and the concept of neurosecretion. Endocrinologist 2003;13:73 5. [2] Klavdieva MM. The history of neuropeptides 1. Front Neuroendocrinol 1995;16:293 321. [3] Harris GW. Neural control of the pituitary gland. I. The neurohypophysis. Br Med J 1951;2:559 64. [4] Sandyk R. Stephen Walter Ranson (1880 1942): a pioneer in hypothalamic research. Int J Neurosci 1994;77:85 8. [5] Scharrer E. Neurosecretion. X. A relationship between the Paraphysis and the Paraventricular Nucleus in the Garter Snake (Thamnophis sp.). Biol Bull 1951;101:106 13. [6] Berkeley. The finer anatomy of the infundibular region of the cerebrum including the pituitary gland. Brain 1894;17:515 47. [7] Popa GT, Fielding U. Hypophysio-portal vessels and their colloid accompaniment. J Anat 1933;67:221, 227 32. [8] Harris GW. Electrical stimulation of the hypothalamus and the mechanism of neural control of the adenohypophysis. J Physiol 1948;107:418 29. [9] Guillemin R. Somatostatin: the beginnings, 1972. Mol Cell Endocrinol 2008;286:3 4. [10] Schally AV, Saffran M, Zimmermann B. A corticotrophinreleasing factor: partial purification and amino acid composition. Biochem J 1958;70:97 103. [11] Sheehan HL, Kovacs K. Neurohypophysis and hypothalamus. In: Bloodworth Jr. JMB, editor. Endocrine pathology, general and surgical. Baltimore: Williams & Wilkins; 1982. pp. 45 99. [12] Scheithauer BW. The hypothalamus and neurohypophysis. In: Kovacs K, Asa SL, editors. Functional endocrine pathology. 2nd ed. Boston: Blackwell Science; 1991. pp. 171 296. [13] Rodriguez EM, Blazquez JL, Guerra M. The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus: the former opens to the portal blood and the latter to the cerebrospinal fluid. Peptides 2010;31:757 76. [14] Ambach G, Palkovits M, Szentagothai J. Blood supply of the rat hypothalamus. IV. Retrochiasmatic area, median eminence, arcuate nucleus. Acta Morphol Acad Sci Hung 1976;24:93 119. [15] Broadwell RD, Balin BJ, Salcman M, Kaplan RS. Brain-blood barrier? Yes and no. Proc Natl Acad Sci USA 1983;80:7352 6. [16] Page RB. The anatomy of the hypothalamo-hypophyseal complex. In: Knobil E, Neill J, Ewing LL, Greenwald GS, Markert CL, Pfaff DW, editors. The physiology of reproduction. New York, NY: Raven Press; 1988. pp. 1161 233. [17] Knigge KM, Scott DE. Structure and function of the median eminence. Am J Anat 1970;129:223 43.

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[153] Marques P, Skorupskaite K, Rozario KS, Anderson RA, George JT. Physiology of Gnrh and Gonadotropin Secretion. In: De Groot LJ, Beck-Peccoz P, Chrousos G, Dungan K, Grossman A, Hershman JM, Koch C, McLachlan R, New M, Rebar R, Singer F, Vinik A, Weickert MO, editors. South Dartmouth (MA): Endotext; 2000. [154] George JT, Seminara SB. Kisspeptin and the hypothalamic control of reproduction: lessons from the human. Endocrinology 2012;153:5130 6. [155] Hrabovszky E. Neuroanatomy of the human hypothalamic kisspeptin system. Neuroendocrinology 2014;99:33 48. [156] Dungan HM, Clifton DK, Steiner RA. Minireview: kisspeptin neurons as central processors in the regulation of gonadotropinreleasing hormone secretion. Endocrinology 2006;147:1154 8. [157] de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972 6. [158] Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr. JS, Shagoury JK, et al. The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349:1614 27. [159] Goodman RL, Lehman MN, Smith JT, Coolen LM, de Oliveira CV, Jafarzadehshirazi MR, et al. Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B. Endocrinology 2007;148:5752 60. [160] Topaloglu AK, Reimann F, Guclu M, Yalin AS, Kotan LD, Porter KM, et al. TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction. Nat Genet 2009;41:354 8. [161] Plant TM. 60 YEARS OF NEUROENDOCRINOLOGY: the hypothalamo-pituitary-gonadal axis. J Endocrinol 2015;226: T41 54. [162] Grachev P, Millar RP, O’Byrne KT. The role of neurokinin B signalling in reproductive neuroendocrinology. Neuroendocrinology 2014;99:7 17. [163] Shughrue P, Scrimo P, Lane M, Askew R, Merchenthaler I. The distribution of estrogen receptor-beta mRNA in forebrain regions of the estrogen receptor-alpha knockout mouse. Endocrinology 1997;138:5649 52. [164] Bethea CL, Brown NA, Kohama SG. Steroid regulation of estrogen and progestin receptor messenger ribonucleic acid in monkey hypothalamus and pituitary. Endocrinology 1996; 137:4372 83. [165] Simerly RB, Chang C, Muramatsu M, Swanson LW. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 1990;294:76 95. [166] Hu L, Gustofson RL, Feng H, Leung PK, Mores N, Krsmanovic LZ, et al. Converse regulatory functions of estrogen receptoralpha and -beta subtypes expressed in hypothalamic gonadotropin-releasing hormone neurons. Mol Endocrinol 2008;22:2250 9. [167] Plant TM. Gonadal regulation of hypothalamic gonadotropinreleasing hormone release in primates. Endocr Rev 1986;7:75 88. [168] Brann DW, Mahesh VB. Excitatory amino acids: function and significance in reproduction and neuroendocrine regulation. Front Neuroendocrinol 1994;15:3 49. [169] Smith MJ, Jennes L. Neural signals that regulate GnRH neurones directly during the oestrous cycle. Reproduction 2001;122:1 10. [170] Goodman RL, Coolen LM, Anderson GM, Hardy SL, Valent M, Connors JM, et al. Evidence that dynorphin plays a major role in mediating progesterone negative feedback on gonadotropinreleasing hormone neurons in sheep. Endocrinology 2004; 145:2959 67.

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[171] Navarro VM, Gottsch ML, Wu M, Garcia-Galiano D, Hobbs SJ, Bosch MA, et al. Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse. Endocrinology 2011;152:4265 75. [172] Schneider JE. Energy balance and reproduction. Physiol Behav 2004;81:289 317. [173] Pralong FP. Insulin and NPY pathways and the control of GnRH function and puberty onset. Mol Cell Endocrinol 2010;324:82 6. [174] Uenoyama Y, Tsukamura H, Kinoshita M, Yamada S, Iwata K, Pheng V, et al. Oestrogen-dependent stimulation of luteinising hormone release by galanin-like peptide in female rats. J Neuroendocrinol 2008;20:626 31. [175] Gundlach AL. Galanin/GALP and galanin receptors: role in central control of feeding, body weight/obesity and reproduction? Eur J Pharmacol 2002;440:255 68. [176] Crown A, Clifton DK, Steiner RA. Neuropeptide signaling in the integration of metabolism and reproduction. Neuroendocrinology 2007;86:175 82. [177] Breen KM, Karsch FJ. New insights regarding glucocorticoids, stress and gonadotropin suppression. Front Neuroendocrinol 2006;27:233 45. [178] Ubuka T, Son YL, Tsutsui K. Molecular, cellular, morphological, physiological and behavioral aspects of gonadotropininhibitory hormone. Gen Comp Endocrinol 2016;227:27 50. [179] Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev 2000;80:1523 631. [180] Lerant A, Herman ME, Freeman ME. Dopaminergic neurons of periventricular and arcuate nuclei of pseudopregnant rats: semicircadian rhythm in Fos-related antigens immunoreactivities and in dopamine concentration. Endocrinology 1996;137:3621 8. [181] Chronwall BM, Millington WR, Griffin WS, Unnerstall JR, O’Donohue TL. Histological evaluation of the dopaminergic regulation of proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary, involving in situ hybridization and [3H]thymidine uptake measurement. Endocrinology 1987;120:1201 11. [182] Lindley SE, Gunnet JW, Lookingland KJ, Moore KE. Effects of alterations in the activity of tuberohypophysial dopaminergic neurons on the secretion of alpha-melanocyte stimulating hormone. Proc Soc Exp Biol Med 1988;188:282 6. [183] Asa SL, Kelly MA, Grandy DK, Low MJ. Pituitary lactotroph adenomas develop after prolonged lactotroph hyperplasia in dopamine D2 receptor-deficient mice. Endocrinology 1999;140:5348 55.

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[184] Vallar L, Meldolesi J. Mechanisms of signal transduction at the dopamine D2 receptor. Trends Pharmacol Sci 1989;10:74 7. [185] Suzuki S, Yamamoto I, Arita J. Mitogen-activated protein kinase-dependent stimulation of proliferation of rat lactotrophs in culture by 3’,5’-cyclic adenosine monophosphate. Endocrinology 1999;140:2850 8. [186] Florio T, Pan MG, Newman B, Hershberger RE, Civelli O, Stork PJ. Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity. J Biol Chem 1992;267:24169 72. [187] Grattan DR. 60 YEARS OF NEUROENDOCRINOLOGY: the hypothalamo-prolactin axis. J Endocrinol 2015;226: T101 22. [188] Denef C, Manet D, Dewals R. Dopaminergic stimulation of prolactin release. Nature 1980;285:243 6. [189] Hill JB, Nagy GM, Frawley LS. Suckling unmasks the stimulatory effect of dopamine on prolactin release: possible role for alpha-melanocyte-stimulating hormone as a mammotrope responsiveness factor. Endocrinology 1991;129:843 7. [190] Arey BJ, Burris TP, Basco P, Freeman ME. Infusion of dopamine at low concentrations stimulates the release of prolactin from alpha-methyl-p-tyrosine-treated rats. Proc Soc Exp Biol Med 1993;203:60 3. [191] Milenkovic L, Parlow AF, McCann SM. Physiological significance of the negative short-loop feedback of prolactin. Neuroendocrinology 1990;52:389 92. [192] Arbogast LA, Voogt JL. Prolactin (PRL) receptors are colocalized in dopaminergic neurons in fetal hypothalamic cell cultures: effect of PRL on tyrosine hydroxylase activity. Endocrinology 1997;138:3016 23. [193] Toney TW, Pawsat DE, Fleckenstein AE, Lookingland KJ, Moore KE. Evidence that prolactin mediates the stimulatory effects of estrogen on tuberoinfundibular dopamine neurons in female rats. Neuroendocrinology 1992;55:282 9. [194] Demarest KT, Duda NJ, Riegle GD, Moore KE. Placental lactogen mimics prolactin in activating tuberoinfundibular dopaminergic neurons. Brain Res 1983;272:175 8. [195] Molitch ME. Drugs and prolactin. Pituitary 2008;11:209 18. [196] Merkle FT, Maroof A, Wataya T, Sasai Y, Studer L, Eggan K, et al. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells. Development 2015; 142:633 43. [197] Wang L, Meece K, Williams DJ, Lo KA, Zimmer M, Heinrich G, et al. Differentiation of hypothalamic-like neurons from human pluripotent stem cells. J Clin Invest 2015;125:796 808.

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C H A P T E R

3 Adrenocorticotrophin Carmen L. Soto-Rivera and Joseph A. Majzoub

INTRODUCTION

CORTICOTROPH BIOLOGY Embryology

Adrenocorticotrophin (ACTH) is the anterior pituitary mediator of the hypothalamic
pituitary
adrenal axis that regulates responses to a variety of stressors, including hypoglycemia, psychological stressors such as fear, and physical stressors such as hypovolemia. ACTH is the 39-amino-acid product of proopiomelanocortin (POMC), which is principally synthesized in anterior pituitary corticotrophs, neurons of the mediobasal hypothalamus, and skin melanocytes. In each of these cell types, POMC is processed to several melanocortins: ACTH (in the pituitary), α-MSH (in the skin and hypothalamus), and β-endorphin (in all three). ACTH, in response to hypothalamic corticotrophinreleasing hormone, is secreted and binds to the adrenal melanocortin 2 receptor (MC2R), a G protein-coupled receptor that signals through cyclic AMP to stimulate cortisol production and secretion. The main action of cortisol is to maintain adequate body fuel supplies and blood pressure during times of stress. In response to ultraviolet light exposure, skin α-MSH is released and binds to MC1R, which stimulates production of melanin to increase skin pigmentation. α-MSH in the hypothalamus, in response to leptin, is released from neurons to act through MC4R receptors in hypothalamic paraventricular nuclei to decrease appetite and increase energy expenditure. Diseases involving ACTH are due to pituitary or ectopic tumors that secrete excessive ACTH, faulty transcription of the POMC gene, abnormal cleavage of the POMC precursor, or defects in signaling of POMC products via (at least) MC1R, MC2R, or MC4R. MC2R and MC4R require an accessory protein for their functions, melanocortin receptor 2 accessory protein (MRAP), and MRAP2, respectively, and mutations in these proteins impair associated receptor function.

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00003-9

The anterior pituitary forms via invagination of pharyngeal stomodeum in the region of contact with the diencephalon. By week 5 of human gestation, this invagination, termed Rathke’s diverticulum, has formed, but the downward migration of the diencephalic diverticulum, destined to be the neurohypophysis, has not yet commenced. It is at this time that adrenocorticotrophin (ACTH) is first detectable by immunocytochemistry in the collection of cells within Rathke’s diverticulum which are furthest from contact with the diencephalon [1]. By 8 weeks of gestation, ACTH is detectable by radioimmunoassay (RIA) of both fetal pituitary tissue and fetal blood [1]. The hypophyseal portal vascular system forms between 8 and 14 weeks gestation, dating the earliest time after which hypothalamic corticotrophin-releasing factors may function to regulate fetal pituitary ACTH [1]. By 14 weeks gestation, release of ACTH from human fetal corticotrophs is highly responsive to exposure to corticotrophin-releasing hormone (CRH) in vitro. The intensity of immunohistochemical ACTH staining in the anterior pituitary increases progressively from the mid-first through the end of the second trimester. In contrast, it is only after 21 weeks gestation that ACTHpositive cells begin to appear in the pars intermedia of the human fetal pituitary, defined as that region between Rathke’s cleft anteriorly and the neurohypophysis posteriorly. ACTH-containing cells in this region are epithelial-like, in contrast to the large, angular, ovoid appearance of corticotrophs in the anterior pituitary [1]. Corticotrophs expand postnatally by selfduplication of differentiated cells, rather than by expansion of progenitors [2].

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© 2017 Elsevier Inc. All rights reserved.

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Adult Anatomy Corticotrophs were initially identified by their basophilic staining. This has been subsequently found to be due to the presence of complex sugars in corticotrophs, principally those containing α-L-fucose and complex N-glycosyl-protein, as well as terminal β-galactose. Corticotrophs constitute between 10% and 20% of the cell population of the anterior pituitary [3], and occur either singly or in clumps. They are most numerous in the midsagittal region of the pituitary (median wedge) but also occur in the lateral wings of the gland. Although the adult human pituitary does not contain an intermediate lobe, the junctional zone between the anterior and posterior lobes is known as the zona intermedia. This region, derived from the portion of Rathke’s diverticulum posterior to Rathke’s cleft, contains scattered cells immunopositive for ACTH. Some of these ACTH-containing cells extend into the posterior pituitary, a feature known as basophilic invasion [3]. These areas of apparent corticotroph migration from the zona intermedia into the posterior pituitary occur focally along the border between these two regions. In humans, in addition to the sellar pituitary, a pharyngeal pituitary is located in either the sphenoid sinus or within the sphenoid bone, and consists of pituitary-like tissue approximately 2
5 mm by 0.2 mm in size connected to the gland by trans-sphenoidal vessels. Only 1
2% of pharyngeal pituitary cells contain immunoreactive ACTH, in contrast to approximately 14% of the cells in the sellar pituitary [4]. The pharyngeal pituitary is thought to arise as a rest of tissue resulting from the normal migration of cells from Rathke’s pouch to the sella turcica. There have been several reports of Cushing disease due to corticotroph adenomas arising in the pharyngeal or sphenoid pituitary [4,5].

Molecular Signals in Development Tremendous progress has been made in recent years concerning molecular mechanisms controlling pituitary organogenesis and pituitary cell-lineage specification in animal models. A complex network of transcriptional events mediated by extrinsic and intrinsic signals has been implicated in the determination and a stereotypical spatio-temporal differentiation of the five trophic cell types of the mature anterior pituitary gland (see chapter: Pituitary Development). Intrinsic Signals Murine fibroblast growth factor (FGF), Notch, bone morphogenetic protein (BMP), and WNT pathways affect induction and growth of the pituitary primordium. Prophet of Pit1 (PROP1), POU Class 1 Homeobox 1 (POU1F1)(PIT1), and pituitary-restricted transcription factor (TPIT) (TBX19) have pituitary-specific actions

[6,7]. Rpx (for Rathke’s pouch homeobox), also known as Hesx1 (for homeobox gene expression in embryonic stem cells) is the earliest known marker for the pituitary primordium [8]. Hesx1/Rpx expression ultimately becomes restricted to Rathke’s pouch, and downregulation of Hesx1/Rpx in the pouch coincides with the differentiation of pituitary-specific cell types. Mice lacking Rpx have abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum [1,9]. These abnormalities are reminiscent of defects associated with a heterogeneous group of human disorders known as septo-optic dysplasia (SOD). Deficits in SOD include optic nerve hypoplasia, abnormalities of the midline brain structures, and abnormalities of the hypothalamic
pituitary axis. Patients commonly present with endocrinopathies including hypoglycemia and adrenal crisis, which may signal growth hormone, ACTH, or thyroid-stimulating hormone (TSH) deficiency [1]. Dattani and colleagues have identified two siblings with SOD who are homozygous for missense mutations within the HESX1 homeodomain which prevents it from binding target DNA. The siblings identified with HESX1 mutations presented with hypoglycemia hours after birth and demonstrated panhypopituitarism, substantiating a role for HESX1 in human pituitary development. The corticotroph lineage appears to diverge relatively early from the other anterior pituitary cell types. Expression of Pit-1, a POU domain transcription factor, is restricted to the anterior pituitary and required for development of thyrotrophs, lactotrophs, and somatotrophs. Accordingly, patients with mutations in Pit-1 have normal HPA axis function. Prophet of Pit-1 (Prop-1) is required for Pit-1 expression, and mutation of its gene is associated with deficiencies in development of thyrotrophs, lactotrophs, and somatotrophs in the Ames dwarf mouse and in humans. In a small number of patients with mutations in PROP1, modestly impaired ACTH secretion has been reported [1]. A corticotroph-restricted transcription factor, TPIT (also known as TBX19), interacts with pituitary homeobox 1 and is required for POMC transcription. TPIT autosomal recessive mutations cause the syndrome of congenital isolated ACTH deficiency (IAD), characterized by low plasma ACTH and cortisol levels and preservation of all other pituitary hormones. Approximately 100 patients have been identified with IAD, which can present in the neonatal period as severe or partial ACTH and cortisol deficiency, often associated with neonatal seizures, hypoglycemia, and hypothermia, or later in life [10,11]. Although approximately two-thirds of patients with IAD have loss-of-function mutations in TPIT, several patients with this syndrome have been reported with a normal gene sequence [12]. Patients with early-onset ACTH deficiency who in addition have infections due

I. HYPOTHALAMIC
PITUITARY FUNCTION

CORTICOTROPH BIOLOGY

to common variable immunodeficiency, termed DAVID (deficit in anterior pituitary function and variable immune deficiency) syndrome, may have mutations in NFKB2 [13,14]. In contrast to IAD patients, these patients may also have anatomic pituitary anomalies, as well as other anterior pituitary hormone deficiencies of TSH, gonadotrophin, and growth hormone. The addition of these additional pituitary deficiencies, plus developmental abnormalities, has been termed GOLIATH syndrome, and has been associated with an intronic variant in IKBKE [15]. Extrinsic Signals The oral ectoderm, from which Rathke’s pouch forms, and the neural ectoderm of the ventral diencephalon, from which the hypothalamus arises, are in direct contact at the time of the formation of Rathke’s pouch [1]. Classical explant experiments have indicated that inductive signals arising from mesenchyme and neural ectoderm adjacent to Rathke’s pouch are required for pituitary organogenesis and cell line specification. The necessity for extrinsic signals for development of corticotrophs is supported by the demonstration that POMC expression in ectoderm explants requires coculture with mesoderm [1]. Extrinsic signals that promote pituitary organogenesis and cause cell-line specification include bone morphogenic proteins (BMP4, BMP2), fibroblast growth factor 8 (FGF8), Sonic hedgehog (SHH), and WNT5a [1]. Constitutive activation of the Notch cascade in mice inhibits differentiation of both corticotrophs and melanotrophs and results in suppression of transcription factors required for Pomc expression [16]. While the precise signals and interactions required for pituitary corticotroph specification are incompletely understood, it is clear that signals from the mature hypothalamus like CRH and arginine vasopressin (AVP) are not required for corticotroph specification. Importantly, the anterior pituitary appears to develop normally in a CRH null mouse [17]. Furthermore, deletion of the class III POU transcription Brn-2 results in failed maturation of AVP, CRH, and oxytocinproducing neurons of the hypothalamus and failed maturation of the posterior pituitary with no apparent defect in the maturation of any anterior pituitary cell type [18]. Treating either mouse [19] or human [20] embryonic stem cells with SHH and other extrinsic factors results in self-aggregation of functional ACTHsecreting corticotrophs, growing in self-organizing Rathke’s pouch-like three-dimensional structures.

Non-ACTH Peptides Within Corticotrophs Several neuropeptides colocalize with ACTH within corticotrophs, although in most cases it is not

49

clear whether this is due to synthesis or binding of the peptide within the cell. Neurophysin (but not vasopressin) colocalizes with ACTH in both normal and adenomatous corticotrophs. Neurophysin immunocytochemical staining is especially prominent in corticotrophs in the zona intermedia which appear to project into the posterior pituitary. Chromogranin A has been described in the majority of corticotroph adenomas, although only a fraction of these patients have elevated circulating plasma chromogranin A levels. Galanin is present in all normal corticotrophs as well as in the majority of corticotroph adenomas which have been examined [21]. Galanin has also been found in those corticotrophs in the zona intermedia which appear to be migrating into the posterior pituitary. It has also been described in corticotrophs which have undergone Crooke’s hyalinization. Calpastatin, an inhibitor of the calciumdependent cysteine proteases, calpain I, and calpain II, has been found in all ACTH-containing cells of the anterior pituitary, including those in the median wedge as well as those extending into the posterior pituitary. Vasoactive intestinal peptide has been found in corticotroph adenomas, but only rarely in normal corticotrophs [21]. Normal corticotrophs contain small amounts of cytokeratin, whereas corticotrophs which have undergone Crooke’s hyalinization are markedly positive for this protein. Corticotrophs appear to have few structural characteristics associated with neuronal cell types, as they are negative for neurofilament, vimentin, glial fibrillary acidic protein, and desmon. Likewise, rodent corticotrophs in the zona intermedia, unlike those in the pars intermedia, appear not to be innervated by neurons, since they do not stain with any of these neuron-specific markers. Similarly, synaptophysin, a 38-kDa integral membrane glycoprotein found in presynaptic vesicles of neurons stains only weakly in corticotrophs. These findings, together with a lack in humans of an anatomically discrete intermediate lobe with large numbers of α-MSH-containing cells, has led most investigators to conclude that there is no functional counterpart of the rodent pituitary intermediate lobe in humans.

ACTH and Related Peptide Expression Outside of Corticotrophs Although most ACTH is synthesized in anterior pituitary corticotrophs, the hormone is also expressed in several nonpituitary human tissues, both within and outside of the central nervous system. Within the central nervous system, ACTH and its related peptides are mostly expressed in cell bodies of the infundibular nucleus of the basal hypothalamus (analogous to the

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PITUITARY FUNCTION

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3. ADRENOCORTICOTROPHIN

rodent arcuate nucleus). The cell bodies of these hypothalamic ACTH neurons are located at the base of the third ventricle, adjacent to the median eminence and pituitary stalk. These neurons project to limbic, diencephalic, mesencephalic, and amygdaloid sites. Lesser amounts of ACTH and related peptides are found in substantia nigra, periventricular gray matter, and hippocampus. Interestingly, hypothalamic POMC expression occurs in areas also known to express the orexigenic neuropeptides NPY and AGRP and projects to many of the same hypothalamic targets [22]. The rodent arcuate nucleus prominently expresses leptin receptors, implicating POMC products in regulation of appetite and energy homeostasis (see melanocortin receptors). Brain areas containing ACTH also coincide with areas mediating stimulation analgesia, suggesting that expression of ACTH, or another product of the POMC gene (such as β-endorphin) in these sites may regulate pain perception. Outside of the central nervous system, ACTH and other POMC gene products, including α-MSH and β-endorphin, are synthesized in a large number of human tissues, including in descending order of abundance, adrenal, testis, peripheral mononuclear cells, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum [23]. In most of these tissues, POMC peptides are translated from truncated messenger RNAs lacking signal peptide sequences, suggesting that they cannot be secreted extracellularly, raising the question of their functional significance. However, adrenal and testis in addition express full-length POMC mRNA, suggesting that these tissues may also secrete POMC-related peptides [23]. Recently, several additional cell types have been shown to produce POMC peptides including monocytes, astrocytes, gastrin-producing cells of the gastrointestinal tract, keratinocytes, skin melanocytes, and atrial myocytes.

Ectopic ACTH-Secreting Tumors Ectopic ACTH secretion is a very uncommon cause of Cushing’s syndrome. It is characterized by cushingoid features (88%), skin pigmentation (88%), profound hypokalemia (88%), hypertension (75%), diabetes/ impaired glucose tolerance (75%), hyperlipidemia (69%), and severe infection (44%) [24]. Several neuroendocrine neoplasms arise in the bronchopulmonary tract, including small-cell neuroendocrine carcinomas, carcinoids, well-differentiated neuroendocrine carcinomas, and intermediate cell neuroendocrine carcinomas. These neoplasms express neuroendocrine markers including chromogranins and synaptophysin. Thirtyfour percent of small-cell carcinomas of the lung show immunoreactivity to one or more peptide hormones,

and patients with peptide-positive small-cell lung carcinomas have a shorter mean survival than patients with nonreactive tumors. ACTH is the most common and abundant hormone present, and is reported in 24% of small-cell carcinomas. However, 56% of small-cell lung cancer cell lines secrete significant levels of ACTH precursors, with little, if any, processing to ACTH. Oat-cell lung carcinomas may also produce ACTH, leading to Cushing’s syndrome. Carcinoid tumors often produce more than one hormone, and can be responsible for the ectopic ACTH syndrome. Bronchial carcinoids occasionally contain ACTH and related opioid peptides, which does not alter the overall favorable prognosis of these tumors. Cushing’s syndrome caused by ACTH secretion by pulmonary tumorlets has been described [25]. Upon radiologic imaging, such tumorlets, which may be over 100 in number, present a very unusual appearance. Most nonpituitary POMC-secreting tumors do not produce CRH. However, there are several reports of neoplasms causing Cushing’s syndrome by cosecretion of both ACTH and CRH [26]. Some patients with ectopic ACTH syndrome due to lung cancer have tumors that secrete ACTH in response to CRH. Bronchial carcinoid tumors may contain CRH and ACTH, and be associated with high plasma ACTH and CRH levels and Cushing’s syndrome, or with normal plasma CRH levels. Cushing’s syndrome has been associated with ectopic ACTH secretion from a unilateral adrenal pheochromocytoma, and from bilateral pheochromocytomas in a case of multiple endocrine neoplasia type 2A. Several other ectopic tumors that secrete ACTH include adenoid cystic carcinoma of the lung, renal cell carcinoma, neuroendocrine tumor of the nasal roof, and ACTH-producing tumor metastatic to the liver. In general, POMC mRNA from nonpituitary tumors responsible for the ectopic ACTH syndrome is identical to normal, and to POMC mRNA derived from pituitary tumors. However, some tumors express a larger POMC mRNA species, increased from 0.3% of overall POMC mRNA in normal pituitary glands to up to 35
40% in the tumor, and transcribed from an alternative upstream promoter. Pancreatic islet cell tumors responsible for Cushing’s syndrome have been demonstrated to contain ACTH, β-endorphin, and POMC mRNA [27].

Corticotroph Neoplasms Corticotroph ACTH-secreting adenomas, resistant to negative feedback of cortisol, cause Cushing disease [28]. Pituitary adenomas constitute about 15% of intracranial tumors [3]. About one-third of secretory

I. HYPOTHALAMIC
PITUITARY FUNCTION

PROOPIOMELANOCORTIN GENE

pituitary adenomas produce ACTH. Their presentation in children is distinctly different from that in adults [29]. Ten to fifteen percent of pituitary adenomas are plurihormonal, some of which secrete ACTH. When discovered, ACTH-producing adenomas are often functional, small, highly vascular, and prone to hemorrhage. Corticotroph adenomas tend to be located in the central portion of the adenohypophysis, in the “mucoid wedge,” and form micronodular aggregates. Invasive adenomas are more frequently found among undifferentiated, extremely laterally localized, or large adenomas, and recur more frequently than noninvasive tumors. There is no correlation between the size of an adenoma and the cortisol level or rate of recurrence. Most adenomas responsible for Cushing disease or Nelson’s syndrome are microadenomas. ACTHproducing adenomas are usually monoclonal, but may be polyclonal [30,31]. Plurihormonal adenomas are usually polyclonal. ACTH immunoreactivity in an abnormal pituitary is usually due to a single functional adenoma, but can be associated with nodular hyperplasia or a silent corticotrophic adenoma. Diffuse or nodular corticotroph hyperplasia, which could result from an ectopic CRH-producing tumor [26], or from Addison disease [32,33], can rarely cause a pituitary adenoma. Primary treatment of Cushing disease is surgical [28,34], with recurrences managed either by repeat surgery, radiation including proton beam [35], or medical therapy using pasireotide, steroidogenic inhibitors, or glucocorticoid receptor (GR) antagonists [36,37]. Small-molecule therapeutics directed at corticotroph proliferation, such as R-roscovitine, a cyclin E inhibitor, are in development [38,39]. Approximately 5% of all surgically removed pituitary adenomas are silent corticotroph adenomas, defined as those that stain positive for ACTH but for unknown reasons are not associated with any symptoms or signs of Cushing’s syndrome [40]. The biochemical structure and ultrastructure of ACTH-secreting pituitary adenoma cells differ from nonadenomatous pituitary cells. The cells may look normal, but be increased in number. Cells are oval to polygonal in shape, with eccentric spherical nuclei and well-developed rough endoplasmic reticulum. Crooke’s hyaline is characteristic of ACTH-producing tumors, is associated with either endogenous or exogenous hypercortisolism, and is due to massive accumulation of intermediate cytoplasmic filaments that are normally present in small numbers [3]. Clinically, an increased number of Crooke’s cells is correlated with a longer postoperative replacement dose of cortisol requirement. Pituitary adenomas may produce other products in addition to ACTH. Some ACTH-producing corticotroph adenomas contain a form of gastrin that is smaller than gastrin found in the normal adenohypophysis.

51

7B2 is a secretory granule-associated protein, which may be involved in proconvertase activation, which is sometimes secreted by ACTH-producing tumors, but is secreted in the highest levels from nonfunctional pituitary tumors. Rarely, both Cushing disease and Nelson syndrome are preceded by generalized glucocorticoid resistance due to a mutation in the GR. In these cases, the high rate of ACTH secretion, stimulated by the generalized glucocorticoid resistance, may lead to adenoma formation, perhaps following a second oncogenic transformation. A variety of genes are over- and underexpressed in human corticotroph adenomas, many involved in cell differentiation or cell cycle control [41]. About one-third of corticotroph adenomas lose expression of BRG1, which in mice is associated with increased cyclin E expression and loss of the cell cycle inhibitor p27 (Kip1) expression, which may contribute to cell proliferation [42]. Corticotroph adenomas also express high levels of TBX19/TPIT, as expected, as well as PROP1 [43]. Approximately one-third of functioning corticotroph adenomas have somatic loss-of-function mutations in the ubiquitin-specific protease USP8, which may lead to enhanced POMC promoter activity [41]. Pituitary carcinomas are exceedingly rare, and are associated with extracranial metastases, including liver and lung. Pituitary carcinomas cannot be histologically differentiated from adenomas [3], and many pituitary carcinomas produce Cushing disease [3]. In patients with Cushing disease due to a pituitary carcinoma, the primary tumor and metastases stain immunochemically for ACTH, β-LPH, β-endorphin, and α-MSH, and production of both CRH and ACTH from a pituitary carcinoma has been described.

PROOPIOMELANOCORTIN GENE ACTH is derived from a 266-amino-acid precursor, proopiomelanocortin (POMC), so named because it encodes opioid, melanotrophic, and corticotrophic activities [44]. The human POMC gene is a single-copy gene located on chromosome 2p23. It and its paralogous genes encoding the highly homologous opioid peptides, preproenkephalin A and preproenkephalin B (dynorphin), are all located on different chromosomes. These three genes likely arose via sequential duplications of a common ancestral gene, and are represented in vertebrates as distant as jawless chordates, with different species having different complements of mature peptides arising from each gene [45]. Gene promoters encoding POMC, preproenkephalin A, and preproenkephalin B are quite divergent, reflecting the different patterns of expression and regulation among the three genes [45].

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POMC Gene Structure The human POMC gene is 8 kilobases (kb) long (Fig. 3.1) and consists of a promoter of at least 400 basepairs (bp) at the 5’ end of the gene, followed by three exons, 86 (exon 1), 152 (exon 2), and 833 (exon 3) bp long, and two introns, 3708 (intron 1) and 2886 (intron 2) bp in length. Exon 1 is not translated. Perhaps because of this, exon 1 of the human and other mammalian POMC genes are less than 50% identical. The initiator methionine is located 20 bp into exon 2, and is followed by a 26-amino-acid hydrophobic signal peptide. Except for the signal peptide and 18 amino acids of the amino-terminal

glycopeptide, the majority of the POMC precursor is encoded by exon 3 [44]. Exon 2 is close to 90% identical between the POMC genes of humans and other mammals. Within exon 3 of POMC are located all known peptide products of the POMC gene, including N-terminal glycopeptide, γ-MSH, joining peptide (JP), ACTH, α-MSH, corticotrophin-like intermediate lobe peptide (CLIP), β-lipotrophin (β-LPH), β-MSH, and β-endorphin. Within exon 3, the regions encoding the N-terminal glycopeptide, α-MSH, ACTH, and β-endorphin, are greater than 95% identical between humans and other mammals. In contrast, JP, the region between the N-terminal glycopeptide and ACTH, is very poorly conserved

(A) POMC gene

γ1-MSH

α-MSH

β-MSH

CLIP γ-LPH

Lys-Lys β-endorphin1-26/7

β-endorphin

PC1

PC2

PC1

γ3-MSH

(C)

Lys-Arg

-LPH

ACTH Lys-Lys

Lys-Arg

Joining peptide Arg-Arg

(B) POMC peptide

Arg-Lys

N-terminal glycopeptide

Exon 3

Lys-LysArg-Arg

Signal peptide

intron

Exon 2

Lys-Arg

intron

Exon 1

Lys-Arg

Promoter

ACTH112–150

Pituitary

SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEFKR

Brain

SYSMEHFRWGKPV-NH2

CLIP130–150 PVKVYPNGAEDESAEAFPLEF-NH2

PC1

PC1

PC2

-MSH112–124

-LPH153–240

Pituitary

ELTGQRLREGDGPDGPADDGAGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKDKRYGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE

-endorphin210–240

-LPH153–207

Brain

ELTGQRLREGDGPDGPADDGAGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKD

YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE

FIGURE 3.1 Structure of proopiomelanocortin (POMC) gene and peptide products. (A) Schematic diagram of the POMC gene, consisting of three exons (rectangles) separated by two introns (thin lines). Translated regions are shown in black with the corresponding peptide regions indicated by dotted lines. (B) The 240-amino-acid precursor is formed after removal of the 26-amino-acid N-terminal signal peptide. The POMC precursor can be divided into three domains: (1) N-terminal glycopeptide and joining peptide; (2) adrenocorticotrophin (ACTH); and (3) β-lipotrophin (β-LPH). These are further cleaved by prohormone convertase enzymes, PC1 and PC2, at arginine-arginine, argininelysine, or lysine-lysine residues, to produce site-specific expression. The POMC gene is highly conserved except in the joining peptide and latter portion of γ3-MSH, denoted by dashed lines. (C) Pituitary- and brain-specific peptides are cleaved from the POMC precursor. Amino acids at proteolytic cleavage sites are indicated in bold, underlined letters (lysine K, arginine R). The glycine (G) residue in white indicates a C-terminal amidation site. MSH, melanocyte-stimulating hormone; CLIP, corticotrophin-like intermediate lobe peptide; α-MSH, α-melanocyte-stimulating hormone.

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among mammals, which has suggested to some workers that it does not encode a biologically important function [44]. The promoter of the human POMC gene contains typical TATA and CAAT boxes 28 and 62 b respectively, upstream from the transcription start site. Using in vitro transcription and transfection of the human POMC gene into heterologous cells [46], the POMC gene promoter contains DNA elements which mediate increased transcription by cyclic adenosine monophosphate (cAMP) and decreased transcription by glucocorticoids. Although these elements have not been precisely localized in the human POMC gene promoter, they are present within the 700 bp 50 to the transcription start site [46]. The rat POMC gene promoter has been studied more extensively. Drouin and coworkers using DNA-mediated gene transfer into transgenic mice and tissue culture cells, found that the DNA sequences needed for corticotroph-specific expression and negative transcriptional regulation by glucocorticoid are contained within 543 bp of the transcription start site. Multiple synergistic DNA elements have been reported to be necessary for correct pituitary-specific expression of the gene, including NeuroD, Pitx1, Pitx2, and Tpit as transcription factors which cooperate to cause corticotroph- and melanocyte-specific expression of POMC within this promoter region [10,47,48]. Pitx1, Pitx2, and Pitx3 also have major developmental roles outside of the pituitary (reviewed in [48]). Approximately 60 nucleotides upstream of the Pitx1 and Tpit binding elements is the binding site for the bHLH transcription factor NeuroD1 [49], also involved in pituitary-specific expression. The transcription factor, Etv1/Er81, acts synergistically with Tpit to enhance Pomc transcription [50]. Another enhancer highly conserved across vertebrates is located seven kilobases upstream from the Pomc transcription start site. It contains a Tpit response element and slightly favors corticotroph over melanocyte expression of Pomc [51]. Expression of Pomc within the hypothalamus depends largely on two other enhancers, termed neuronal POMC enhancers 1 and 2 (nPE1 and nPE2). nPE1 and nPE2 are located 12 and 10 kilobases, respectively, upstream from the POMC transcription start site [52]. They have partially redundant function to control POMC neuronal expression during development and postnatally [53]. Nuclear factor kappa beta, encoded by NFKB2, activates hypothalamic neuronal transcription of POMC via a binding site approximately 100 nucleotides upstream from the transcriptional start site [54]. Mutations in NFKB2 cause DAVID syndrome, associated with ACTH deficiency [14].

53

POMC MRNA Transcription, Splicing, and Polyadenylation In addition to the major transcriptional initiation site present in corticotrophs, at least six other start sites have been found in several nonpituitary tissues, including human adrenal, thymus, and testes [55]. These sites are all between 41 and 162 nucleotides downstream from the 5’ end of exon 3. The mRNAs transcribed from these sites thus would be intronless, and the only truncated molecules that might be translated would be devoid of a signal peptide, and therefore could not be secreted. Tissues containing these shorter forms of POMC mRNA, including adrenal, testis, spleen, kidney, ovary, lung, thyroid, and gastrointestinal tract, express ACTH, N-terminal glycopeptide1
61, and β-endorphin [23]. These truncated mRNAs are capable of being translated both in cell-free translation systems and in heterologous cells transfected with the appropriate fragment of the human POMC gene, although peptide products are not secreted. A canonical polyadenylation signal is present in human POMC mRNA 23 bases upstream from the poly (A) addition site.

POMC Biosynthesis and Processing The human POMC precursor has the potential to encode several overlapping peptides of biological importance (Fig. 3.1). Within the precursor, these peptides are separated from one another by two or more basic amino acids which serve as recognition sites for prohormone cleavage enzymes. POMC-derived peptides contain potential signals for amidation, glycosylation, acetylation, and phosphorylation. Because the nomenclature of POMC proteolytic products has been derived from both peptide-mapping studies as well as molecular biological studies in which putative peptides had been predicted from inspection of nucleotide sequences [44], the terminology is often confusing. To avoid confusion, amino acid (aa) positions in this chapter are numbered as superscripts with reference to the 240-aa-long human POMC precursor, formed after removal of the 26-aa-long signal peptide. The 240-aa POMC precursor can be considered to be composed of three domains (Fig. 3.1). Domain I (aa 1
111), the N-terminal domain, encodes the 76-aa-long N-terminal glycopeptide1
76 within its first 78 aa, and the 30-aa-long C-terminal joining, or hinge, peptide (JP79
108) within its last 33 aa. The middle Domain II (aa 112
152), encodes the 39-aa ACTH112
150 peptide, which may be further processed to α-melanocyte-stimulating hormone (MSH)112
124. The C-terminal Domain III, termed β-LPH153
240, is 88-aa long. It contains

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3. ADRENOCORTICOTROPHIN

within it the 31-aa-long β-endorphin210
240. Several other products have been identified, although evidence for their existence and/or biological importance in man is not clear [44]. These include gamma1MSH51
61, gamma2-MSH51
62, and gamma3-MSH51
76 in Domain I, CLIP130
150 in Domain II, and gammaLPH153
206 and β-MSH191
206 in Domain III (Fig. 3.1). Glycosylation of POMC Within the ER, POMC undergoes initial glycosylation. Human POMC is glycosylated solely at two sites in the N-terminal glycopeptide1
76 of Domain I. Carbohydrate is added via an O-linked glycosylation to Thr45 and via an N-linked glycosylation to Asn65. C-Terminal Amidation of POMC Three human POMC products undergo C-terminal amidation [44]. These include N-terminal glycopeptide1
61, JP79
108, and α-MSH112
124. These three products are also present in their Gly-extended forms, which may be incompletely processed intermediates. C-terminal amidation is common among neuropeptides, and is usually essential for bioactivity. This reaction is mediated by a bifunctional enzyme consisting of peptidylglycine-α-amidating monooxygenase (PAM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) activities, which transfer the amino group of a C-terminal Gly to the carboxyl group of the adjacent amino acid. Human PAM/PAL exists in both a membrane-bound and free cytoplasmic form. N-Terminal Acetylation of POMC Two human POMC products undergo N-terminal acetylation: α-MSH and β-endorphin. In humans α-MSH exists predominantly in the nonacetylated form [44]. N-terminal acetylation of ACTH1
13-amide to form α-MSH results in increased melanotrophic activity and decreased corticotrophic activity. Proteolytic Processing of POMC POMC gives rise to several smaller, biologically active peptide products generated by posttranslational cleavage of POMC by trypsin-like prohormone convertase endopeptidase enzymes which cleave the precursor on the C-terminal side of regions of two or more basic aa residues [44]. These basic amino acids are subsequently removed by carboxypeptidase activity. Some POMC proteolytic products subsequently undergo amidation at their C-terminus or acetylation at their N-terminus, as described above. Posttranslational processing of POMC exhibits a remarkable degree of tissue-specificity, which has been postulated to be due to the differential distribution of processing enzymes in the various tissues which synthesize POMC (see below).

PROTEOLYTIC PROCESSING ENZYMES

All proteolytic processing of human POMC occurs at either lys-arg or arg-arg residues (Fig. 3.1). Every lys-arg and arg-arg site within the human precursor is capable of being cleaved in vivo, whereas in the mouse and rat, additional arg-lys and lys-lys sequences at the N-termini of gamma1-MSH and β-MSH, respectively, appear to be utilized [44]. It is likely that proteolytic digestion of human POMC at all sites is mediated by either of two structurally related endopeptidases, prohormone convertase 1/3 (PC1/3) or prohormone convertase 2 (PC2), encoded by PCSK1 and PCSK2, respectively. These enzymes, best studied in rodents thus far, are part of a seven-member family of subtilisin/kexin-like mammalian proteinases, and are distributed specifically within endocrine cells and neurons. Both enzymes are capable of cleaving neuropeptide precursors, including POMC, proinsulin, and proglucagon, at dibasic sites, and each appears to manifest distinct preferences for different sites within the same precursor prohormone, with PC2 able to cleave at a wider selection of available dibasic sites than PC1/3 [44]. Tissue distribution of PC1/3 and PC2 mRNAs is distinctly different. PC1/3 is abundant in B20% of anterior pituitary cells (presumably including corticotrophs), in all intermediate lobe pituitary cells (of the rodent), and in the supraoptic nucleus of the hypothalamus [56]. In contrast, PC2 is absent from pituitary corticotrophs, but is highly expressed in rodent pituitary intermediate lobe, multiple sites within the central nervous system, including cerebral cortex, hippocampus, and thalamus, and in pancreatic islet cells [56]. The differential tissue-specific distribution of these enzymes matches well with known tissue-specific differences in POMC proteolytic processing (see below), suggesting that PC1/3 is responsible for POMC cleavage products found in anterior pituitary corticotrophs, whereas PC2 cleaves POMC in the pituitary intermediate lobe (of lower mammals) and in the brain. This suggestion is supported by studies of differential POMC processing by PC1/3 and PC2 [56]. Patients with mutations in POMC-processing enzymes have disease phenotypes predicted from loss of function of POMC products [57]. Thus, a patient with severe childhood obesity and hyperproinsulinemia with postprandial hypoglycemia was identified with compound heterozygous mutations in PCSK1 [58]. The patient presented with multiple endocrine abnormalities including impaired glucose tolerance and postprandial hypoglycemia attributed to hypersecretion of proinsulin given its partial insulin-like action and long biological half-life. Hypogonadotropic hypogonadism with primary amenorrhea, but normal development of secondary sexual characteristics, were reported. Ovulation was

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PROOPIOMELANOCORTIN GENE

induced with exogenous gonadotropins, and the patient delivered healthy quadruplets. The pregnancy was complicated by gestational diabetes requiring insulin treatment. She also exhibited mild adrenal cortical insufficiency with complaints of fatigue and excessive daytime somnolence reversed by glucocorticoid administration. Adrenal cortical insufficiency was attributed to defective POMC processing with elevated levels of serum ACTH precursors confirming a role for PC1/3 in human POMC processing [58]. Subsequently, several patients have been described with similar, severe homozygous or compound heterozygous loss-of-function mutations in PCSK1 associated with obesity, hypogonadotrophic hypogonadism, diabetes insipidus, and type 1 diabetes [59
61], and several common variants in the gene are strongly associated with obesity [62,63]. Following the generation of peptide products by prohormone convertase enzymes, the C-terminal basic amino acids are removed by carboxypeptidase activities. Carboxypeptidases E (Cpe) is required for excision of paired dibasic residues of various peptide prohormone intermediates, including those derived from proinsulin and POMC. The mutation in a strain of hyperproinsulinemia, late-onset fat/fat obese mouse, was mapped to the carboxypeptidase E gene. A missense mutation has been identified in the Cpefat allele and these mice demonstrated a 20-fold decrease in Cpe enzymatic activity in pituitaries and isolated islets. A morbidly obese female from a consanguineous family exhibited homozygosity for a truncating mutation of the CPE gene. Obesity, abnormal glucose homeostasis, and hypogonadotrophic hypogonadism in this patient phenocopies the Cpe knockout mouse, demonstrating the important role of CPE in the processing of POMC and other peptides in humans [64]. The sorting of POMC into secretory granules is probably mediated by a signal patch, located within the tertiary structure of the molecule, which directs it to the granules. The presence of all POMC processed products in equimolar amounts within secretory granules suggests that this sorting precedes proteolytic cleavage of POMC. This suggestion is supported by data which demonstrate that initiation of proteolytic processing of POMC begins in the trans-Golgi system and continues in secretory vesicles and is also consistent with data demonstrating localization of PC1/3 and PC2 within the TGN and/or dense core secretory granules. Since POMC as well as PC1/3 and PC2 all contain signal peptides and are presumably colocalized throughout the endoplasmic reticulum, prohormone convertase activity must be inhibited in the endoplasmic reticulum and cisGolgi regions. Proconvertase activity may be regulated by the local intracellular environment and/or by control of catalytic activation of prohormone convertase.

55

Prolylcarboxypeptidase has been identified as the enzyme responsible for inactivation of α-MSH by removal of the C-terminal valine [65]. Loss of function mutations in this enzyme have not been described in humans but are predicted to result in leanness, as has been found in mice [66]. 7B2 is a small, hydrophobic acidic protein originally isolated from porcine and human anterior pituitary glands that is widely distributed in neuroendocrine tissues and found to associate specifically with PC2 [67]. The 27-kDa 7B2 precursor protein is cleaved to a 21-kDa protein and a small carboxy-terminal peptide (CT peptide). Interaction of 7B2, particularly the 21-kDa fragment, with proPC2 appears necessary for the generation of mature and active PC2 in the transGolgi region. The 7B2 CT peptide is a nanomolar inhibitor of PC2 in vitro, but its role in vivo has not been defined. The role of 7B2 in activating proPC2 has been confirmed in vivo in 7B2 null mice. 7B2 null mice are devoid of PC2 activity and deficiently process islet hormones and POMC resulting in hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. These mice also demonstrate profound intermediate lobe ACTH hypersecretion with minimal conversion of this peptide to α-MSH, resulting in a severe Cushing’s syndrome that causes death by 9 weeks of age. Curiously, PC2 null mice demonstrate similar islet cell dysfunction resulting in hypoglycemia, but do not produce a Cushingoid syndrome. This discrepancy suggests additional functional roles for 7B2 which are further suggested by localization of 7B2 in regions of the brain lacking PCSK2. The suggestion that 7B2 may represent one member of a family of related convertase inhibitor proteins has been proposed with the identification of the protein proSAAS [68]. ProSAAS is a 26-kDa granin-like neuroendocrine peptide precursor isolated from rodents and humans with structural similarity to 7B2 including a proline-rich sequence in the first half of the molecule and a C-terminal peptide (SAAS CT peptide) following a dibasic cleavage sequence [68]. Overexpression of proSAAS in AtT-20 cells reduces the rate of POMC processing and the SAAS CT peptide is a nanomolar competitive inhibitor of PC1/3, but not PC2 [68]. A 2.9-kDa peptide fragment of chromogranin A, termed serpinin, identified as a secreted product from a corticotroph cell line, may promote secretory granule biogenesis [69]. TISSUE SPECIFICITY OF POMC PROCESSING

In human corticotrophs, POMC is processed predominantly into N-terminal glycopeptide1
76, JP79
108, ACTH112
150, and β-LPH153
240 (Fig. 3.1) [44]. Much smaller amounts of α-MSH112
124, CLIP130
150,

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β-endorphin210
240 [70], and a truncated form of N-terminal glycopeptide1
61, also known as “big” gamma-MSH, are also present [44]. There is no evidence for cleavage after arg50, and therefore no evidence for the presence of gamma1-MSH51
61 in the human pituitary. JP79
108 exists as both a monomer and homodimer, most likely joined via disulfide bonding between the single Cys87 of two molecules. A function for N-terminal glycopeptide has also not been assigned, although one intriguing study reported that it is capable of stimulating aldosterone release from adrenal cells. Whereas the production of distinct POMC peptide derivatives is clearly segregated between the anterior and intermediate lobes of the rodent, in the human, small, acetylated POMC peptide derivatives colocalize with larger deacetylated POMC peptides in corticotrophs of the anterior pituitary, suggesting that the strict dichotomy between corticotroph and melanotrope POMC processing observed in rodents and other species does not extend to human pituitaries [44]. Levels of desacetyl-α-MSH are elevated in pituitary corticotrophs and plasma of patients with Addison disease, Cushing disease, and Nelson syndrome [44]. Desacetyl-α-MSH has approximately 75% of the melanotrophic activity as does α-MSH, whereas ACTH is only 5% as potent as α-MSH in this regard. Because the serum levels of ACTH are 50- to 100-fold higher than levels of desacetyl-α-MSH in patients with Cushing disease, Addison disease, and Nelson syndrome, it is likely that the hyperpigmentation associated with these disorders is largely due to the melanotrophic effect of ACTH, and not MSH. Thus, in human anterior pituitary corticotrophs, the POMC precursor is predominantly cleaved at limited Lys-Arg sites into two peptides in Domain I (N-terminal glycopeptide1
76 and JP79
108), one peptide in Domain II (ACTH112
150), and one peptide in Domain III (β-LPH153
240) (see Fig. 3.1). Human POMC is also expressed in several extrapituitary brain sites, predominantly in the arcuate nucleus of the anterior hypothalamus. In these extrapituitary locations, POMC is processed to a greater extent than in anterior pituitary. Brain ACTH112
150 is cleaved to α-MSH112
124 and CLIP130
150, due to expression of PC2, such that the amount of α-MSH relative to ACTH is 300-fold higher in hypothalamus, telencephalon, and mesencephalon than it is in the anterior pituitary [44]. As in the anterior pituitary, α-MSH is almost exclusively present in the desacetyl form. Adding additional levels of complexity to the question of tissue specificity of POMC processing are results obtained from dopamine D2 receptor (D2R)-deficient mice. Similar to tonic inhibitory dopaminergic control of prolactin expression in lactotrophs, POMC expression in the rodent intermediate lobe is under inhibitory dopaminergic control mediated via D2 receptors.

D2R-deficient mice demonstrate mild intermediate lobe hyperplasia accompanied by upregulation of both PC1/3 and PC2. These mice present with unexpectedly high levels of ACTH with corresponding adrenal hypertrophy and increased corticosteroid secretion. The altered prohormone convertase levels in these mice suggest the possibility of dynamic regulation of prohormone processing within specific tissues.

POMC Mutations Leading to POMC Deficiency Several unrelated individuals have been identified with genetic defects in the POMC gene [71,72]. The first patient was a compound heterozygote for two mutations in exon 3 which interfere with appropriate synthesis of ACTH and α-MSH (Fig. 3.1). The second patient was homozygous for a mutation in exon 2 which abolishes POMC translation. Both patients presented with adrenal insufficiency, early-onset obesity, fair skin, and red hair pigmentation. The brother of patient one had died at the age of 7 months with hepatic failure following severe cholestasis and was found to have bilateral adrenal hypoplasia in the postmortem examination. Other anterior pituitary-derived hormones were normal, and heterozygous parents were normal in both families. Approximately 10 additional patients have been reported with homozygous or compound heterozygous nonsense or frameshift mutations [73,74]. This syndrome can present with normal skin and hair pigmentation, despite complete ACTH and glucocorticoid deficiency due to a homozygous frameshift mutation [75]. A 4-month-old boy homozygous for a POMC missense mutation, R145C in POMC, and at amino acid 8 of ACTH and α-MSH, presented with hypoglycemia, obesity, red hair, severe cortisol deficiency, and very elevated ACTH. His mutant ACTH and α-MSH were immunoreactive but bioinactive [76]. In the family of an affected obese child homozygous for a pathogenic POMC frameshift mutation, heterozygous carriers, but not normal members, were obese, suggesting either haploinsufficiency or a dominant-negative mechanism of disease [77]. Two severely obese children without adrenal insufficiency were described with a heterozygous mutation in POMC which disrupts the dibasic cleavage site between β-MSH and β-endorphin, suggesting this may interfere with normal processing of the POMC precursor and lead to obesity [78]. Two additional heterozygous missense mutations in POMC have been reported in unrelated patients with early-onset obesity, without adrenal insufficiency, red hair, or fair skin. These mutations are located in a region of the POMC N-terminus that may be involved in sorting to the regulated secretory pathway [79]. Among over 300 French obese children, one heterozygous missense mutation in both an obese child and her obese father,

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REGULATION OF THE HPA AXIS

present within α-MSH, abolished in vitro activation of MC4R [80]. Hypermethylation of POMC may be associated with obesity [81]. In the absence of ACTH due to these POMC gene mutations, adrenal glands are hypotrophic and patients present with adrenal insufficiency. Unlike in the pituitary, neuronal ACTH is further cleaved into α-MSH, which binds MC4R to decrease food intake and increase energy expenditure. α-MSH also activates MC1R, which contributes to skin and hair pigmentation. In the absence of α-MSH due to these POMC gene mutations, patients are hyperphagic, obese, and have fair skin with red hair pigmentation. Humans with polymorphic variants in MC1R also have fair skin and red hair pigmentation [82]. No symptoms related to β-endorphin deficiencies have been noted. IAD of the pituitary due to other causes is also rare and most commonly appears to be acquired in later life, more commonly in men. Most cases have been reported in Japan, and are often associated with other autoimmune endocrinopathies, including Hashimoto’s thyroiditis and type 1 diabetes mellitus. It is likely that only pituitary ACTH, and not hypothalamic α-MSH, is affected in these patients, as they are not obese and do not have red hair, unlike patients with POMC gene mutations [71]. Mutations in TPIT/TBX19 also lead to IAD. Consistent with sites of expression, these patients have congenital, secondary adrenal insufficiency, but not obesity or red hair. A POMC-deficient mouse was produced whose phenotype is similar to the human POMC-deficient syndrome and confirms the known functions of melanocortins [83]. The phenotype includes obesity, increased body length, yellow pigmentation, deficits in sebaceous gland function and thermoregulation, and adrenal hypoplasia and glucocorticoid deficiency. Adrenal glands could not be identified. In addition to undetectable corticosterone levels, aldosterone levels were also undetectable. The mutant mice lost 40% of their excess weight after 2 weeks of treatment with a stable α-MSH agonist. Although some of this weight loss was clearly attributable to decreased food intake, these same authors have clearly shown that α-MSH administration exerts additional lipolytic effects and results in increased energy expenditure. Consistent with the lack of aldosterone dependence upon ACTH, these mice have normal aldosterone function [84].

57

paraventricular nucleus (PVN), acts via the type 1 CRH receptor to increase cAMP content in anterior pituitary corticotrophs. CRH is responsible for both the increase in POMC transcription and peptide synthesis, as well as for the rise in intracellular calcium which results in ACTH secretion [46] via the cAMP/protein kinase A (PKA) pathway [85]. CRH-induced rise in cAMP activates signaling cascades that lead to expression of Nur factors, which act on NurRE as a fast-response system to modulate POMC expression in response to stress [46]. In AtT-20 cells, CRH leads to dephosphorylation of the orphan nuclear receptor Nur77 (nerve growth factorinduced protein IB [NGFI-B]), resulting in increased transcription of POMC [86]. CRH also recruits activators from the steroid receptor coactivator family, which function as Nur coactivators [87]. CRH might also mediate stimulation of POMC transcription via the POMC-CRH responsive element (PCRH-RE), which binds PCRH-RE binding protein [46]. Negative effects of glucocorticoids upon POMC gene transcription are thought to be mediated by a glucocorticoid
GR complex. GR does not bind POMC sequences directly; but interacts with Nur factors through protein
protein interactions that repress transcription [88,89]. BRG1, which is constitutively present at the POMC promoter before Gc/GR activation and the histone deacetylase HDAC2, are required for GR repression of transcription of POMC [48]. Glucocorticoid stimulates, rather than inhibits, POMC gene expression in the arcuate nucleus of the hypothalamus, the site of α-MSH production, suggesting POMC involvement in inhibition of appetite by glucocorticoids in the postabsorptive state, when glucocorticoid levels are high [90]. Vasopressin decreases both basal and CRHstimulated POMC mRNA levels in anterior pituitary cells. β-Adrenergic catecholamines, similar to CRH, also increase POMC mRNA levels in corticotrophs via a cAMP mechanism. Insulin-induced hypoglycemia causes increased POMC mRNA content in rat anterior pituitary corticotrophs [91], but whether this is secondary to an increase in hypothalamic CRH, vasopressin, or catecholamines is not known. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) causes a decrease in POMC mRNA levels in intermediate, but not anterior pituitary corticotrophs [21].

Ontogeny of ACTH Regulation REGULATION OF THE HPA AXIS POMC Gene Regulation In human anterior pituitary corticotrophs, POMC mRNA levels are increased by CRH and inhibited by glucocorticoids. CRH, produced by neurons in the

Fetal and Neonatal Regulation of ACTH Secretion In utero, maternal cortisol influences alterations of fetal heart rate, movement, and behavioral states. By 35 weeks of gestation, there is a circadian rhythm in fetal behavioral states that is altered if maternal diurnal variation in ACTH and cortisol secretion is

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abolished by maternal administration of triamcinolone. There is evidence for a 24-hour or circadian rhythm of plasma cortisol concentration in the term neonate that results from fetal adrenal cortisol secretion controlled by the fetal circadian system [92]. The adrenal circadian rhythm of the newborn infant is first entrained by maternal stimuli in utero and is then transiently reentrained at birth and during the first 5 days of life, possibly in response to birth-related stimuli which might aid a safe transition of the fetus to the extrauterine life [93]. After this period, diurnal cortisol changes in the newborn infant are unsynchronized with the external clock time for several months until the adrenal clock is finally entrained to the day
night cycle. Minimally and severely stressed neonates in the neonatal intensive care unit, born between 23 and 38 weeks of gestation, have a significant diurnal rhythm in cortisol and endorphin secretion, although ACTH levels do not vary significantly. There is progressive maturation of the circadian system after birth, with day
night (diurnal) rhythms in activity and hormone secretion developing sometime between 1 and 3 months of age [94], although some authors believe it does not occur until after 6 months. Recent studies show that the infant diurnal rhythm is responsive to light and that low-intensity lighting can help regulate the developing clock. Regulation of ACTH Secretion During Adrenarche, Puberty, and Adulthood Normal children between the ages of 1 year and 16 years do not differ from adults in ACTH, β-endorphin, and cortisol responses to CRH, and the responses of boys do not differ from girls. Some other adrenal steroids and adrenal androgens demonstrate basal and stimulated variation with age. Nonetheless, there are both maturational and sex differences in morning cortisol measurements which are higher in Tanner stage 5 than Tanner stage 1 children, and higher in sexually mature males than in equally mature females [95]. CRH-stimulated androstenedione to 17hydroxyprogesterone ratio increases with sexual maturation, suggesting that the 17,20-desmolase activity increases with puberty. The dehydroepiandrosterone response to CRH increases as children progress from stage 1 to stage 5 of puberty, and by stage 5 of puberty, dehydroepiandrosterone levels do not differ from adults [96]. In 1947, Albright coined the term adrenarche to denote the developmental increase in adrenal androgens that occurs several years before the onset of gonadal maturation [97]. Dehydroepiandrosterone (DHEA) levels rise with adrenarche [98]. Adrenal androgen secretion may be sufficient for development of some secondary sexual changes including the development of pubic and axillary hair and the maturation of sebaceous glands. A condition in which axillary and

pubic hair develop prematurely as a result of early adrenal androgen secretion has been termed premature adrenarche, and mechanisms controlling adrenarche remain obscure. Hypotheses include involvement of ACTH, estrogens, prolactin, gonadotrophins, growth hormone, glucocorticoids, androgens, and other POMCderived products as modulators of adrenal androgen secretion. ACTH is widely accepted as a modulator of adrenal androgen secretion although, after administration of corticotrophin, increases in DHEA and dehydroepiandrosterone sulfate (DHEAS) tend to be modest. Furthermore, increased adrenal androgens occurring in adrenarche are not accompanied by an increase in serum cortisol levels, leading to the suggestion that factors other than ACTH are responsible for adrenarche. However, cortisol production rates do increase contemporaneously with increased adrenal androgen secretion, suggesting a possible common link due to ACTH stimulation of both steroid pathways [99]. Consistent with this, patients with ACTH deficiency due to hypopituitarism have delayed or absent adrenarche [100]. Patients with genetic MC2R defects provide definitive evidence of the participation of ACTH and this receptor in the process of adrenarche [101]. Dissection of the physiology of adrenarche has proven difficult as the only animals showing an adrenarche similar to that of humans are great apes [102]. Cortisol inhibits 3-beta hydroxysteroid dehydrogenase type 2 in adrenal cells [103]. This could result in a decline in cortisol production, a rise in ACTH, and stimulation of 17,20-lyase, which is also known to increase during adrenarche [104]. It is possible that adrenarche may be triggered by a rise in intraadrenal cortisol, which inhibits 3-beta hydroxysteroid dehydrogenase type 2, causing cortisol secretion to fall, with a rise in ACTH, DHEA, and DHEA sulfate [103].

Hormonal and Pharmacological Regulators of ACTH Secretion of ACTH from the corticotrophs of the anterior pituitary is mediated by several factors (Fig. 3.2). CRH and vasopressin are the primary secretagogues for ACTH, although a number of other agents may also affect its release, while glucocorticoids are the major negative regulators of ACTH secretion. Once a ligand has bound to its receptor, release of ACTH from the corticotroph is mediated by second messengers through one of four signal transduction pathways, involving either PKA, protein kinase C (PKC), glucocorticoids, or the Janus kinase/ signal transducers and activators of transcription (STAT) system. These pathways result in changes in the phosphorylation pattern of specific cellular proteins, and/or in intracellular calcium levels,

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Catecholamines IL1β + IL-6

CRH AVP

TNFα + LIF ACTH

IL1β

+

IL-6 TNFα

s rti

Co

+

ol

CRH Adrenal

IL1β

+

IL-6 TNFα

FIGURE 3.2 Regulation of ACTH secretion.

impacting on ACTH synthesis and release. Circulating ACTH then binds to receptors, primarily in the adrenal gland, leading to steroid biosynthesis. Corticotrophin-Releasing Hormone CRH STIMULATION OF ACTH SECRETION

CRH binding to the type 1 CRH receptor stimulates adenylate cyclase activity which increases cAMP content in anterior pituitary corticotrophs. CRH is responsible for both increased POMC transcription and ACTH secretion [46] via the cAMP/PKA pathway [85,105]. To trigger ACTH secretion, CRH sustains depolarization of the corticotroph cell membrane, possibly mediated by suppression of the background TREK-1 current, which then activates voltage-gated calcium channels, leading to persistent calcium elevation and ACTH release in a monophasic pattern [106
109]. Perfused human fetal pituitaries and human fetal pituitary cells in culture secrete ACTH in response to CRH. In dispersed rat anterior pituitary cells, CRH stimulates a ninefold increase in ACTH release that is sustained for as long as cells are exposed to CRH. In addition to stimulating ACTH expression and release, CRH can also directly stimulate glucocorticoid secretion from the adrenal gland. MODULATORS OF CRH RELEASE

CRH is the most important physiologic ACTH secretagogue. Stressors, endogenous circadian rhythms, and glucocorticoids influence CRH release. In the rat, afferent inputs to the PVN may mediate the action of stressors by controlling CRH release. Sources of neuronal

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afferents to the hypothalamus include the amygdala and hippocampus of the limbic system, and brainstem regions involved in autonomic functions. In rats, acetylcholine, norepinephrine, angiotensin II, and possibly CRH itself, increase CRH concentrations in the hypophyseal portal plasma. On the other hand, β-endorphin and GABA inhibit the ACTH response to stress. In humans and other mammals, the impact of angiotensin II on basal or CRH-stimulated ACTH release is unclear. Infusion of angiotensin II alone in humans does not increase ACTH release. However, angiotensin II has a synergistic effect with CRH stimulating ACTH release in vivo. Angiotensin II potentiates CRH-stimulated ACTH release from cultured anterior pituitary cells, although it is less effective than AVP, and enhances CRH-induced cAMP. Angiotensin II and AVP do not potentiate each other’s effect on ACTH release, suggesting that they act via the same mechanism. CRH RECEPTORS

In human pituitaries, CRH binds to sites in the anterior lobe with a distribution that correlates with that of corticotrophs. CRH receptors in the anterior pituitary gland are low-capacity, high-affinity receptors, with a Kd for CRH binding of about 1 nM. Two CRH receptor genes have been identified in humans and other mammals, with a third additional one described in the catfish [85]. The type 1 receptor is expressed predominantly in anterior pituitary corticotroph cells, whereas the type 2 receptor is more widely distributed in the brain and periphery, particularly in cardiovascular tissue [85]. The type 1 receptor is a Class B G proteincoupled receptor (GPCR) that signals primarily through Gsα, activates adenylyl cyclase to form cyclic AMP, and results in activation of PKA [110]. CRHR1 binds and is activated by both CRH as well as the CRH-like peptide, urocortin [85]. This receptor mediates CRH actions at the corticotroph. CRH type 1 receptor can also couple to Gq, mostly in peripheral cells or cell lines, and activate phospholipase C to promote calcium mobilization via inositol triphosphate and PKC activation through diacylglycerol. In addition, the type 1 receptor mediates fear and anxiety behaviors following stressors, even in CRH-deficient mice [111]. Mice with deletion of the CRH type 1 receptor gene show reduced fear and anxiety [85]. These data suggest that a CRH-related peptide, possibly urocortin or another unknown member of the CRH family, mediates fear responses via the CRH type 1 receptor. The CRH type 2 receptor binds urocortin with greater than 20-fold higher affinity compared with CRH. The receptor may be involved in blood pressure control as the hypotensive cardiovascular response to infused urocortin is abolished in CRH type 2 receptor-deficient mice [85]. This mechanism

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may underlie the hypotension observed during the CRH stimulation test. AVP AVP STIMULATION OF ACTH SECRETION

AVP is synthesized in the same parvocellular hypothalamic PVN neurons which express CRH, and is coreleased with CRH at the median eminence into the portal hypophyseal system. However, a substantial amount of AVP in the portal blood is released from supraoptic nuclei projections in the median eminence. Posterior pituitary AVP may also reach the anterior pituitary through connecting portal vessels, raising the possibility that increased posterior pituitary vasopressin secretion, in response to hyperosmolality, may also stimulate ACTH secretion. AVP acts on VP-1b receptors, resulting in generation of inositol 1,4,5-triphosphate (IP3) through the phospholipase C pathway and activation of PKC. Increased ACTH secretion resulting from AVP signaling is described as a “spike and plateau” pattern, where the initial spike occurs in response to calcium release from intracellular stores leading to exocytosis [109,112
114], and the plateau results from activation of PKC and voltage-gated calcium channels. Desmopressin 1-desamino-8-D-arginine vasopressin (DDAVP), the synthetic version of vasopressin, can be used during bilateral inferior petrosal sinus sampling to stimulate ACTH release. Ten micrograms of desmopressin produce an exaggerated ACTH response that can result in similar sensitivity and specificity as CRH [115]. MODULATORS OF AVP RELEASE

AVP secretion is under the control of selective osmoreceptors that increase AVP concentration when plasma osmolality increases by 1
2%, and baroreceptors which modulate AVP release in response to changes in arterial volume. AVP is also released in response to physiological stress and perhaps psychological stress, although the mechanism is less clear. AVP release may be caused by osmotic-mediated changes in the shape of vasopressinergic neurons, possibly involving Trpv1 channels [116]. Opiates inhibit CRH- and AVP-stimulated ACTH release, and different opiate agonists differentially affect CRH- versus AVP-stimulated release. In humans, morphine blunts CRH-stimulated ACTH release without decreasing AVP or catecholamine levels. AVP RECEPTORS

A single population of specific AVP receptors has been identified in rat anterior pituitaries, which are distinct from CRH receptors. Most corticotrophs express AVP receptors, since 80% of ACTH-secreting

cells in the pituitary bind AVP. Anterior pituitary AVP receptors are distinct from the V2 renal receptors and the V1 hepatic/pressor receptors. This has led to the classification of hepatic/pressor receptors as V1a receptors and anterior pituitary receptors as V1b, or V3, receptors. V1a, V1b, and V2 receptors can be distinguished by their patterns of recognition of AVP analogues. V1a binding sites in the rat anterior pituitary have a Kd of about 1 nM, and the minimal effective dose of AVP is 0.1 nM. DDAVP (desmopressin), an AVP analogue with V2 receptor affinity, has an insignificant effect on plasma ACTH levels, though it does increase, but is not additive to, CRH-stimulated ACTH release. The genes for all three vasopressin receptors (V1, V2, and V1b) have been identified. They are highly related members of the seven-transmembrane, G protein-coupled, receptor family. V1b receptor mRNA is highly expressed in anterior pituitary corticotrophs, and is coupled to stimulation of POMC gene expression and ACTH secretion via the PKC pathway. SYNERGISM BETWEEN CRH AND AVP

AVP is a weaker stimulus for ACTH secretion than CRH, but is essential to achieve a maximum ACTH response during stress [112], as it potentiates CRH action. During periods of stress and in the presence of CRH, AVP causes up to 30 times higher ACTH release compared to CRH effects alone [117
119]. AVP causes a transient hyperpolarization in corticotrophs by activation of the SK channels during intracellular Ca release, followed by a sustained depolarization due to PKC-dependent suppression of the TREK-1 channels. When the cell is maximally stimulated by CRH, AVP further suppresses the TREK-1 current, resulting in a more robust depolarization that potentiates ACTH secretion [120]. Despite continued exposure to AVP, ACTH secretion eventually decreases to the plateau phase of CRH-stimulated secretion. In cells exposed to AVP before CRH, CRH does not potentiate AVPstimulated ACTH secretion. Cytokines Insights into the development and regulation of the HPA axis have come from the discovery that leukemia inhibitory factor (LIF) has an important role in these events [46]. LIF is a cytokine expressed in corticotrophs and folliculostellate cells beginning as early as 14 weeks gestation. LIF stimulates transcription of POMC and expression of ACTH [46]. In many tissues, including pituitary, LIF expression is upregulated by inflammatory stimuli. The LIF receptor is a member of the class I cytokine receptor superfamily which heterodimerizes with gp130. In common with other family members, the LIF receptor signals through the Jak-STAT pathway, particularly utilizing Jak1 and STAT-346.

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As with other receptors coupled via these mediators, SOCS-3 inhibits POMC expression following its simulation by LIF. Mice which overexpress a LIF transgene develop Cushing’s syndrome [121]. Their pituitary glands have corticotroph hyperplasia and multiple Rathke-like cysts lined by ciliated cells. Mice with targeted deletion of Lif have secondary adrenal insufficiency. LIF may interact with CRH and CREB to regulate POMC transcription [46]. Humans with Stuve
Wiedemann syndrome, a rare autosomal recessive disorder caused by mutations in the leukemia inhibitory factor receptor (LIFR) gene, have cortisol secretion patterns and response to low-dose cosyntropin tests suggestive of central adrenal insufficiency [122]. Other cytokines can stimulate ACTH release, primarily by direct or indirect stimulation of hypothalamic CRH secretion [123,124]. IL-1α, IL-1β, IL-6, LIF, and TNF-α, and their receptors, are expressed in the hypothalamus and pituitary and mediate communication between the immune system and the HPA axis. A direct pituitary effect of cytokines may occur, as exemplified in CRH-knockout mice, where injection of lipopolysaccharide induces systemic inflammation and results in increased plasma ACTH and corticosterone concentrations [125]. Interleukin-1β stimulates ACTH release in conscious rats by acting on the hypothalamus to induce CRH secretion. Interleukin-1 does not cross the blood
brain barrier, but activates noradrenergic neurons in the brainstem and hypothalamus, which may stimulate CRH secretion, especially in the median eminence. Prostaglandins may be involved in the response to interleukin-1, since ibuprofen, which blocks the formation of prostaglandins, blocks endotoxin-induced ACTH release in humans [126]. Tumor necrosis factor is a potent secretagogue for ACTH, and when administered to human subjects, leads to increased ACTH, cortisol, and AVP, but inhibits CRH-, AVP-, and angiotensin II-stimulated ACTH secretion. Tumor necrosis factor may stimulate ACTH secretion by stimulating hypothalamic CRH release. However, there is evidence that the site of action of tumor necrosis factor is peripheral to the pituitary and hypothalamus. In rats and humans, interleukin-6 leads to ACTH secretion via CRH-dependent and CRH-independent pathways, most likely via a prostaglandin-dependent pathway. Met-enkephalin analogues inhibit ACTH secretion, and controversy exists as to whether inhibition occurs at the hypothalamus or pituitary. In humans CRH-induced ACTH release is almost completely abolished with met-enkephalin analogue pretreatment. Cytokines also have direct effects on adrenal steroid production. In cultured NCI-H295R cells, IL-1

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increases basal production of cortisol, androstenedione, DHEA, and DHEAS through stimulatory effects on STAR, CYP17A1, and HSD3B2 expression. IL-1 also augments the effect of other inflammatory cytokines (TNF-α and LIF) on steroid production. This has yet to be proven in normal human adrenal cells [124]. Oxytocin In humans, low-dose oxytocin perfusion decreases plasma ACTH and cortisol levels, and completely inhibits CRH-stimulated ACTH release. Oxytocin acts via a similar mechanism as AVP, i.e., through PKC, and binds competitively to AVP receptors in the anterior pituitary, but is much weaker than AVP at stimulating ACTH release. Glucocorticoids Glucocorticoids are the primary negative regulators of ACTH secretion [127]. After binding to GRs, the glucocorticoid
GR complex translocates to the nucleus to regulate gene transcription and cellular function. Glucocorticoids also have nongenomic effects that occur more rapidly than genomic ones, including negative glucocorticoid feedback on neuronal activity in the hippocampus and on CRH release from CRH neurons [128]. Glucocorticoids act on corticotrophs to inhibit secretion of ACTH induced by AVP and CRH, synergism between CRH and AVP, and substances that provoke production of inositol phosphates and cAMP. Glucocorticoids’ negative impact on ACTH regulation is also due to their inhibition of the principal stimulators of ACTH, CRH, and AVP. Negative feedback can be defined as long, short, or ultrashort, depending on the location and nature of the hormone mediating the feedback. For glucocorticoids, long feedback refers to effects of adrenal glucocorticoids on ACTH secretion at the pituitary and in the hypothalamus. Short feedback refers to the effect of pituitary ACTH to inhibit CRH release. In normal subjects, ACTH administration does not affect CRH levels, most likely because of negative effects of cortisol present prior to ACTH administration. However, in patients with elevated CRH levels due to Addison disease or hypopituitarism, ACTH decreases circulating CRH and β-endorphin levels, suggesting that ACTH inhibits CRH secretion. ACTH may act in the median eminence or in the hypothalamus to inhibit CRH release. Long negative feedback by glucocorticoids plays an important role in limiting HPA axis activation. In the anterior pituitary, glucocorticoid inhibition of ACTH secretion in vitro is mediated via GRs, and lack of glucocorticoid effect in the intermediate lobe of the pituitary most likely occurs because functional receptors are not present in these cells. The negative effect of

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glucocorticoids on CRH-, AVP-, angiotensin II-, and norepinephrine-stimulated ACTH release is biphasic, which may reflect initial inhibition of ACTH release, followed by inhibition of POMC biosynthesis. Glucocorticoids also inhibit POMC secretion, gene transcription, and mRNA levels in the anterior pituitary. In humans, negative feedback by glucocorticoids on ACTH secretion manifests within 30
60 minutes. When endogenous cortisol is suppressed with metyrapone, effects of exogenous glucocorticoids on the morning ACTH rise, or on CRH-stimulated ACTH release, are not seen initially but appear 30 minutes after glucocorticoid administration. In addition, glucocorticoids have little effect on the initial CRH-induced increase in ACTH release, but decrease CRH-induced ACTH release after 60 minutes. Glucocorticoid-mediated negative feedback can be further subdivided into fast and delayed, intermediate and slow feedback. Rapid inhibition after corticosterone exposure and equally rapid recovery of CRH-stimulated ACTH secretion after glucocorticoid withdrawal are consistent with involvement of rapid feedback on generation of ACTH pulses. In animals, the initial fast negative effect of glucocorticoids occurs within seconds to minutes, involves inhibition of stimulated ACTH and CRH release, not synthesis, and occurs during the period when plasma glucocorticoid levels are increasing. Cortisol given to patients at the start of surgery attenuates the surgery-induced ACTH rise, and may be an example of fast feedback. Delayed feedback has intermediate and slow components [129]. Intermediate feedback is the component of delayed feedback due to inhibition of ACTH release, but not synthesis, and may be important after short durations of glucocorticoid exposure, or after noncontinuous, repeated exposures [129]. Intermediate delayed feedback develops after 45
120 minutes, and maximal inhibition occurs 2
4 hours after administration of one dose of glucocorticoids. Unlike ACTH, CRH synthesis as well as release may be affected by intermediate feedback [129]. The slow component of delayed feedback is most important after long exposures to moderately high doses of glucocorticoid, and is a function of the total dose of glucocorticoids, the glucocorticoid level achieved, and the amount of time since the steroid was given [129]. Slow feedback occurs after more than 24 hours of exposure to glucocorticoids and can persist for days. POMC biosynthesis is inhibited, leading to inhibition of basal and stimulated ACTH secretion [129], and intracellular ACTH decreases, implying decreased synthesis. Cortisol modulates pituitary responsiveness, and the corticotroph is dependent on CRH stimulation to maintain ACTH secretion. Glucocorticoid inhibition of

ACTH secretion may recover more quickly than does CRH secretion from the hypothalamus. Secondary adrenal insufficiency due to long-term glucocorticoid therapy may in part be due to continued suppression of hypothalamic CRH secretion. Adrenalectomized patients on exogenous glucocorticoid therapy have a blunted ACTH response to CRH that normalizes after several CRH boluses, suggesting that lack of stimulation of the corticotroph by CRH suppresses the ACTH response. On the other hand, corticotrophs of patients recovering from trans-sphenoidal surgery for Cushing disease are profoundly unresponsive to CRH, which cannot be attributed solely to deficient CRH priming. Glucocorticoids increase, and adrenalectomy decreases, the amount of GABA in the hypothalamus and the hippocampus. Inhibition of CRH release by glucocorticoids might be mediated by an increased GABA activity of the hippocampus and hypothalamus. Glucocorticoids inhibit AVP secretion. In most studies, patients with hypopituitarism or primary adrenal insufficiency are unable to maximally dilute their urine in response to a water load, and this is corrected by glucocorticoid administration [130]. However, it is unclear whether elevated AVP levels or lack of glucocorticoids is responsible for the inability to maximally dilute the urine in the hypocortisolemic state. Glucocorticoids inhibit nitric oxide synthase, and nitric oxide is capable of stimulating the insertion of the water channel, aquaporin 2, in the luminal membrane of the renal collecting cell. This may provide an explanation for why glucocorticoid deficiency is associated with decreased free water clearance. Other Hormones and Pharmacological Regulators of ACTH Secretion In humans, catecholamines have little direct effect on pituitary ACTH secretion. Peripheral catecholamines, increased by a variety of stresses, do not cross the blood
brain barrier to reach the hypothalamus, but do reach the pituitary, yet do not increase basal or CRH-stimulated plasma ACTH levels. This suggests that increased peripheral levels of epinephrine and norepinephrine generated during stress are probably not responsible for increased ACTH, and that catecholamines do not act directly on the pituitary to stimulate ACTH release. In rodents, central catecholamines stimulate ACTH release, and the effects of catecholamines on ACTH secretion appear to be mediated via secretion of CRH into the hypophyseal portal circulation.

Physiological Regulation of ACTH Secretion Physiologically a number of factors interact to determine the final pattern of ACTH release, including

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circadian rhythms, stress, and negative feedback by glucocorticoids. These factors impact on each other in an integrated fashion to control ACTH release. The stage of development, from fetal life through puberty, and pregnancy and parturition, also impacts on ACTH secretion. Finally, the immune system interacts with the HPA axis, adding another facet to the complexity of ACTH release. Secretion Dynamics of ACTH In Vivo ACTH secretion is characterized by a mix of pulses and basal (nonpulsatile) hormone release. Pulses occur in a relatively high frequency (12
30 events per day) and the half-life of circulating plasma ACTH is short, from 14 to 35 minutes [131]. Frequent 10 minute sampling reveals 40 ACTH pulses in 24 hours [131]. Blood ACTH rises by an average of 24 pg/mL per pulse [131]. β-Endorphin secretion parallels pulsatile ACTH release. Pulsatile, basal, and total 24-hour ACTH secretion and mean ACTH concentrations are all increased with elevated body mass index (BMI). Cortisol levels and BMI are associated with a greater amount of ACTH secreted per burst, but the frequency of pulses and their waveform are independent of BMI. The regularity of cortisol (but not ACTH) secretion and the synchrony of cortisolACTH feedback both decrease with age. ACTH secretion is also affected by gender. Men have higher basal, pulsatile, and total ACTH secretion when compared to women. Total 24-hour ACTH secretion, even when normalized for BMI, is higher in men. Cortisol levels do not differ by gender, and higher pulsatile ACTH secretion in men results from a greater mass per burst rather than more frequent secretory bursts [131]. Sensitivity of the adrenal cortex, or availability of ACTH to the adrenal cortex, may be greater in females. Alternatively, males and females may have different set points for cortisol feedback. Gender, age, and BMI do not appear to affect the frequency or duration of the ACTH pulses or interpulse regularity. Spontaneous ACTH and cortisol pulses correlate highly. There is a strong relationship between the magnitude of concomitant ACTH and cortisol pulses, particularly if a 15-minute phase delay in cortisol secretion is allowed for [131]. Not all spontaneous ACTH and cortisol pulses are concomitant: approximately 50
75% of spontaneous ACTH pulses are followed by a cortisol pulse, whereas approximately 60
90% of spontaneous cortisol pulses are preceded by an ACTH pulse. Circadian Regulation of ACTH Secretion An endogenous circadian rhythm to the pulsatile pattern of ACTH secretion leads to a circadian rhythm of glucocorticoid release. The function of this circadian

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rhythm in cortisol secretion is not known, although one hypothesis suggests that the early morning rise in cortisol causes a delayed-phase rise in insulin resistance, which may play a role in altered glucose metabolism [132]. For instance, since the brain does not require insulin for glucose uptake, peripheral insulin resistance might cause a rise in glucose levels, leading to greater brain uptake. The circadian rhythm is generated in the suprachiasmatic nucleus, and signals travel via efferent inputs to the PVN to modulate CRH release. This circadian rhythm is due to variation in ACTH pulse amplitude, but not frequency [133]. The amount of ACTH secreted per pulse varies by 3.8-fold over a 24-hour period [133]. Basal ACTH and cortisol levels parallel each other and are the highest upon awakening in the morning between 0600 hour and 0900 hour, decline through the day to intermediate levels at 1600 hour, and are lowest between 2300 hour and 0300 hour [133]. From 2300 hour to 0200 hour, there is a quiescent period of minimal secretory activity, corresponding to the nadir of ACTH and cortisol levels. Secretion of ACTH and cortisol abruptly increases in the early morning. The diurnal secretory pattern is similar for free and total cortisol, although the relative increase in free cortisol is about 1.5 times greater than the relative increase in total cortisol. Alterations in feeding and sleep impact on cortisol secretion. Cortisol briefly increases postprandially, especially after the midday meal. Exercise or administration of ACTH at 1000 hour leads to a rise in cortisol levels and blunts the midday cortisol surge, and at 1400 hour leads to a subsequent rise in cortisol. Overall, the major features of the diurnal cortisol pattern persist under conditions of complete fasting, continuous feeding, or total sleep deprivation. However, the circadian rhythm of cortisol secretion fully adapts to permanent changes in environmental time and the sleep
wake pattern. This adaptation requires about 3 weeks, the limiting factor being the time it takes for the quiescent secretion period to fully adapt. The acrophase adapts much more quickly and is partially synchronized after 1 day and totally synchronized after 10 days. There is circadian regulation of the sensitivity of the adrenal cortex response to ACTH and CRH. Injection of ACTH or CRH at the time of the endogenous morning cortisol peak results in the lowest incremental increase but the highest total cortisol level. Conversely, injection of ACTH or CRH at the nocturnal cortisol nadir produces the largest incremental increase but the lowest total cortisol level. Factors governing the circadian rhythm in ACTH release in humans are not clear. On the one hand, it may be regulated by a diurnal rhythm in CRH secretion. The highest CRH levels occur at 0600 hour (7.0 pg/mL) and the lowest at 1800 hour and 2200

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hour (about 1.8 pg/mL), which parallels ACTH and cortisol secretion patterns. Serotoninergic and cholinergic pathways may play a role in the CRH circadian rhythm, and display circadian periodicity in their hypothalamic concentrations. On the other hand, when the pituitary is exposed to constant levels of CRH 30-fold higher than those found in portal hypophyseal blood, such as during CRH infusion and in pregnancy [134], the ACTH and cortisol circadian rhythms persist. CRH clearly plays a role in the ACTH rhythm, as CRH-deficient mice have an absent or markedly attenuated diurnal rise in ACTH [135]. However, in these mice, constant infusion of CRH restores the ACTH rhythm. This indicates that changes in CRH amplitude are not necessary to drive the ACTH rhythm, but that a tonic level of CRH is required to maintain ACTH responsiveness [136] to circadian cues. Many peripheral cells express circadian clocks, and glucocorticoids are required for clock function in several, but not all, peripheral tissues [137]. Physiological Regulators of ACTH Secretion Glucocorticoid release mediated by ACTH plays a major role in the stress response. An interaction exists between stress-mediated ACTH release, which leads to glucocorticoid secretion, and glucocorticoid-mediated negative feedback, which inhibits further ACTH and glucocorticoid release. A number of stressful stimuli elicit ACTH secretion, and most physical stressors activate the HPA axis. The magnitude of ACTH and cortisol rise is dependent upon the nature of the stress, its magnitude including the rapidity of its appearance, and the time of day it is experienced. In general, stressors have a larger impact on ACTH release when they develop rapidly, are of high magnitude, and occur during the circadian nadir in ACTH release. HYPOGLYCEMIA

In humans, insulin-induced hypoglycemia is associated with a five- to sixfold increase in plasma ACTH levels [138], from a basal level of about 40 pg/mL to a peak of 250 pg/mL at 45 minutes [138]. Cortisol levels increase over twofold, from a basal level of about 11 μg/dL to a peak of about 25 μg/dL at 60
90 minutes [138]. Insulin-induced hypoglycemia causes a four- to fivefold greater increase in ACTH secretion than CRH alone, and a 1.3-fold greater increase than AVP plus CRH. AVP may play a more direct role in the initial phase of ACTH response to hypoglycemia than CRH. AVP levels increase 2.4- and 2.5-fold at 30 and 45 minutes after insulin administration. Mean plasma CRH levels rose 1.7-fold with a peak at 45 minutes indicating a dynamic role for CRH as well as in the response to hypoglycemia [138]. When AVP levels are raised

endogenously by saline infusion or lowered to undetectable levels by waterloading, hypoglycemiainduced AVP increase is greater after saline, even though saline blunts the hypoglycemic response to insulin [139]. Catecholamines also increase in response to hypoglycemia. Epinephrine appears to play more of a role than norepinephrine, increasing at least 13-fold at 30 minutes after insulin, whereas norepinephrine increases 2.4-fold at 60 minutes. There is contradicting evidence of the effect of this catecholamine increase upon ACTH secretion in humans. EXERCISE

Exercise increases ACTH and β-endorphin levels, and the response is dependent on the intensity of exercise and the level of training. Exercising to exhaustion, or exercise of short duration and high-intensity, increases ACTH, β-endorphin, and cortisol levels. Hypercortisolism occurs in highly trained athletes, who require a higher level of oxygen consumption to stimulate ACTH release, and have elevated basal ACTH levels. Physical exercise and stress both lead to analgesia in man. ACTH and cortisol are significantly higher after high-intensity intermittent exercise compared with continuous moderate-intensity exercise in men [140]. In rats with diet-induced insulin resistance, there are decreased POMC expression levels and ACTH production with a concomitant reduction in serum glucocorticoid levels. In these animals, moderate exercise prevents induction of antioxidant enzymes and autophagy, and prevents the decrease in pituitary ACTH synthesis and release associated with increased insulin resistance [141]. Naloxone reverses exercise-induced analgesia from certain types of pain, suggesting a role for endorphins. Dexamethasone reverses exercise-induced analgesia from other types of pain, like dental pain, that are not reversed by naloxone, suggesting a role for ACTH, although dexamethasone also suppresses β-endorphin release. STARVATION

Essential bodily functions, such as reproduction, growth, and immune responses, require adequate energy from food. The ability to survive famine through various adaptive changes, from increased food seeking to decreased metabolic function, is evolutionarily advantageous. The HPA axis is activated during starvation. In humans and animal models, all hormones in the HPA axis (CRH, ACTH, and cortisol) are increased following acute food deprivation [142]. Cortisol induces glucose release via stimulation of glycogenolysis, gluconeogenesis, and lipolysis, which provides interim energy during fasting. Cortisol also stimulates hypothalamic AGRP, which stimulates

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appetite and likely causes “rebound hyperphagia” once food becomes available after a period of starvation [143]. The circadian peak of cortisol coincides with morning awakening and mobilizes glucose reserves for activity after overnight fasting. Nocturnal animals have circadian rhythmicity in which the corticosterone peak corresponds to nighttime awakening and activity. High levels of cortisol also correlate with increased food-seeking behaviors in both food-deprived animal models and human dieters. Following prolonged food deprivation, however, levels of CRH and ACTH are decreased, likely because sustained cortisol or corticosterone elevation provides negative feedback to inhibit CRH and ACTH [142]. Activation of the HPA axis correlates with inhibited reproductive function and decreased bone and muscle density, suggesting a role in stress-induced inhibition of nonessential functions. Anorexia nervosa is associated with hypercortisolemia, without features of Cushing’s syndrome. Cortisol levels inversely correlate with BMI, fat mass, and fasting glucose and insulin concentrations, suggesting an adaptive mechanism to maintain glucose homeostasis during low energy availability. In adolescent girls, hypercortisolemia results from increased pulsatile secretion, appears to be a direct consequence of undernutrition, and is associated with decreased markers of bone formation [144]. HYPOTENSION AND HYPOVOLEMIA

Lower body negative pressure in humans simulates acute hemorrhage, and increases ACTH secretion to peak values of 60
250 pg/mL at 2
10 minutes after cessation of the stimulus, and ACTH increase is reversed by dexamethasone [145]. In animals, hemorrhage stimulates ACTH secretion primarily mediated by CRH and, to some degree, by AVP. Hypovolemia increases portal blood CRH, AVP, epinephrine, and oxytocin, whereas hypotension, also a component of hemorrhage, induced by nitroprusside, increases portal blood CRH only.

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significant decrease in cortisol inactivation [146]. Deep anesthesia and postoperative analgesics can attenuate physiologic responses to stress and, depending on the anesthetic used, effects on the HPA axis may differ. In adults undergoing cardiac surgery with cardiopulmonary bypass (CPB), when anesthesia is induced with etomidate, there is a significant fall in the cortisol values during bypass and further significant fall after weaning off CPB to approximately 50% from baseline, a phenomenon that is not replicated when using propofol. On the contrary, propofol-treated patients exhibit almost double baseline cortisol values at all time-points after surgery. At 24 hour serum cortisol returned to almost twice the baseline values in etomidate-treated patients and remained high in the propofol group, suggesting that adrenal suppression by etomidate is transient [147]. Etomidate inhibits adrenal steroidogenesis and decreases the ACTH response of the adrenal gland [148,149]. BRAIN DEATH

Almost 80% of brain-dead patients have circulatory failure associated with heart, lung, kidney, liver, and pancreas dysfunction. Animal and human models of brain death show that cortisol levels are increased at 5 minutes and then decline progressively to sub-baseline levels. Plasma thyroid hormone levels also fall within 1 hour after brain death and are undetectable within 9 and 16 hours and vasopressin is undetectable within 6 hours [150]. Adrenal stimulation by synthetic ACTH injection (250 μg) revealed adrenal insufficiency (defined as plasma cortisol level inferior to 18 μg/dL at time of injection and/or a change in plasma cortisol level less than 9 μg/ dL) in 78% of brain-dead patients in the CORTICOME study [151]. Low-dose steroid treatment during braindeath resuscitation of potential organ donors is associated with a more than 20% decrease in the amount of vasopressors needed to control circulatory failure, shorter duration of vasopressor support, and more frequent weaning of norepinephrine before aortic clamping [151].

SURGICAL STRESS

Surgery induces a large increase in plasma ACTH levels due to increased sensitivity of the adrenal cortex to ACTH. Fentanyl, an opiate agonist, attenuates the ACTH response to surgery. Evidence in adrenalectomized primates suggests that supraphysiological doses of glucocorticoids are not necessary for the animal to withstand surgical stress, but that a minimal level is necessary. Pediatric surgery results in a distinct postoperative increase in median cortisol production, which has been described to be more than threefold in children undergoing major cardiac surgery and at least twofold after conventional surgery, and might be accompanied by a

CRITICAL ILLNESS

The physiologic response to stress consists of release of catecholamines from the adrenal gland and activation of the HPA axis. CRH and AVP are released from the hypothalamus to stimulate ACTH production and ultimately activate the cortisol biosynthetic pathway, with cortisol release following just minutes after ACTH secretion [152]. Chronic release of ACTH results in increased transcription and posttranscriptional regulation of genes involved in steroid synthesis, increases the number of cholesterol uptake receptors, and upregulates 3-hydroxy-3-methylglutaryl coenzyme A (HMGCR, HMG-CoA).

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A reductase, HMCGR increases cholesterol availability and promotes overexpression of its own receptor to increase responsiveness of the adrenal cells to ACTH [152]. Critical illness is a model of severe stress where cortisol levels are markedly increased. Although these high cortisol levels had been attributed primarily to increased production in response to HPA activation, recent studies have revealed that there might be a dissociation between ACTH and cortisol levels, suggesting alternate explanations for the rise in serum cortisol. Neuropeptides from the adrenal medulla and circulating cytokines have been proposed as extrapituitary stimulators of the adrenal cortex, among others. However, even during critical illness, cortisol production over 24 hours is only moderately increased during daytime and even lower than healthy controls overnight. If cortisol production is only moderately increased during critical illness, the significantly elevated blood levels need to be otherwise explained. Decreased cortisol clearance was reported in critically ill patients, not dependent on the severity or type of illness, suggesting that cortisol metabolism is highly attenuated, leading to accumulation of tissue glucocorticoids, likely due to reduced expression and activity of cortisolmetabolizing enzymes in liver and kidney [153,154]. Cortisol buildup would exert negative feedback on ACTH secretion and may explain the dissociation between ACTH and cortisol levels seen in previous studies of critically ill patients. More studies are needed to elucidate the effect of low ACTH levels in adrenal gland structure and function during the prolonged phase of critical illness. The moderate increase in cortisol production during critical illness questions the optimal therapeutic dose of steroids (“stress-dose”) required for patients deemed adrenally insufficient. Current practice relies on 200 mg of hydrocortisone for adults and 50
100 mg/m2/day in children, which is approximately 10-fold higher than needed for healthy subjects and 3
6 times higher than critically ill patients, and also do not account for decreased metabolism and clearance [155
157]. This further suppresses ACTH and could lead to glucocorticoid accumulation in susceptible tissues, leading to lean tissue wasting, increased risk of myopathy, and longer intensive care requirements [158]. Critical illness-related corticosteroid insufficiency, or relative adrenal insufficiency, refers to inadequate cortisol levels and/or inadequate response to ACTH stimulation for the severity of illness. It is still a controversial topic amongst critical care and endocrinology providers. The most widely accepted definition is a random cortisol of less than 10 μg/dL or a delta cortisol (change from baseline) of less than 9 μg/dL after

250 μg of ACTH. There is insufficient evidence to prove that corticosteroid treatment for these patients lowers ICU and in-hospital mortality, though studies are ongoing to further define the role of steroid treatment, particularly in patients with sepsis [159]. Furthermore, larger studies are needed to determine the significance of free cortisol levels in the critically ill, which often do not correlate directly with total cortisol, possibly due to decreased plasma concentrations of cortisol carriers (cortisol-binding globulin and albumin), and increased neutrophil elastase activity, and tissue resistance to cortisol [160,161]. COSYNTROPIN ADMINISTRATION

The insulin tolerance test (ITT) is considered the gold standard for assessing adrenal function, and consists of provoking insulin-induced hypoglycemia that should result in HPA activation and consequently, cortisol release. This is a labor-intensive test that requires very close monitoring and carries many inherent risks due to hypoglycemia. The cosyntropin stimulation test, on the other hand, uses a synthetic analogue of ACTH to stimulate cortisol production in the adrenal gland. The conventionally used dose of 250 μg is considered to be supraphysiological and, although reliable to diagnose primary adrenal insufficiency, can fail to identify subjects with defective ACTH secretion (central adrenal insufficiency) that have not yet developed adrenal atrophy. A low-dose cosyntropin test using only 1 μg of the drug has been proposed as a more physiological study with promising results [162
164]. The ITT is usually conducted after an overnight fast using 0.1
0.15 U/kg IV regular insulin. Peripheral venous blood for measurement of serum glucose and cortisol is sampled immediately before insulin is injected (0 minutes) and at 30, 60, 90, and 120 minutes thereafter; a cortisol level of 15 ng/dL was proposed as the optimal cut-off value for the ITT, which is the 95th percentile in the normal control subjects [165]. HIGH-DOSE (HDT 5 250 µG) VERSUS LOW-DOSE (LDT 5 1 µG) COSYNTROPIN TEST For the HDT, blood

samples are usually obtained 30 and 60 minutes after ACTH injection. The peak cortisol response of the LDT usually occurs at 20
30 minutes in normal subjects but can be delayed in subjects with adrenal insufficiency. When comparing ITT, HDT, and LDT, none of 28 healthy controls had lower cortisol levels than 18 ng/dL for the LDT and 20 ng/dL for the HDT [165]. For patients with suspected central adrenal insufficiency, optimal cut-off values for peak cortisol using the LDT and HDT were 15.8 and 17.4 ng/dL, respectively. The sensitivity of the HDT was 6% lower than the LDT at all cut-offs, but the specificity and PPV were 15
20% higher in the HDT. Peak cortisol

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levels of the ITTs were lower than those of the HDT or LDT when the three tests were performed in the same study subject, and similarly the peak cortisol level of the HDT was higher than the LDT when the two tests were performed in the same subject. Cortisol responses were attenuated in patients with one or more anterior pituitary hormone deficiencies, such as gonadotrophins, growth hormone, and TSH, suggesting that different cut-off values should be applied in these patients. Cut-off values established by ROC analysis were 16 ng/dL for the LDT and 17 or 18 ng/dL for the HDT, and from the normative data 18 ng/dL for the LDT and 20 or 21 ng/dL for the HDT. In patients with suspected or proven pituitary disease, using a cortisol cut-off level of 18 ng/dL (500 nmol/L), the 250 μg cosyntropin test achieved a sensitivity of 100% and a specificity of 90%. At a higher cortisol cut-off level of 22 ng/dL (600 nmol/L), it kept a sensitivity of 100%, with a specificity of 77% [162]. The low-dose (1 μg) cosyntropin test on the other hand, achieved a sensitivity of 100% and a specificity of 93.3% using a cortisol cut-off level of 18 ng/dL (500 nmol/L). A cortisol cut-off level of 22 ng/dL (600 nmol/L) lowered the specificity to 80%. Patients who failed the ITT also failed the lowdose test [162]. At a cortisol cut-off of 22 ng/dL (600 nmol/L), the low-dose cosyntropin test is 100% sensitive but has a false-positive (failure) rate of 11% (patients that “fail” the test with neither clinical nor biochemical evidence of HPA axis impairment), highlighting the importance of using these tests in conjunction with clinical evaluation. Peak cortisol responses after three tests do not seem to vary by age and gender [165]. Both high-dose and low-dose cosyntropin tests are safer than ITT for clinical decision-making with regard to hydrocortisone replacement therapy. Many practitioners choose to perform a combination of low-dose cosyntropin stimulation followed by a high-dose test. Patients whose clinical data and low-dose cosyntropin stimulation results remain inconclusive, might require an ITT for further evaluation, though even the latter is not 100% diagnostic. PSYCHOLOGICAL AND EMOTIONAL STRESS

Psychological and emotional stress play a role in the hormonal stress response. ACTH levels are high in patients awaiting an insulin tolerance test (ITT). During physical exercise, psychological and physical stress may act synergistically to increase β-endorphin and ACTH levels. Posttraumatic stress disorder (PTSD) has been associated with low daily cortisol concentrations, particularly in the morning [166] and with GR hypersensitivity [167]. Low levels of salivary cortisol immediately after an accident predict severity of PTSD

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symptoms after 6 months [168]. High predeployment GR number in peripheral blood cells, low FKBP5 mRNA expression, and high GILZ mRNA expression, were all independently associated with an increased risk of developing PTSD symptoms 6 months after deployment to war zones [169]. Recent genome-wide association studies have failed to show an association between GR gene SNPs and PTSD [170] but it is associated with PACAP and its receptor PAC1 [171].

EFFECTS OF SECRETED POMC-DERIVED PEPTIDES IN PHYSIOLOGY AND DISEASE Melanocortin receptors are a family of seventransmembrane spanning, GPCRs activated by melanocortin derivatives of POMC including α-MSH and ACTH. Activation of all five receptors results in adenylate cyclase activity and cAMP production. MC4R signals through a GPCR-independent pathway involving the potassium channel, Kir7.1 [172]. Cloning of the melanocyte MSH receptor (melanocortin 1 receptor [MC1R]) [173] and the adrenal ACTH receptor (melanocortin 2 receptor [MC2R]) [173] were quickly followed by the cloning of three additional family members. The five known melanocortin receptors show distinct tissue distributions throughout the nervous system and periphery and distinct selectivity for the various melanocortin peptides. Prior to the cloning of this receptor family, actions of melanocortins were primarily known through effects of MSH on pigmentation and effects of ACTH on glucocorticoid secretion from the adrenals. However, many additional roles, including cognitive and behavioral effects, effects on the immune system, and effects on the cardiovascular system have also been attributed to the melanocortins. With the cloning of this family of receptors, the physiologic roles of ACTH, MSH, and other melanocortin derivatives are beginning to be elucidated. The pharmacology of melanocortin receptor activation with a large number of natural and synthetic melanocortin peptides is the subject of extensive investigation. All five melanocortin receptors are activated by ACTH. However, MC2R binds only ACTH and is not activated by other melanocortin peptides [174]. The synthetic agonist 4-norleucine,7-D-phenylalanine-α-MSH (NDP-MSH) is the most potent agonist of MC1R, MC3R, MC4R, and MC5R [175]. The endogenous non-ACTH melanocortin peptides generally bind the melanocortin receptors with an order of potency MC1R . MC3R . MC4R . MC5R when expressed in COS cells and measurements are obtained in competition with NDP-MSH [175]. γ-MSH is relatively selective for MC3R over MC4R and MC5R [175]. Whether differences in melanocortin receptor specificity for

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different melanocortin ligands are physiologically relevant is unknown. Interestingly, Org 2766 and BIM 22015, two ACTH4-10 analogues, have no activity at any of the cloned MCRs, but have potent effects on central and peripheral nervous systems suggesting the possibility of undiscovered melanocortin receptors.

ACTH Adrenal Gland The critical role of ACTH in regulating the synthesis and secretion of steroids from the adrenal gland has long been recognized [176]. In adrenocortical cells, ACTH regulates lipoprotein uptake by receptormediated endocytosis from the plasma to lipid droplets by stimulation of lipoprotein receptors. Within the lipid droplets, ACTH regulates hydrolysis of cholesterol esters by activation of cholesterol esterases or suppression of cholesterol acyltransferase, through cAMPdependent protein kinase. ACTH stimulates the transport of cholesterol to the mitochondria, principally via stimulation of steroidogenic acute regulatory protein (StAR) [177]. The rate-limiting step in steroidogenesis is the side-chain cleavage of cholesterol to pregnenolone, and is catalyzed by cytochrome P450 side-chain cleavage enzyme in the inner membrane of mitochondria of the adrenal, probably on the matrix side. ACTH stimulation results in long-term and short-term effects on steroid hormone biosynthesis in the mitochondria. Long term, ACTH leads to increased amounts of steroid hormone enzymes by increasing transcription of these genes. StAR is the key protein which regulates cholesterol transport into the mitochondrion [177]. Mutations in this protein result in defects in adrenal and gonadal steroidogenesis, which had been previously attributed to defects in side-chain cleavage enzyme activity [177]. Beginning several hours after ACTH administration, ACTH increases the levels of steroidogenic enzyme mRNAs in primary cultures of human adrenals by several-fold, including cholesterol side-chain cleavage enzyme, 17-α-hydroxylase/17,20-lyase, 11-β-hydroxylase/18-hydroxylase/18-methyl-oxidase, and 21hydroxylase cytochrome P-450 enzyme. ACTH has a positive regulatory effect on its own receptors, and on the cAMP response to binding of ACTH to the receptor. With sustained stimulation, downregulation does occur, but physiologically this effect is minor since ACTH causes proliferation as well as steroid secretion. In addition to its prominent role in regulating adrenal steroidogenesis, ACTH exerts profound trophic effects upon the adrenal. Hypophysectomy results in adrenal atrophy and ACTH replacement restores adrenal gland weight in a dose-dependent manner. While

the role of ACTH in adrenal hypertrophy is well established, its role in adrenocortical mitogenesis and hyperplasia is incompletely understood. The absence of ACTH induces apoptotic cell death in the adrenal cortex. Prolonged ACTH administration blocks apoptosis and also increases adrenal DNA content in the rat and ACTH increases mRNA levels for c-fos and β-actin, proteins involved in cellular proliferation. However, ACTH paradoxically inhibits mitosis of adrenocortical cells in culture. ACTH-antiserum administered to intact rats caused a highly significant decrease in corticosterone levels, but had no effect on adrenal weight. Furthermore, ACTH inhibits the rapid compensatory proliferation of the remaining adrenal that normally occurs after unilateral adrenalectomy. Another anterior pituitary-derived candidate for stimulation of adrenal proliferation is the 28-aminoacid N-terminal POMC peptide (N-POMC) [178]. This peptide is mitogenic in vitro and in vivo for the adrenal cortex, and N-POMC antiserum significantly diminishes adrenal mitotic activity after enucleation. Nek2 has been described to be involved in this process [179]. Compensatory adrenal growth that occurs after unilateral adrenalectomy may be mediated by a neural reflex that includes afferent neurons originating from the disrupted adrenal gland, the ventromedial nuclei of the hypothalamus, and efferent neurons innervating the remaining gland. Melanocortin 2 Receptor—the ACTH Receptor Early in the study of receptor biology, Haynes demonstrated the action of ACTH in generating cAMP in adrenal cells. The ACTH receptor was the first receptor shown to bind its ligand with high affinity and specificity. In human adrenal glands, the ACTH receptor has a Kd of 1.6 nmol/L and about 3500 sites/cell. The ED50 of ACTH for cAMP production is 0.11 nmol/L, 20-fold less than the Kd for binding. The ED50 of ACTH for cortisol production is 2 pmol/L, 720-fold less than the Kd for ACTH binding, and 35-fold less than the concentration of ACTH needed to obtain a half-maximal increase in cAMP production. Only a small percentage of ACTH receptors needs to be occupied to achieve a maximal effect on steroidogenesis, which occurs at an ACTH concentration of 0.01 nmol/L. Because the adrenal gland may express more than one of the five melanocortin receptors whose functions may overlap, it is not possible to assign the biochemical characterizations to a specific melanocortin receptor. The human ACTH receptor (MC2R) was cloned based on its homology to MC1R. In situ hybridization with the adrenal gland of the rhesus monkey demonstrated expression in the zona glomerulosa and fasciculata cells, and a weaker signal in the zona reticularis.

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ACTH

N

C

N

C

ACTH

C

N

Plasma membrane

N

MRAP antiparallel homodimer N C

C C N C

Endoplasmic reticulum C N C

N MRAP

C N

N MRAP–MC2R complex

C

MC2R

N

FIGURE 3.3

Interaction between MRAP and MC2R. The melanocortin 2 receptor accessory protein, MRAP, is required to traffic MC2R to the plasma membrane, where it interacts with ACTH. MRAP was localized to the endoplasmic reticulum and plasma membrane, and both Cand N-termini of MRAP were found to face externally in the membranes. MRAP is the first eukaryotic protein known to function as an antiparallel homodimer. In this conformation, MRAP stably binds MC2R, and the complex is trafficked to the plasma membrane. In the plasma membrane, MC2R interacts with and responds to ACTH. Knock down of MRAP confers the inability of MC2R to respond to ACTH. In human patients, MRAP mutations are responsible for approximately 25% of cases of familial glucocorticoid deficiency.

Initial attempts at characterizing the ligand specificity and cAMP signal generation in response to ACTH and other melanocortins was confounded by either poor levels of expression or the presence of endogenous melanocortin receptors in transfected cells. The human MC2R has been stably transfected into the Y6 cell line, a mutant derived from the mouse Y1 adrenocortical cell line, that fails to express endogenous MC2R [180]. Y6 cells alone demonstrated no cAMP response to micromolar ACTH, and MC2R transfected cells displayed an EC50 of 6.8 nmol/L [180]. Genome-wide array studies of patients with familial glucocorticoid deficiency (FGD) but without MC2R mutations revealed a new protein linked to the disease, melanocortin 2 receptor accessory protein (MRAP) required to traffic MC2R to the plasma membrane [181], where it binds ACTH (Fig. 3.3). MRAP is a 19-kDa single-transmembrane domain protein and maps to chromosome 21q22.1 in humans [181]. The MRAP gene consists of six exons, and the last two exons may be alternatively spliced to encode MRAP-α or MRAP-β isoforms. Coimmunoprecipitation studies showed that MC2R is physically associated with MRAP. Heterologous cells

only responded to ACTH when MRAP and MC2R were transfected together, not when MC2R was transfected alone [181]. Mouse Y1 cells, one cell line known to endogenously respond to ACTH, was shown to express MRAP. When MRAP was knocked down by RNAi, these cells lost ACTH responsiveness [182]. MC2R was localized to the endoplasmic reticulum and plasma membrane. In transfected CHO cells, markers for both the N- and C-termini of the MRAP protein were localized externally [183]. Endogenous MRAP also presented both N- and C-termini externally in adrenal cells. Half of MRAP was glycosylated at a single endogenous N-terminal glycosylation site, and mutant MRAP with potential glycosylation sites on both sides of the membrane were glycosylated only at one domain. MRAP forms an antiparallel homodimer that stably complexes with MC2R. MRAP was the first antiparallel homodimeric membrane protein identified in eukaryotic cells. MRAP was initially thought to be highly specific for MC2R and did not increase cell surface expression of other melanocortin receptors, β2-adrenergic receptors, or TSH-releasing hormone receptors [183]. MRAP coprecipitates with MC5R and blocked MC5R dimerization and cell surface localization [184].

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FAMILIAL GLUCOCORTICOID DEFICIENCY SYNDROME TYPE 1

The FGD syndrome is a rare autosomal recessive syndrome originally described by Shepard et al. in 1959 [185]. Patients typically present in early childhood with symptoms resulting from their glucocorticoid insufficiency, including hyperpigmentation, hypoglycemia, lethargy, and weakness [185]. The clinical course may be complicated by frequent infections [185]. Patients with FGD typically have low or undetectable plasma cortisol and high ACTH levels (often .1000 pg/mL) that cause striking hyperpigmentation, in conjunction with normal aldosterone and plasma renin levels. Rare patients with FGD may have aldosterone deficiency with salt loss, for unknown reasons. Occasionally FGD patients have low-normal cortisol values, which respond subnormally to exogenous corticotrophin. Patients with FGD do not present with symptoms related to salt wasting, dehydration, or electrolyte disturbances, as the renin
aldosterone axis is preserved. The preservation of the renin
aldosterone axis clearly distinguishes this syndrome from childhood Addison disease. FGD also shares symptoms with Allgrove, also called triple-A, syndrome, but Allgrove can be distinguished by its presentation of alacrima, achalasia, and neurological defects in addition to adrenal insufficiency. A feature of FGD is that many of the patients are reported to be unusually tall [185]. The mechanism is unclear, but excessive growth is suppressed upon glucocorticoid replacement, suggesting that glucocorticoid deficiency or ACTH excess may be the cause. Melanocortin receptors are present in bone and the growth plate, and one hypothesis is that excess ACTH stimulates growth via these receptors. The adrenal glands are atrophic, and only occasional cortical cells remain in the zona glomerulosa with no remnants of the zona fasciculata or reticularis, but the adrenal medulla appears normal [185]. Adrenal atrophy substantiates the physiologic relevance of ACTH’s trophic action on the adrenal gland. An examination of 11 patients with FGD revealed a discrepancy between partial glucocorticoid deficiency and significantly diminished DHEAS secretion confirming a significant contribution for ACTH (or at least for MC2R) in the onset of adrenarche [101]. A number of different homozygous or compound heterozygous missense and nonsense mutations in MC2R have been reported in patients with FGD, and, in all cases, these mutations cosegregate with disease in the affected families [186]. Cell-based studies suggest that some mutants interfere with ligand binding, whereas others interfere with coupling of the receptor to downstream cyclic AMP generation [174]. FGD caused by MC2R mutations is

classified as FGD type 1, which accounts for approximately 25% of FGD. For many years, there were no animal models of FGD. The Mc2r knockout mouse was first generated and characterized in 2007 [187]. Like human patients with FGD, these mice had high levels of ACTH and undetectable levels of the corticosterone. Adrenal histology in these animals showed atrophied zona fasciculata but intact zona glomerulosa. Mc2r knockout mice also presented with neonatal hypoglycemia, which frequently led to death. However, they did not present with excessive longitudinal growth. These mice also differed from humans in that they were deficient in aldosterone and aldosterone synthase. ACTH is thought to regulate zona glomerulosa development in rodents more so than in humans [174]. FAMILIAL GLUCOCORTICOID DEFICIENCY SYNDROME TYPE 2

ACTH-MC2R signaling in adrenal cells requires the presence of the MC2R accessory protein (MRAP) [188,189]. MRAP traffics MC2R from the endoplasmic reticulum to the plasma membrane, where MC2R interacts with ACTH. In the absence of MRAP, MC2R remains sequestered in the endoplasmic reticulum [183], and cells are unable to bind and respond to ACTH. Mutations in MC2R account for roughly 25% of FGD. FGD without MC2R mutations is called FGD type 2. Whole-genome SNP analyses of families with FGD but no MC2R mutations demonstrated that mutations in MRAP were linked to FGD type 2 [181]. Sequence analysis revealed a slice site mutation that led to a defective protein [181], and further sequencing of FGD type 2 families revealed similar mutations. Subsequently several other patients with FGD type 2 and MRAP mutations have been reported [190
192]. Patients with missense MRAP mutations, possibly less severe than those reported initially, may present with late-onset adrenal insufficiency [193]. Mutations in MRAP account for approximately 20% of all cases of FGD [194]. OTHER CAUSES OF FAMILIAL GLUCOCORTICOID DEFICIENCY SYNDROME

In addition to MC2R and MRAP, mutations in several other genes can cause FGD [195]. These include STAR [196], required for cholesterol transport and adrenal steroidogenesis, MCM4 [197,198], required for DNA repair and genome stability, and NNT [199
201], which protects cells from reactive oxygen stress. Most of these other causes include variable degrees of mineralocorticoid deficiency, depending on the extent of pathological involvement of the zona glomerulosa and aldosterone production [202]. The

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ACTH

diagnosis of FGD depends on finding marked hyperpigmentation associated with very high plasma ACTH (often .1000 pg/mL), low cortisol, and (usually) normal electrolytes. The diagnosis can be obscured by acute illness [192]. Surprisingly, patients with FGD type 1 may have severe salt-losing adrenal insufficiency [203], even though patients with other forms of secondary adrenal insufficiency do not. Moreover, a patient with FGD type 1 due to a mutation in MC2R had normal pigmentation due to a second mutation in MC1R [204]. Increasingly, whole-exome sequencing (WES) is becoming a valuable tool to identify patients with known and novel genetic causes of FGD. In one report, WES successfully identified a genetic cause in 17 of 43 patients with undiagnosed FGD [205], and a second comprehensive analysis of rare genetic causes of adrenal insufficiency including 95 patients in eight families, in whom congenital adrenal hyperplasia, adrenoleukodystrophy, and autoimmune adrenal insufficiency were excluded, found the most commonly involved genes were MC2R (25), NROB1/DAX1 (12), STAR (11), CYP11A1/Side-chain cleavage (nine), MRAP (nine), NNT (seven), ABCD1 (two), NR5A1/SF1 (one), and AAAS/Aladin (one) [206]. ACTH-Independent Activation of ACTH Receptor Pathways Several rare causes of Cushing’s syndrome are due to ACTH-independent pathological activation of MC2R/ACTH receptor pathways [207]. In the McCune
Albright syndrome, a mutation in the GTPase region of the stimulatory alpha subunit G protein, Gsα, can result in constitutive activation of PKA in the adrenal cortex, leading to hypersecretion of cortisol and adrenal adenoma formation [208]. Ectopic expression of other seven-transmembrane GPCRs, including those which bind gastric inhibitory polypeptide, luteinizing hormone, vasopressin, and catecholamines, in the adrenal cortex, may occur [209]. In this situation, Cushing’s syndrome results from the cognate hormone binding to the ectopically expressed receptor, leading to stimulation of adenylate cyclase, activation of cyclic AMP, and cortisol hypersecretion. Patients with Carney complex (Cushing’s syndrome due to primary pigmented nodular adrenal disease, myxomas of the cardiac atria and other tissues, and freckling of the skin) were found to have mutations in the regulatory subunit of PKA, R1α [210]. Many subsequently discovered mutations in this gene have been associated with Carney complex [211,212]. Both germline and somatic mutations in the catalytic subunit of PKA are associated with the majority of bilateral and unilateral cortisol-secreting adenomas, respectively [213,214]. In addition, germline and somatic mutations in several

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other genes in the PKA pathway have been found to cause adrenal tumors and Cushing’s syndrome [207]. Mutations in the phosphodiesterases, PDE11A and PDE8B, leading to increased cAMP levels, are associated with Cushing’s syndrome due to primary pigmented nodular adrenal disease. Outside of the PKA pathway, bilateral macronodular adrenal hyperplasia can be due to autosomal dominant, loss-of-function mutations in the tumor suppressor, ARMC5 [215,216], which can also cause primary hyperaldosteronism [217]. Mutations in the β-catenin gene (CTNNB1) are associated with adrenocortical adenomas producing both cortisol [218] and aldosterone [219]. Thus Cushing’s syndrome due to adrenal adenomas or hyperplasia is often caused by somatic or germline mutations in genes in the cAMP/PKA pathway, through which MC2R, the physiological regulator of cortisol secretion, acts [220].

Nonadrenal Actions of ACTH ACTH binds with high affinity to rat adipocytes and has potent lipolytic effects. High levels of MC2R mRNA expression have been demonstrated in all murine adipose tissues examined, but MC5R mRNA expression was also found in a subset of these tissues. Both MC2R and MC5R mRNA were identified in the 3T3-L1 cell line after these cells were induced to differentiate into adipocytes. The physiologic importance of the actions of melanocortins on adipose tissue is unclear. Primate adipose tissues have been reported to be insensitive to the lipolytic actions of ACTH. Human skin cells express MC2R along with mRNA for three obligatory enzymes of steroid synthesis, the cytochromes P450scc (CYP11A1), P450c17 (CYP17), and P450c21 (CYP21A2). An equivalent of the HPA axis composed of locally produced CRH, CRH receptor, POMC, and cortisol likely operates in mammalian skin as a local response to stress [221]. Recently, it was demonstrated that ACTH can induce DNA synthesis and cell proliferation in an oral keratinocyte cell line [222]. Stress suppresses immune function. Following an infection or most immunization procedures, and the presence of bacterial endotoxin, a stress-like response of the pituitary occurs, leading to the release of ACTH and cortisol which tends to suppress the immune system. However, in humans, there is no clear evidence that the rise in cortisol following an immune or inflammatory stimulus plays a significant role in modulating the subsequent immune response. In fact, prior to the treatment of patients with primary adrenal insufficiency, there were no substantive reports that these patients suffered from immune dysfunction. Receptors

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for CRH, ACTH, and glucocorticoids most likely mediate effects of these hormones on the immune system. ACTH receptors on human peripheral monocytes have been characterized, and GRs are present on human lymphocytes. Glucocorticoids inhibit many aspects of immune function, establishing a negative feedback loop between the immune and neuroendocrine systems. Glucocorticoids block lymphocyte activation, block the production and action of interleukin-2, interleukin-1, gamma-interferon, tumor necrosis factor, and prostaglandins, and interfere with the interaction of certain effector molecules with target cells. In some neurological diseases, particularly infantile spasms (West syndrome), ACTH rather than glucocorticoid has been used therapeutically. Retrospective analyses suggest that both are equally effective [223], but glucocorticoid to be superior, suggesting ACTH acts by increasing endogenous cortisol levels [224].

α-MSH Skin MELANOCORTIN 1 RECEPTOR

The MC1R was initially cloned from primary melanoma tumors and is expressed in primary human melanocytes. Other cutaneous cells including keratinocytes and dermal fibroblasts have also been reported to express MC1R, although the presence of MC1R in keratinocytes could not be confirmed in other studies. MC1R is expressed in keratinocytes and is induced by calcium and UV light treatments [225]. Corresponding to skin expression, activation of melanocyte MC1R, via adenylate cyclase, stimulates tyrosinase activity, and is the rate-limiting enzyme in melanogenesis. Activation of tyrosinase results in an increased proportion of eumelanin (brown-black pigment) formation over pheomelanin (red-yellow pigment) formation resulting in increased pigmentation. Mutations and variant alleles in the MC1R gene have been linked to variation in mammalian pigmentation. The extension locus has long been known to regulate pigmentation in mammalian species. The extension locus has been shown to encode the mouse MC1R [226]. The recessive yellow allele (e) at this locus results from a frameshift that produces a prematurely terminated, nonfunctioning receptor [226]. The somber (Eso and Eso-3j) alleles and tobacco darkening (Etob) alleles, which have dominant melanizing effects, result from point mutations that produce constitutively active or hyperactive receptors [226]. The human MC1R is highly polymorphic, and mutant alleles have been associated with fair skin and blond or red hair [227]. Analysis of five naturally

occurring common variants of MC1R associated with fair skin and red hair have revealed decreased stimulation of cAMP synthesis with no changes in or only slightly reduced α-MSH binding. In addition, MC1R variants determine sun-sensitivity in humans with dark hair. Melanocytes, which express POMC and several of its processed peptides, are derived from the neural crest [228]. It is likely that α-MSH produced via POMC expression in melanocytes is responsible for the pigmenting effects of ultraviolet light, and together with variations in MC1R discussed above, for the different degrees of skin pigmentation observed among ethnic groups. Ultraviolet light induces POMC/ α-MSH production in melanocytes in a p53-dependent manner [229]. Humans with MC1R variants associated with light skin and red hair are more prone to develop melanomas. When skin containing these variants is exposed to ultraviolet light, AKT is induced in a BRAF-dependent manner [230] to drive oncogenic transformation [231]. Addictive properties of tanning [232] have been attributed to the synthesis and release of β-endorphin from melanocytes in response to ultraviolet light exposure [233]. Blood levels of β-endorphin are elevated after light exposure in mice and blunt pain perception in an opioid receptor-dependent fashion [233]. MELANOCORTIN 5 RECEPTOR

Expression of the fifth melanocortin receptor has been recognized in the brain and at low levels in many tissues including the adrenal glands, skin, adipocytes, skeletal muscle, kidneys, lung, stomach, liver, spleen, thymus, lymph nodes, mammary glands, ovary, pituitary, testis, and uterus. In situ hybridization studies showed that within the adrenal, MC5R is predominantly expressed in the aldosterone-producing zona glomerulosa cells. High levels of MC5R mRNA expression have been documented in the secretory epithelia of exocrine and endocrine glands including Harderian, preputial, lacrimal, sebaceous, and prostate glands and pancreas [234]. Melanocortins have been reported to affect a number of exocrine glands. Removal of the neurointermediate lobe of the pituitary reduces sebaceous lipid production, and this reduction is restored by administration of α-MSH, which acts through MC5R in this tissue [235,236]. Exogenous ACTH and MSH increase lacrimal gland secretion. Deletion of the murine MC5R resulted in loss of detectable binding of [125I]NDP-MSH to Harderian, lacrimal, and preputial glands, and skeletal muscle indicating that MC5R is the predominant melanocortin receptor in these tissues [234]. Development of the MC5R-null mouse confirmed a physiologic role for the melanocortins in regulating exocrine gland function; the mice demonstrate severe deficits in water repulsion and thermoregulation as a

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ACTH

result of decreased sebaceous lipid production [234]. MC5R also appears to be essential for hormonally regulated release of porphyrins from the Harderian gland [234]. The preputial gland is a specialized sebaceous gland implicated in pheromone production. Exogenous α-MSH stimulates the release of a preputial odorant into the urine of male mice which stimulates aggressive attacks. Possibly supporting this, MC5Rdeficient mice have decreased aggression [237,238] and increased defensive behavior [239]. The existence of a hypothalamic
pituitary
exocrine axis might provide a mechanism by which stress could alter behavior via regulation of olfactory cues [234]. Brain MELANOCORTIN 4 RECEPTOR

The MC4R is localized to the brain and, in contrast to the MC3R, its expression has been documented throughout the central nervous system including the cortex, thalamus, hypothalamus, brainstem, and spinal cord [240]. In humans, the MC4R has not been detected in any peripheral tissue, although this melanocortin receptor subtype appears to play a central role in weight regulation. Mice homozygous for a Mc4r-null allele demonstrate autosomal dominant, maturity-onset obesity, hyperphagia, hyperinsulinemia, and hyperglycemia [241]. Heterozygotes have an intermediate phenotype. The MC4R-null mouse also demonstrates increased linear growth, a feature unique to the agouti yellow mice and the MC4R-null mouse among rodent obesity models. The role of MC4R in weight regulation in humans has been confirmed with identification of individuals with dominantly inherited obesity segregating with mutations in the MC4R gene that result in frameshift errors [242]. MC4R haploinsufficiency may be a frequent, but incompletely penetrant, cause of human obesity [242
244]. Incomplete penetrance is highlighted by the absence of obesity in individuals with large deletions of chromosome 18q, a region that spans the MC4R gene [245]. A dominant-negative effect of mutant MC4R has been proposed, but cotransfection studies of mutant and wild-type MC4R in vitro showed that mutants affected neither signaling nor cell surface expression of wild-type MC4R. There is a strong correlation between an MC4R mutation’s impact on receptor trafficking/signaling and the severity of the obesity and hyperphagic phenotype [246]. Single nucleotide polymorphisms near MC4R have been identified in association with obesity, and insulin resistance in several genome-wide association studies [247]. Surprisingly, patients [59] and mice [248] with severe obesity due to MC4R mutations have lower blood pressure and cardiovascular morbidity than obese persons without MC4R mutations,

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suggesting that hypertension is melanocortindependent, consistent with the stimulation of sympathetic activity by melanocortin pathways. MC4R agonists have been developed as possible antiobesity therapeutics, for treatment of patients with idiopathic obesity [249]. These could be useful in patients with surgical or genetic causes of α-MSH deficiency, as well as in those with haploinsufficiency of MC4R expression. Mutations in β-MSH in obese, hyperphagic humans suggest that this, in addition to α-MSH, may be an active MC4R ligand [250]. Human genome analysis has identified a single MRAP paralogue, MRAP2, which also interacts with MC2R. Though MRAP2 bears only 27% sequence homology to MRAPα, there is greater interspecies conservation of MRAP2 than MRAP, and it is believed that MRAP2 most closely resembles the ancestral protein. There is 87% sequence homology between human and mouse MRAP2 [251]. MRAP2 is a 205-amino-acid single-transmembrane domain protein that is primarily expressed in the human adult adrenal gland and ventromedial hypothalamus in the brain, a site of MC3R and MC4R expression. Severely obese humans with heterozygous loss-of-function mutations in MRAP2 have been described, along with obese mice with knockout of the Mrap2 gene [252]. MRAP2 interacts with all melanocortin receptors [253] and enhances the response of MC4R to α-MSH, both by lowering basal cAMP-generating activity, as well as by increasing receptor response to ligand [252]. Mrap2 stimulates food intake through an effect on the prokineticin receptor 1 [254]. Whether MRAP and MRAP2 are coexpressed and interact in adrenocortical cells and central neurons to regulate common functions is an area of active investigation [189,253,255,256]. The agouti gene locus was identified over half a century ago as a genetic locus that controls the amount and distribution of eumelanin (brown/black) and pheomelanin (yellow/red) pigmentation in the mammalian coat [257]. However, analyses of mutations at the agouti gene locus have occupied investigators for nearly a century. The lethal yellow mutation at the agouti locus was the first murine embryonic lethal mutation and the first murine obesity syndrome to be characterized [258]. Agouti is a small 131-amino-acid protein that is secreted by dermal papillae cells and acts to block melanocortin action on follicular melanocytes at MC1R in the mouse [259,260]. The recombinant murine agouti protein is a potent nanomolar competitive-antagonist for melanocortin receptors at MC1R and MC4R, relatively weaker at MC3R, and only a micromolar inhibitor of MC5R [259] and represents the first known endogenous antagonist for a G protein-coupled receptor. In wild-type rodents, agouti expression is restricted to the hair follicle [260]. The human agouti gene, which is 85% identical to the

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mouse gene, is expressed much more widely, including in adipose tissue, testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin. The human agouti protein, the agouti-signaling protein (ASIP), displays a similar pharmacologic profile for antagonism of melanocortin receptors as compared to the murine agouti protein, except that ASIP may display both competitive and noncompetitive antagonism at MC4R and also appears to act as a noncompetitive antagonist of MC2R. The agouti-related protein (AGRP) gene was identified based on its homology to agouti and is expressed in the hypothalamus, adrenal medulla, and at low levels in the testis, lung, and kidney [261]. In contrast to divergent expression patterns of murine and human agouti, expression patterns of murine and human AGRP appears identical. Within the hypothalamus, AGRP expression is confined to the arcuate nucleus and AGRP-immunoreactive terminals paralleled POMC-immunoreactive terminals projecting from the arcuate nucleus [22]. AGRP is a selective, nanomolar competitive-antagonist of MC3R and MC4R clearly implicating it as the endogenous melanocortin antagonist involved in energy homeostasis [262]. Whereas germline deletion of Agrp has only a mild impact to retard weight gain in older mice, ablation of the gene in adult mice is associated with a profound decrease in feeding and death [263]. MELANOCORTIN 3 RECEPTOR

Expression of the third cloned melanocortin receptor, MC3R, has been identified in the brain, placenta, gut, and heart, with the highest densities of MC3R mRNA in the hypothalamus and limbic system [226]. MC3R mRNA was found in the arcuate nucleus [226], the site of hypothalamic POMC expression. MC3R and MC4R are visualized within the central nervous system [264], in the nucleus accumbens shell, the medial preoptic area, and the ventromedial nucleus of the hypothalamus. In the lateral septum and the olfactory tubercle, both MC3R and MC4R are present. A physiologic role for MC3R has not yet been identified. In a murine model of experimental gout, systemic treatment of mice with ACTH4
10 inhibited neutrophil accumulation [265]. This effect was blocked by the melanocortin receptor type 3/4 antagonist SHU9119. MC3R, but not MC4R, mRNA was detected in murine macrophages suggesting that MC3R may play a role in modulating inflammation. A mouse with targeted deletion of the MC3R gene [266] has a normal body weight and an increased fat-to-lean weight ratio due to increased efficiency of converting ingested food into stored fuel. Thus, a function of melanocortins acting via MC3R may be to promote the conversion of energy in food into either lean body mass or forms of energy

other than fat. This raises the possibility that in humans, mutations in MC3R may contribute to the “thrifty” genotype, as is found in Pima Indians [267]. MRAP appears to increase coupling of α-MSH to MC3R [268], but Mrap2 does not appear to have a similar effect on Mc3r function [252]. Overall, MC3R seems to have a modest effect on energy metabolism, at least in mice [269].

β-Endorphin The major source of β-endorphin is the POMC neurons in the arcuate nucleus of the hypothalamus, along the third ventricle, with a smaller fraction observed in the caudal brainstem. β-Endorphin is an opioid neuropeptide, produced by cleavage of the C-fragment (amino acids 61
91) from the precursor molecule β-lipotrophin, with a molecular weight of 3465 g/mol [270,271]. It produces analgesia by binding to opioid receptors (mu subtype) at both pre- and postsynaptic nerve terminals. In the peripheral nervous system, binding of β-endorphin to mu receptors results in inhibition of the release of tachykinins (i.e., substance P) which are important in the transmission of pain [272]. In the central nervous system, binding of mu-opioid receptors, instead of inhibiting substance P, inhibit the release of GABA, resulting in excess production of dopamine, which is associated with pleasure [272]. Studies in rats suggest that immune cells produce opioid peptides derived from both POMC and proenkephalin. Opioid medications bind opioid receptors and mimic natural endorphins. Acute use of exogenous opiates inhibits the production of endogenous opiates, while chronic use inhibits the production of both endogenous opiates and mu-opioid receptors, therefore increasing the risk for opioid-induced hyperalgesia, tolerance, and addiction. β-Endorphin has a role in modulating food intake, mainly in the introductory/ appetitive, goal-directed phase of feeding behavior, and to a lesser degree than α-MSH. β-Endorphin also has a role in the control of sexual behavior, reward mechanisms, and effects of meditation [270].

Additional Potential Melanocortin Actions Additional physiologic roles for melanocortins have been proposed that do not clearly correlate with known functions of specific melanocortin receptors as surmised from human or mouse mutants. Intracerebroventricular administration of ACTH or α-MSH elicits excessive grooming behavior, yawning, stretching, and penile erection. In a double-blind, placebo-controlled crossover study, a cyclic α-MSH analogue initiated erections in

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REFERENCES

men with psychogenic erectile dysfunction [273]. This effect is mediated via melanocortin receptors in the forebrain and spinal cord [274]. Melanocortin analogues do not have hypotensive side-effects found in the phosphodiesterase inhibitors used for this purpose [275]. Roles for melanocortin actions in neuromuscular development, promotion of the regeneration of crushed nerves, and CNS protection from injury have also been postulated [276]. Melanocortins have a potential role in fetal growth and brain development. Centrally administered MSH is a potent antipyretic agent and, in an endogenous pyrogeninduced fever model in rabbits, is approximately 25,000-fold more potent than acetaminophen [277]. Physiological roles for melanocortins in maintenance of cardiovascular homeostasis have also been proposed. Peripheral or central administration of γ-MSH causes tachycardia and pressor effects [278]. Central administration of α-MSH results in bradycardia and depressor effects [278]. α-MSH may prevent pancreatic islet cell graft survival via downregulation of Toll-like receptors [279]. Finally, α-MSH has been reported to modulate human chondrocyte activation induced by proinflammatory cytokines [280].

MEASUREMENT ASSAYS FOR ACTH (RIA, IRMA, LC/MS, ETC.) ACTH was one of the first substances to be measured by RIA [281]. In pioneering work using polyclonal antisera, Yalow described the measurement of ACTH and related peptides in normal persons and those with ectopic ACTH production by lung cancer. These studies provided among the first data that ACTH was synthesized from larger precursors, which were termed “big” and “big-big” ACTH. The ACTH RIA can be performed on unextracted plasma, but suffers from a limit of sensitivity of B25 pg/mL, and is therefore often unable to detect levels of plasma ACTH in the normal basal range. Radioimmunoassay remained the standard method for the measurement of plasma ACTH until the development of the immunoradiometric assay (IRMA). ACTH IRMAs employ two antibodies, one or both monoclonal, against ACTH. The solution-phase antibody is radiolabeled, and the solid-phase antibody is linked to a bead or other solid support. In general, the ACTH IRMA on unextracted plasma compares very favorably with RIA, being more sensitive, more reproducible, and more rapid [282]. Most IRMAs have lower limits of detection of 3
5 pg/mL and coefficients of variation of less than 10% up to 5000 pg/mL. Depending on antigenic specificity of the chosen antibodies, the ACTH IRMA may detect only intact ACTH, both ACTH and POMC precursor peptides, or

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only POMC precursors. It is essential to know the sequence specificity of any IRMA in clinical use, for some, unlike most polyclonal RIAs, are incapable of detecting ACTH precursors which may be secreted by lung carcinomas. Despite the wide availability of the ACTH IRMA, results from different laboratories are difficult to compare because of lack of agreement on a suitable ACTH reference standard. An immunofluorometric assay for ACTH has also been developed. Its sensitivity and accuracy are similar to those of the IRMA. Its principal advantage is speed and use of nonradioactive label which is stable for over 1 year. A direct comparison of IRMA and RIA tests for ACTH found them to be comparable [282]. Furthermore, a nonradioactive electrochemiluminescent ACTH immunoassay was found to be precise and reliable in combination with reduced turnaround time [283]. An assay for cosyntropin (1
24 ACTH) in urine by tandem mass spectrometry has been developed to detect illegal doping activity in athletes. Given the size of the peptide and the presumed rapid metabolism and clearance from circulation, this technique requires immunoaffinity purification, subsequent liquid chromatographic separation, and finally mass spectrometric detection to provide enough sensitivity and specificity [284]. Plasma assay of POMC products other than ACTH, including β-endorphin and N-terminal glycopeptide, has been suggested as an adjunct in the evaluation of the HPA axis. In general, levels of these hormones parallel those of ACTH in various HPA axis abnormalities. However, except for use as a screening test for lung carcinoma associated with preferential secretion of N-terminal glycopeptide1
61 or pro-ACTH1
150, such tests are much less helpful than the ACTH IRMA because of the low concentration of other POMC peptide fragments compared with ACTH in most physiologic and pathologic settings.

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C H A P T E R

4 Growth Hormone Vivien S. Bonert and Shlomo Melmed

INTRODUCTION

trophoblasts [3]. GH1 codes for a 22-kDa protein consisting of 191 amino acids (Fig. 4.1). Approximately 10% of pituitary GH circulates as a 20-kDa spliced variant lacking amino acid residues 32 46, as well as less abundant shorter variants [4]. GH2 is expressed by placental syncytiotrophoblasts during the second and third trimesters of gestation and encodes a 22-kDa protein secreted form detected in maternal circulation from midpregnancy [5,6]. The role of placental GH2 is unknown, however, the rise in maternal serum

Growth hormone (GH), secreted by anterior pituitary somatotroph cells, binds to hepatic GH receptors, initiating several intracellular signaling pathways resulting in the generation of insulin-like growth factor (IGF)-1, cytoskeletal changes, alterations in glucose metabolism, and modulation of cell proliferation genes. IGF-1, synthesized primarily in the liver, mediates most of the growth-promoting actions of GH. Metabolic actions of GH also affect carbohydrate, protein, and lipid metabolism and alter body composition. GH and IGF-1 influence body composition, cardiovascular function, muscle strength, and exercise performance. Clinical evaluation of GH excess or deficiency requires estimation of GH responses to inhibitory or stimulatory factors during provocative testing. GH replacement therapy has beneficial effects in GH-deficient children with short stature and adults with GH deficiency. Shortand long-term actions of GH have been evaluated for potential beneficial effects in the aging population and for enhanced athletic performance by athletes, with lack of proven efficacy.

GROWTH HORMONE GENE STRUCTURE The human growth hormone (GH) genomic locus spans approximately 66 kb and contains a cluster of five highly homologous genes located on the long arm of human chromosome 17 at bands q22 24. The 5’ to 3’ arrangement includes GH1, chorionic somatomammotropin hormone (CSH) 1, CSH2, GH2, and CSH4 [1], all of which have the same basic structure, comprising five exons separated by four introns [1,2]. GH1 gene is transcribed in anterior pituitary somatotrophs, while CSH1 and CSH2 are expressed in placental

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00004-0

FIGURE 4.1 Amino acid structure of human GH. GH is a 191amino-acid single-chain 21.5-kDa polypeptide with two intramolecular disulfide bonds. Fifteen percent of GH is deleted from amino acid (32 46) and is secreted as a 20-kDa protein.

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correlates with a fall in GH1 concentrations, suggesting the possibility of a feedback loop on the maternal hypothalamic pituitary axis [5]. Postpartum, GH2 levels drop rapidly and are undetectable after 1 hour [5]. GH may also be expressed at low abundance in peripheral tissues, including breast and colon [7].

Somatotroph Development and Differentiation Somatotrophs comprise up to 45% of pituitary cells and are located predominantly in the lateral wings of the anterior pituitary gland, which contains 5 15 mg of GH. Acidophil cells are the progenitors for both GH-producing somatotrophs and prolactin (PRL)producing lactotrophs. Expression of the α-subunit transcript in the hypophyseal placode within pharyngeal ectoderm prior to formation of Rathke’s pouch defines the onset of pituitary organogenesis [8,9]. Evidence from transgenic mice studies suggests that most PRL-expressing cells arise from GH-producing cells [10]. Specifically, GH expression in somatotroph cells develops in a time-and-space-dependent manner. The anterior pituitary develops from Rathke’s pouch during the early stages of embryonic development [11], and specific cytodifferentiation pathways lead to differentiated hormone-producing cells. Ablation of somatotrophs by expression of GHdiphtheria toxin and GH-thymidine kinase fusion genes inserted into the germ line of transgenic mice also results in elimination of the majority of lactotrophs; however a small percentage of lactotrophs escape destruction [10,12]. This suggests that the majority of PRL-producing cells arise from postmitotic somatotrophs. The transcription factors Prophet of Pit1 (PROP-1) and POUIFI determine somatotroph and lactotroph growth, differentiation, and commitment to expressing the GH or PRL gene product [8] (Fig. 4.2). The Pit-1 gene transcript and POUIF1 protein are expressed in somatotrophs, lactotrophs, and thyrotrophs [8]. Pit-1 actions are complemented by other factors required to achieve physiologic patterns of cell-specific gene activation [8]. Inherited syndromes of GH deficiency or GH action may be attributed to several cellular defects, including mutations of transcription factors, the GH-1 gene, the growth hormone releasing hormone (GHRH) receptor, GH receptor (GHR) signaling, or very rarely IGF-related molecules. PROP-1, a paired homeobox protein, is required for initial commitment of Pit-1 cell lineages [14]. PROP-1 represses Rpx expression, and missense and spliced mutations of PROP-1 leading to loss of DNA-binding or transactivation leads to pituitary failure with short stature and varying degrees of thyroid failure,

hypogonadism, and adrenocorticotrophic hormone (ACTH) deficiency [15]. POUIFI [16] mutations may also lead to pituitary failure. Patients with combined pituitary hormone deficiency have predominantly GH and PRL deficiency, with variable degrees of hypothyroidism [16]. In the absence of Pit-1 the promoter region is inactive, and binding of Pit-1 facilitates interactions with other ubiquitous activators to enhance GH transcription. This model of cooperative interaction likely contributes to tissue-specific expression of the hGH gene by a single cell type-specific activator.

GH SYNTHESIS Somatotrophs comprise up to 45% of pituitary cells and are located predominantly in the lateral wings of the anterior pituitary gland which contains 5 15 mg of GH. The GH molecule, a single-chain polypeptide hormone consisting of 191 amino acids (Fig. 4.1), is synthesized, stored, and secreted by somatotroph cells. The 217-amino-acid GH precursor is synthesized and transported to the endoplasmic reticulum lumen. The 1 26-amino-acid peptide is cleaved and the mature GH molecule transported to the golgi for packaging into secretory vesicles mediated by zinc ions [17]. The crystal structure of human GH reveals four α-helixes; and several structural features confer functional characteristics which determine GH signaling. These included the third α-helix with amphiphilic domains, and a large helical loop [18,19]. Circulating GH molecules comprise at least three monomeric forms and several oligomers. The monomeric moieties include a 22- and 20-kDa form, acetylated 22 K, and two desamido GHs. The 22-kDa peptide is the major physiologic GH component. The 20-kDa GH has a slower metabolic clearance than the 22-kDa form. which accounts for the plasma 20:22 ratio being higher than in the pituitary gland. The 22-kDa and 20-kDa peptides have similar growth-promoting activity. Monomeric GH forms found in the plasma of acromegaly patients are qualitatively similar to those found in normal plasma [20]. Circulating GH is first detectable in fetal serum at the end of the first trimester, peaks at a concentration of 100 150 ng/mL at 20 weeks of gestation, and subsequently falls to 30 ng/mL at birth. GH levels continue to fall during infancy. During childhood, levels are similar to those in adulthood, until puberty, when circulating levels are elevated. GH levels decline after adolescent growth and remain stable until midadulthood, when they decline progressively through old age [21].

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GH SYNTHESIS

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FIGURE 4.2 Hypothalamic pituitary control of GH secretion. Control of the secretion of GH is achieved by hypothalamic GHRH and somatostatin, which traverse the portal vein, somatotroph-specific transcription factors, and negative feedback control of IGF-1. Accurate measurement of pulsatile secretion of GH requires ultrasensitive assays. POU1F1, POU domain class 1 transcription factor 1; Prop-1, Prophet of Pit-1; GHS, growth hormone secretagogues; SRIF, somatostatin. Source: From Melmed [13].

Neuroendocrine Control of GH Central neurogenic control of GH is complex. Neuropeptides, neurotransmitters, and opiates impinge on the hypothalamus and modulate GHRH and SRIF release. The net effect of these complex influences determines the final secretory pattern of GH. Small synthetic molecules termed growth hormone secretagogues (GHS) [22] stimulate and amplify

pulsatile pituitary GH release, via a separate pathway distinct from GHRH/SRIF. GHS, administered alone or in combination with GHRH, are potent and reproducible GH releasers and are useful tools for the diagnosis of GH deficiency [23]. The GHS receptor (GHSR), a heterotrimeric GTPbinding protein (G-protein)-coupled protein [24], comprises seven α-helical membrane-spanning domains and three intracellular and extracellular loops. The

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GHSR is expressed in pituitary somatotroph cells and in both hypothalamic and nonhypothalamic brain regions. The endogenous ligand of GHSR is a 28-aminoacid peptide, ghrelin, isolated from the gastrointestinal tract, and is n-octanoylated at the serine 3 residue [25]. Ghrelin releases GH both in vivo and in vitro, and noctanoylation is essential for GH releasing activity. Ghrelin is expressed in the arcuate nucleus of the hypothalamus, and also in the pituitary gland [25,26]. Ghrelin modulates GH secretion at both a hypothalamic and pituitary level [27], and signals through the GHSR to induce GH release [25]. In vitro, GHRH and ghrelin have additive effects on GH release, whereas in vivo administration of GHRH with GHS/ghrelin is synergistic [28]. GH secretagogues and GHRH act via different mechanisms [28]. Furthermore, GHRP-6 which activates the GHSR, does not elicit GH release following hypothalamic pituitary disconnection [29]. Ghrelin amplifies the GH secretory pattern [30] and enhances GH responsiveness to GHRH [28,31,32]. These observations may be explained by findings that GHRH acts as an allosteric coagonist for the GHSR [33]. Ghrelin thus appears to act with GHRH to regulate GH secretion and energy balance [34]. Functional GHSR is detected in the human pituitary by the fifth week of gestation [35], and transgenic mice with decreased GHSR mRNA expression exhibit reduced GH and insulin-like growth factor (IGF)-1 levels [36], while GHSR knockout mice have lower IGF1 levels and decreased body weight [37]. However, ghrelin-null mice do not develop dwarfism [38]. Missense mutations in the GHSR (TRP2X, Arg237Trp, Ala204Glu), with attenuated ghrelin binding or possibly GHSR constitutive signaling, result in partial isolated GH deficiency [29,39], with incomplete penetrance and a range of phenotypes. Healthy volunteers demonstrate synchronicity between ghrelin and GH pulsatility, suggesting stimulation of GH by ghrelin or possibly coregulation of both by other neuroendocrine factors [40]. As GH secretagogues elicit a synergy with GHRH on GH release, which is minimally altered by age, sex, or adiposity and is devoid of potential side effects (unlike insulin-induced hypoglycemia), this combined test is a useful and safe diagnostic tool in the diagnosis of adult growth hormone deficiency (GHD). Thyrotrophin-releasing hormone (TRH) does not stimulate GH secretion in normal subjects, but induces GH secretion in about 70% of patients with acromegaly, and also in patients with liver disease, renal disease, ectopic GHRH-releasing carcinoid tumors [41], anorexia nervosa [42], and depression. A group of reproductive kisspeptin neuropeptides encoded by the KISS-1 gene, stimulate hypothalamic GnRH neurons, and also induce GH release in peripubertal rats [43]. The physiological relevance in humans has not been elucidated.

Leptin, a 167-amino-acid cytokine product of the ob gene, plays a key role in regulating body fat mass [44], food intake, and energy expenditure. As GH secretion is markedly impaired in obese subjects, leptin may act as a metabolic signal to regulate GH secretion. In the fasting state, leptin levels decrease rapidly, prior to and out of proportion to changes in fat mass [45], triggering a neuroendocrine adaptive response to acute energy deprivation, including decreased reproductive and thyroid hormone levels, increased GH levels that mobilize energy, and reduced IGF-1 levels that may slow growth-related processes [46]. Interactions between leptin and the GH and adrenal axes may be less important in humans than in animal models, as patients with congenital leptin deficiency have normal linear growth and adrenal function [46]. Dopamine, a precursor of epinephrine and norepinephrine, influences GH secretion. GH-deficient children exhibit increased growth velocity after 6 months of L-dopa treatment, while adults increase their serum GH levels from 0 to 5 20 ng/mL within 60 90 minutes after oral L-dopa administration. Similarly, the central dopamine receptor agonist apomorphine, stimulates GH secretion. Norepinephrine increases GH secretion via α-adrenergic pathways and inhibits GH release via β-adrenergic pathways. Insulin-induced hypoglycemia increases GH secretion via an α2-adrenergic pathway, whereas clonidine acts on αl-adrenergic receptors to increase GH secretion. Arginine administration, exercise, L-dopa, and antidiuretic hormone (ADH) facilitate GH secretion by α-adrenergic effects [47]. β-Adrenergic blockade increases GHRH-induced GH release, possibly due to a β-adrenergic effect at the pituitary level or via decreased hypothalamic somatostatin release. β-Adrenergic blockade also enhances GH release elicited by insulin-induced hypoglycemia, ADH, glucagon, and L-dopa [47]. Epinephrine may regulate GH secretion by decreasing somatostatin release. Cholinergic and serotoninergic neurons have been implicated in the etiology of sleep-induced GH secretion. The lateral hypothalamus expresses orexin-A (hypocretin-1) and orexin-B (hypocretin-2) [48], which primarily regulate food intake and modulate the sleep wake cycle and arousal, and also play a role in control of several endocrine axes. Orexin-A is expressed mostly in lactotrophs and to a lesser extent in thyrotrophs, somatotrophs, and gonadotrophs, but is not expressed in corticotrophs. Orexin-B is expressed in most pituitary corticotroph cells [49]. Orexin-A markedly reduces spontaneous GH secretion and GH pulsatility as well as GH response to ghrelin in rats [50] mediated via somatostatinergic neurons [51]. Furthermore, loss of orexin function in knockout mice results in narcolepsy [52], implicating orexins in the

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regulation of arousal and the sleep wake cycle. As GH secretion is linked to the sleep wake cycle and feeding state in humans, pituitary-derived orexins may play a role in coordinating sleep and energy homeostasis. Several gastrointestinal neuropeptides stimulate GH secretion in animal models, including substance P, neurotensin, vasoactive intestinal polypeptide, peptide histidine isoleucine (PHI) amide, motilin, galanin, cholecystokinin, and glucagon [53].

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Age-associated decrease in GH output is due to decreased GH pulse amplitude, with no appreciable changes in GH pulse frequency or nadir levels, implicating reduced GHRH pulse amplitude in the somatopause [65]. Sexually dimorphic GH secretion may also be attributed to GHRH. In males, GH is secreted predominantly at night, with low daytime baseline secretion, whereas females exhibit more daytime GH secretory pulses with higher basal GH levels [66], mirroring increased nocturnal male GHRH secretion, with elevated daytime GHRH levels in females [66].

HYPOTHALAMIC HORMONES Growth Hormone-Releasing Hormone

Somatostatin

Hypothalamic GHRH was characterized from ectopic pancreatic GHRH-secreting tumors causing acromegaly [41,54]. Analysis of one tumor revealed a 44-amino-acid GHRH residue; the other contained 37-, 40-, and 44-amino-acid forms [55]. GHRH (1 40) and GHRH (1 44) are both found in extracts derived from the human hypothalamus. GHRH is secreted from neurons in the hypothalamic arcuate nucleus and premammillary area, with axons that project to the median eminence. The hGHRH gene encodes a 108amino-acid preprohormone for GHRH-44 [56,57], which has a free amino terminal and amidated carboxy terminal residue. The amino terminal appears to bestow biologic activity on the GHRH molecule. There is considerable structural homology between GHRH and several gut peptides, the highest between GHRH and PHI, which have 12 amino acids in common in equivalent positions [58]. Varying degrees of homology exist between GHRH and VIP, glucagon, secretin, and GIP. All of these peptides stimulate GH secretion in various physiologic systems, but with lower potency than GHRH. GHRH binds to a specific receptor on the somatotroph membrane, resulting in increased intracellular 3’, 5’cAMP levels [59]. The GHRH receptor encodes a 47kDa protein of 423 amino acids [60]. GHRH has a selective action on GH synthesis as well as secretion, and stimulates GH gene transcription. GHRH stimulates GH release from both stored and newly synthesized intracellular GH pools. Somatostatin suppresses both basal and GHRH-stimulated GH release, but does not affect GH biosynthesis [61]. GHRH administered to normal adults elicits a prompt increase in serum GH levels, with higher levels occurring in female subjects [62]. Furthermore, GHRH facilitates GH responses to several pharmacological stimuli including levodopa, arginine, clonidine, insulin hypoglycemia, pyridostigmine, and GHRP-6 [63]. GHRH is the principal regulator of pulsatile GH secretion, and age-related decline in GH secretion (somatopause) is likely GHRH-mediated [64].

Somatostatin (SRIF), a cyclic tetradecapeptide, includes quantitatively predominant, but less bioactive SRIF-14, and more bioactive SRIF-28 [67]. The SRIF precursor is a 116-amino-acid prohormone consisting of a 24-amino-acid signal peptide, a 64-amino-acid connecting region, followed by SRIF-28 [68] which incorporates SRIF-14. Prosomatostatin is synthesized in the anterior hypothalamic periventricular nuclei, and is transported by axonal flow to nerve terminals terminating at the hypophyseal portal vessels. SRIF is also expressed in pancreatic islets, gastrointestinal, neural and epithelial cells, and extrahypothalamic central nervous system neurons. SRIF has a short plasma half-life of 2 3 minutes [67] and inhibits GH, ACTH, and TSH release, TRH stimulation of TSH but not PRL, and pancreatic secretion of insulin and glucagon [69]. SRIF-28 binds to pituitary receptors with a threefold greater affinity than SRIF-14. Both SRIF-14 and SRIF-28 block GHRH effects on GH, as well as secretory responses to insulin-induced hypoglycemia, exercise, arginine, morphine, levodopa, and sleep-related GH release. Somatostatin exerts biologic effects through specific membrane-bound high-affinity receptors. Five somatostatin receptor (SSTR) subtypes, termed SSTR1 5 [70] are coupled to guanine nucleotide protein (G), and comprise seven-transmembrane domains. There is 42% to 60% amino acid homology among the five SSTR subtypes. SSTRs mediate responses via cellular effectors including adenylyl cyclase, protein phosphatases, Na1-H1 exchanger, cyclic GMP-dependent protein kinases, phospholipase C, potassium and calcium channels [70]. The human pituitary gland expresses predominantly SSTR1, 2, and 5 [71], whereas human pituitary adenomas express SSTR1, 2, 3, and 5 [71 73]. Somatostatin analogues, used to control GH hypersecretion in acromegaly, bind with varying affinity to SSTR2 and SSTR5 [74,75]. SRIF receptors may also signal constitutively in the absence of ligand, to regulate basal pituitary hormone release [70].

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FIGURE 4.3 Central and peripheral components that regulate the GH axis. NPY, neuropeptide Y; FFA, free fatty acids; GH, growth hormone; IGF1, insulin-like growth factor 1; GHRH, GHreleasing hormone; SRIF, somatotrophin release inhibitory factor. Source: From Murray PG, Higham CE, Clayton PE. 60 YEARS OF NEUROENDOCRINOLOGY: the hypothalamo-GH axis: the past 60 years. J Endocrinol 2015;226(2):T123 40. [78].

GHRH and SRIF Interaction in Regulating GH Secretion SRIF and GHRH secreted in independent pulses from the hypothalamus interact to generate pulsatile GH release. SRIF inhibits GH secretion, while GHRH stimulates GH synthesis and secretion. GH secretion is further regulated by its target growth factor, IGF-1, which participates in a hypothalamic pituitary peripheral regulatory feedback system [76,77] (Fig. 4.3). GH stimulates the liver and other peripheral tissues to produce IGF-1, which exerts a feedback effect on the hypothalamus and pituitary. IGF-1 also induces hypothalamic SRIF release. Specific antibodies directed against GHRH or SRIF have been used to dissect the respective contributions of these two peptides in the generation of GH pulsatility in rats. Anti-SRIF

administration results in elevated baseline GH levels, with intact intervening GH pulses [79]. These studies imply that hypothalamic SRIF secretion generates GH troughs. Anti-GHRH antibodies eliminate spontaneous GH surges and GH pulsatility persists when GHRH is tonically elevated due to ectopic GHRH production by a tumor or during GHRH infusion [80], suggesting that hypothalamine SRIF is also largely required for GH pulsatility. The rat hypothalamus releases GHRH and SRIF 180 out of phase every 3 4 hours, resulting in pulsatile GH levels [79]. GHRH and SRIF also act synergistically, in that pre-exposure to SRIF enhances subsequent somatotroph sensitivity to GHRH stimulation [81]. Hence, during a GH trough period, high SRIF levels likely prime the somatotroph to respond maximally to subsequent GHRH pulses, thus optimizing GH release. In addition, SRIF exerts a central

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inhibitory effect on GHRH release via direct synaptic connections between SRIF-containing axons and GHRH-containing perikarya in the arcuate nucleus.

TABLE 4.1 Adult GH Secretion

GH Autoregulation

24-h secretion (μg/24 h)

Chronic GHRH stimulation downregulates GH release in humans [82] due to somatotroph desensitization. However, loss of GH sensitivity to administered GHRH does not occur in acromegaly [83] or in somatotroph adenomas in vitro, possibly reflecting larger intracellular pools of GH or abnormal signaling. GHRH pretreatment in vitro also leads to a 50% decrease in somatotroph GHRH-binding sites [84]. Feedback loops exist between GH and IGF-1 and the release of SRIF and GHRH (Fig. 4.3). GH stimulates hypothalamic SRIF release in vitro [85], and in vivo, GH administration decreases GH responses to GHRH [86], most likely by increasing hypothalamic SRIF release [87]. GHRH and SRIF also autoregulate their own secretion. GHRH inhibits its own secretion but stimulates SRIF secretion in vitro, while SRIF inhibits its own secretion in vitro [88].

Physiologic Factors Affecting GH Secretion GH secretion from the anterior pituitary gland is pulsatile, and almost undetectable basal levels between bursts [89]. The number of GH pulses detected depends on the frequency of blood sampling. Integrated GH levels are higher in women than in men, and are also enhanced in postmenopausal women following estrogen replacement [89]. In healthy individuals, discrete pulses account for the majority (.85%) of GH release over the 24-hour period [89]. Pathophysiological regulation of GH output is achieved by altering GH secretory-burst size rather than by modulating pulse frequency [53,90]. Burst size is increased by GHRH, decreased by somatostatin, and synergistically augmented in vivo by combined stimulation with GHRH and GHreleasing peptide (GHRP, also known as GH secretagogue), including ghrelin [36,91,92]. GHRH, SRIF, and ghrelin regulate GH secretion, with contribution from multiple secondary regulators, including gender, gonadal sex steroids, visceral fat, pregnancy, puberty, aging, exercise, sleep, amino and fatty acids, glucose, fasting, insulin, IGF-1, and GH feedback [53,90,93] (Table 4.1). Thus, acute hour-by-hour regulators, such as GH pulse-stimulating effects of exercise and GH pulsesuppressing effects of glucose, are superimposed on individual day-to-day baseline GH secretion determined by age, degree of adiposity, nutritional status, physical fitness, and insulin and sex-steroid levels [95].

Interval

Young adult 540 6 44

Obesity

Middle age

2171 6 333 77 6 20

196 6 65

Fasting

Secretory bursts (number in 24 h)

12 6 1

32 6 2

3 6 0.5

10 6 1

GH burst (μg)

45 6 4

64 6 9

24 6 5

10 6 6

From Melmed [94].

Aging Circulating GH levels decrease significantly with aging, and are reduced by 15% to 70% in men and in women older than 60 years [96], with 24-hour integrated GH concentrations in elderly individuals comparable to those observed in young GH-deficient patients. Aging is associated with decreasing GH responses to most single secretagogues, with the exception of insulin-induced hypoglycemia [53], and is likely facilitated by excessive somatostatin release, diminished GHRH secretion, ghrelin deficiency, and/or a relative failure of feedback inhibition of pulsatile GH secretion independent of IGF-1 concentrations [53]. Gender Women have higher mean GH levels throughout the day due to higher incremental and maximal GH peak amplitudes, but show no significant difference in GH half-life, interpulse times, or pulse frequency [97], and manifest less orderly patterns of pulsatile GH release. Higher basal GH levels may underlie higher nadir GH levels seen in normal women after GH suppression with oral glucose [98]. Sexual differences in expression of mouse pituitary somatostatin and SSTR subtypes likely cause differences in the physiological regulation of GH release [99]. Sleep Sleep stimulates GH secretion. Approximately 60 70% of daily GH secretion occurs during early sleep, in association with slow-wave sleep [100], with a major GH secretory pulse occurring shortly after the onset of sleep and coinciding with the first episode of slow-wave sleep. Rapid-eye movement sleep is reduced by approximately 50% after age 50, with significant sleep fragmentation. Increased GHRH, decreased somatostatin, and increased ghrelin levels may mediate nocturnal GH surges, but the mechanism is unknown [101]. “Jet lag” transiently increases the height of GH peaks during the day and night, resulting in a transient increase of 24-hour GH secretion. Jet

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lag also shifts the major GH secretory spike from early to late sleep [102]. Exercise Exercise is a potent inducer of GH secretion and increases GH secretion, probably mediated by cholinergic mechanisms [103]. Intensity and duration of exercise, fitness, gender, and age all influence GH response to exercise [104]. GH excursions during exercise are influenced mainly by muscle exercise power (voluntary work/unit time), estradiol, and insulin concentrations [95]. Stress GH release is stimulated by physical stress, including trauma with hypovolemic shock and sepsis. However, chronic debilitating diseases, including cancer, are not associated with increased GH levels. Increased GHRH release, mediated by adrenergic pathways, is thought to mediate stress-induced GH secretion. Emotional deprivation is associated with suppressed GH secretion, and subnormal GH responses to provocative stimuli have been described in endogenous depression. Nutritional and Metabolic Regulation Nutritional and metabolic factors profoundly influence GH secretion. Chronic malnutrition and voluntary 5-day fasting [105] are associated with elevated GH levels, most likely as a result of direct somatotroph stimulation by decreasing IGF-1 levels relieving negative-feedback inhibition [106]. Both pulse frequency and amplitude of GH secretory peaks increase with fasting. In contrast, obesity decreases both basal and stimulated GH secretion, and the degree of GH attenuation correlates with the amount of total and visceral body fat [107]. Obese subjects demonstrate decreased somatotroph response to GHRH and GH [92], suggesting increased SRIF activity or a direct pituitary suppressive effect of free fatty acids (FFAs). Insulin-induced hypoglycemia stimulates GH release 30 45 minutes after the glucose trough, whereas acute hyperglycemia inhibits GH secretion for 1 3 hours [108], followed by a GH increase 3 5 hours after oral glucose administration. Insulin-induced hypoglycemia forms the basis of the insulin tolerance test (ITT), which is a gold-standard GH provocative test. Diabetic patients with chronic hyperglycemia, however, do not have suppressed GH levels, and many poorly controlled diabetic patients have increased basal and exercise-induced GH levels. Central nervous system glucoreceptors appear to sense fluctuations, rather than absolute glucose levels. Glucose homeostasis is thus not the major determinant of GH secretion, but is overridden by effects of sleep, exercise, stress, and random GH bursts.

A high-protein meal and single amino acids (including arginine and leucine) administered intravenously stimulate GH secretion. Arginine may suppress endogenous somatostatin secretion and thereby stimulate GH secretion [109]. Decreased serum FFA levels cause acute GH release and increased serum FFA blunts the effects of various stimuli on GHRH-stimulated GH release, including arginine infusion, sleep, levodopa, and exercise [110]. In acromegaly patients, dexamethasone suppresses GH secretion [111], and supraphysiologic serum glucocorticoid concentrations retard growth. In Cushing disease, due to an ACTH-secreting adenoma, growth retardation, decreased serum GH [112], and decreased pituitary GH content in tissue surrounding the adenoma are seen [113]. In normal subjects, glucocorticoid administration suppresses GHRH-induced GH release and produces a dose-dependent inhibition of GHRH-stimulated GH secretion, similar to that seen in Cushing’s syndrome, but acute administration induces GH levels [114]. Thus glucocorticoids exhibit short-term stimulatory effects and delayed inhibitory effects on GH secretion. GH-Binding Proteins Circulating GH is attached to two specific GHbinding proteins (GHBPs), one of high affinity and one of low affinity. The 60-kDa high-affinity BP corresponds to the extracellular domain of the hepatic GHR, produced by proteolytic cleavage with receptor ectodomain shedding. Under basal conditions, half of the circulating 22-kDa GH is bound to the high-affinity BP when GH levels are up to 10 15 ng/mL [115], while 20-kDa GH binds preferentially to the low-affinity BP. Binding to plasma GHBP prolongs GH plasma half-life by decreasing GH metabolic clearance rate [116]. The high-affinity BP also inhibits GH binding to surface receptors by competing for the ligand. Thus, GHBP may serve to dampen acute oscillations in serum GH levels caused by pulsatile pituitary GH secretion. High-affinity BP levels are low in the fetus and neonate, rise most rapidly in the first 1 2 years after birth, and are constant throughout adult life, with similar levels found in males and females. Circulating GHBP levels correlate with fat mass, as well as with circulating leptin levels. GHBP levels increase gradually during pregnancy, and peak in the second trimester, declining to normal levels before term. Placental GH levels correlate inversely with serum GHBP levels [117]. GH resistance, demonstrated in Laron dwarfism [118] and in African pygmies, is characterized by decreased plasma levels of high-affinity BP. Other syndromes of growth retardation with low GHBP levels include the “Pygmies” of the Democratic Republic of Congo and “Little Women” of Loja.

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In adult patients with GH deficiency and changes in body composition and increased fat mass, GHBP levels are either normal or increased. GH replacement in adult GHD patients is not associated with changes in GHBP levels. In acromegaly, measuring GHBP levels offers no diagnostic utility.

Peripheral GH Action GH Receptor GH binds to its peripheral receptor and induces intracellular signaling by a phosphorylation cascade involving the Janus Kinase (JAK)/signal transducing activators of transcription (STAT) pathway [19]. GH also acts indirectly by inducing synthesis of IGF-1, the potent growth and differentiation factor. The GHR is a 70-kDA protein member of the class I cytokine/hematopoietin receptor superfamily [119,120]. GHR consists of an extracellular ligand-binding domain, a single membrane-spanning domain, and a cytoplasmic signaling component. The GH ligand complexes with a preformed dimer of two GHR components leading to internal receptor rotation critical for subsequent GH signaling [120]. The activated receptor dimer induces separation of JAK2 sites, and GHR rotation is followed by rapid activation of JAK2 tyrosine kinase, leading to phosphorylation of cytoplasmic signaling molecules, including the GHR itself, and STAT proteins, critical signaling components for GH action [121]. Phosphorylated cytoplasmic proteins are translocated to the cell nucleus where they elicit GH-specific target gene expression by binding to nuclear DNA [122]. STAT1 and STAT5 may also interact directly with the GHR molecule [123]. Other target actions induced by GH include c-fos induction, IRS-1 phosphorylation, and insulin synthesis, cell proliferation, and cytoskeletal changes. As a differentiating and growth factor, IGF-1 is a critical protein induced by GH, and likely responsible for most of the growth-promoting activities of GH [124]. GH-activated STAT5B directly induces IGF-1 transcription [125]. Thus, STAT5B mediates GHinduced somatic growth [126]. IGF-1 itself may also directly regulate GH in a negative feedback loop [124] and GHR trafficking [127]. The liver contains abundant GHRs, and several peripheral tissues, including muscle and fat, also express modest amounts of receptor. STAT5B is required for GH-mediated postnatal growth, adipocyte functions, and sexual dimorphism of GH hepatic actions [19]. Transgenic mice with inactivated STAT5B exhibit impaired growth, with low IGF-1 levels, and are insensitive to injected GH [128]. GHR mutations are associated with partial or complete GH insensitivity and growth failure. These syndromes are associated with normal or high circulating

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GH levels, decreased circulating GHBP levels and low levels of circulating IGF-1. Multiple homozygous or heterozygous exonic and intronic GHR mutations have been described, most of which occur in the extracellular receptor ligand-binding domain. Tissue responses to GH signaling may be determined by the pattern of GH secretion, rather than the absolute amount of circulating hormone. Thus, linear growth patterns, liver enzyme induction, and STAT5B activity may be phenotypically distinct for male animals due to higher rates of GH pulse frequency [129]. STAT5B is sensitive to repeated pulses of injected GH [130], unlike other GH-induced patterns which are desensitized by repeated GH pulsing. Mice harboring a disrupted STAT5B transgene exhibit impaired male pattern body growth [128] with IGF-1 and testosterone levels normally seen in female mice. Thus, sexual dimorphic patterns of GH secretion and GH tissue targeting appear to be determined by STAT5B, as does the requirement for appropriate GH pulsatility to determine body growth [131]. In humans, STAT mutations result in short stature and relative GH insensitivity [132]. Intracellular GH signaling is also abrogated by suppressor of cytokine signaling proteins, which disrupt the JAK/STAT pathway and thus exert a further level of control over the action of GH [133] (Fig. 4.4). Insulin-Like Growth Factors (IGFs) IGF-1 and IGF-2 are single-chain polypeptide molecules with three intrachain sulfide bridges (Fig. 4.5). IGF-1, composed of 70 amino acids, and IGF-2, consisting of 67 amino acids, have a sequence homology of 62%. The IGFs consist of B and A peptide domains (structurally homologous with the insulin B and A chains), a C domain analogous to the connecting (C) peptide of proinsulin, and a D domain. IGF-1 and IGF-2 are single distinct gene loci, localized on chromosome 12 (12q22-q24.1) and chromosome 11 (11p15), respectively. The IGF-1 gene primary transcript can be alternately spliced to different products resulting in IGF-1a (exons 1, 2, 3, 5) or IGF-1b (exons 1, 2, 3, 4). Several IGF-1 mRNA species have been isolated from adult and fetal tissues, and the liver is the main source of circulating IGF-1 levels. The IGF-1 gene is expressed in human fetal connective tissues and cells of mesenchymal origin [135]. This ubiquitous localization of IGFs favors a paracrine/ autocrine function as well as an endocrine function of IGF-1. GH is the major regulator of IGF-1 gene expression in adult liver, heart, lung, and pancreas [136] and acts at the level of IGF-1 transcription. Fetal IGF-1 production is GH-independent, and platelet-derived growth factor and fibroblast growth factor also

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FIGURE 4.4 GH action. GH binds to the GHR dimer, which undergoes internal rotation, resulting in JAK2 phosphorylation (P) and subsequent signal transduction. GH signaling is mediated by JAK2 phosphorylation of depicted signaling molecules or by JAK2-independent signaling including Src/ERK pathways (S42). Ligand binding to a preformed GHR dimer results in internal rotation and subsequent phosphorylation cascades. GH targets include IGF-1, c-fos, cell proliferation genes, glucose metabolism, and cytoskeletal proteins. GHR internalization and translocation (dotted lines) induce nuclear proproliferation genes via importin α/β (Impα/Impβ) coactivator (CoAA) signaling. IGF-1 may also block GHR internalization, acting in a feedback loop. The GHR antagonist, pegvisomant, blocks GHR signaling; SRLs also attenuate GH binding and signaling (not shown). Source: From Melmed S. Acromegaly pathogenesis and treatment. J Clin Invest 2009;119 (11):3189 202.

FIGURE 4.5 Amino acid sequence of human IGF-1. The black amino acids are identical to those in human insulin. The numbering corresponds to the numbering of residues in the proinsulin molecule. IGF-1 consists of a 70-amino-acid single-peptide chain with A, B, C, and D domains. A and B domains are structurally homologous to the A and B chains in the insulin molecule, and the C domain is equivalent to the connecting (C) peptide in proinsulin. Source: From Humbel [134].

stimulate IGF-1 production from human fibroblasts in vitro [137]. ACTH, TSH, LH, and FSH stimulate paracrine production of IGF-1 in their respective target tissues. In addition to GH, nutritional status is an

important regulator of IGF-1 production at all ages [138]. IGFs are found in lymph, breast milk, saliva, and amniotic fluid. IGF-1 levels are low before birth, rise during childhood to high levels during puberty, and decline with age [139]. Multiple cellular actions of IGF-1 are mediated via the IGF-1 receptor, a transmembrane tyrosine kinase cell surface receptor, with high homology to the insulin receptor. IGFs are expressed widely throughout most tissues in the body, are not stored in cellular secretory granules, and are secreted associated with high-affinity circulating IGF-binding proteins (IGFBPs). IGFs play an important role in regulating somatic growth and ensure that growth and development proceed appropriate to nutritional supply. IGF-Binding Proteins IGF-1 and IGF-2 are bound to carrier proteins in the serum. IGF-1 and IGF-2 are complexed to six specific binding proteins in biological fluids [140] (Table 4.2). These proteins are regulated by signals derived from nutritional status, as well as by hormone action [142]. IGFBPs are cysteine-rich proteins, with similar amino acid sequences, and a unique ability to bind IGFs with high affinity (Fig. 4.6). Actions of the IGFBPs include

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TABLE 4.2 Human IGFBPs No. of amino acids

Core molecular mass (kDa)

Chromosomal localization

IGF Modulation of affinity IGF action

IGFBP-I

234

25.3

7

I 5 II

Inhibition and/ Amniotic fluid, serum, placenta, endometrium, milk, urine, or potentiation synovial fluid, interstitial fluid and seminal fluid

IGFBP-II

289

31.4

2

II . I

Inhibition

IGFBP-III 264

28.7

7

I 5 II

Inhibition and/ Serum, follicular fluid, milk, urine, CSF, amniotic fluid, or potentiation synovial fluid, interstitial fluid, and seminal fluid

IGFBP-IV 237

25.9

17

I 5 II

Inhibition

Serum follicular fluid, seminal fluid, interstitial fluid, and synovial fluid

IGFBP-V

252

28.5

5

II . I

Potention

Serum and CSF

IGFBP-VI 216

22.8

12

II . I

Inhibition

CSF, serum, and amniotic fluid

Source in biological fluids

CSF, serum, milk, urine, synovial fluid, interstitial fluid, lymph follicular fluid, seminal fluid, and amniotic fluid

From Rajaram [141].

FIGURE 4.6 Structural features of IGFBPs. N- and C-terminal sequences of six members of the IGFBP family contain regions important for IGF binding, and form a high-affinity binding site. The mid-region of each protein, the linker region, diverges in sequence among the family members, allowing structural modifications enabled by glycosylation, proteolytic cleavage, and phosphorylation. Unique regions are also important for nuclear localization and cellsurface protein interaction. Source: From Clemmons DR. Role of IGF binding proteins in regulating metabolism. Trends Endocrinol Metab 2016;27(6):375 91.

modulation of IGF action and storage of IGFs in extracellular matrices. IGFBP-3, the most common form of binding protein in human circulation, associates the IGF molecule with an 80-kDa acid-labile subunit (ALS) to form a 150 200-kDa complex [140]. Approximately 75% of IGF-1 and IGF-2 circulates in this IGF IGFBP ALS ternary complex, which is stabilized by IGF binding [143]. Complexed IGFs do not readily leave the vascular compartment, and have prolonged half-lives [144] compared to the half-life of free IGF-1, which is less than 10 minutes [145]. A circulating protease acting specifically on IGFBP-3 results in limited cleavage of IGFBP-3, with subsequent decreased binding affinity of IGF-1. There is little detectable IGFBP-3 protease activity in normal serum due to the presence of inhibitors that protect IGFBP-3 from proteolysis. A pregnancy-associated plasma protein A system cleaves IGFBP-4 [146]. Plasma concentrations of IGFBPs are hormonally regulated. IGFBP-I levels are high at birth and decline until puberty [147], and diurnal variation with a

nocturnal peak in serum IGFBP-1 levels occurs [148]. Serum IGFBP-3 levels correlate with IGF-1 and -2 levels, increase in patients with acromegaly, and are lower in hypopituitarism [149]. In contrast, IGFBP-1 levels are elevated in hypopituitarism, [150] decreased in acromegaly, and increased in acromegaly patients receiving octreotide [151]. Malnutrition, insulin-dependent diabetes mellitus, and cirrhosis are associated with suppressed IGFBP-3 levels [152]. IGFBP-1 levels are regulated by insulin. Increased IGFBP-1 levels associated with insulin-dependent diabetes mellitus [140] are normalized by insulin, insulinoma is associated with suppressed IGFBP-1 levels, and the fall in IGFBP-1 levels after glucose ingestion in normal subjects correlates inversely with rising insulin levels [153]. Insulin also increases IGFBP-2 levels [152] and hypophysectomy is associated with elevated rat IGFBP-2 levels, which fall with GH administration [152]. Overall, IGFBPs serve to determine the availability of free IGF-1, which ultimately binds to the IGF receptor.

GH ACTION GH regulates several biologic functions, including intermediary metabolism and homeostasis, and plays a pivotal role in normal postnatal growth and development. Most GH actions are mediated by IGF-1 induced by GH in target tissues, mainly in the liver [154], but also has direct actions [155] (Fig. 4.7). IGF-1 is synthesized independent of GH, under the control of other regulatory factors [156], and may act synergistically with GH, as illustrated by their bone-growthpromoting properties, or antagonistically, as in hepatic glucose metabolism [156].

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FIGURE 4.7

Physiological actions of GH and IGF-1. Actions that involve more direct GH action are depicted on the right, whereas those involving induced IGF-1 are on the left. The GH IGF-1 system is regulated by a negative-feedback loop between hepatic IGF-1 production and pituitary growth hormone secretion. Source: From Brooks AJ, Waters MJ. The growth hormone receptor: mechanism of activation and clinical implications. Nat Rev Endocrinol 2010; 6(9):515 25.

Cell GH actions are mediated by the dimeric GHR, a member of the cytokine receptor superfamily [157], expressed on the target cell plasma membrane [158]. After GH binding to the GHR, intracellular signaling pathways are activated, including JAK2/STAT5 and ERK1/2 [159]. Similarly, IGF-1 acts through the IGF-1 receptor (IGF-1R), a tyrosine kinase receptor, and binding of IGF-1 activates canonical intracellular signaling cascades, including ERK1/2 and PI3K/AKT.

Role of GH/IGF-1 in Growth and Development Throughout the Lifespan Longitudinal bone growth is initiated in the epiphyseal growth plate of long bones, mediated by endochondral ossification [160]. When growth occurs, progenitor chondrocytes in the resting zone of long bone epiphyseal growth plates proliferate and replicate rapidly as clonal populations are arranged in columns. Subsequent differentiation of hypertrophic chondrocytes occurs, and extracellular matrix is secreted, resulting in new cartilage formation (chondrogenesis), leading to bone formation. Chondrocytes in the skeletal growth plate express GHRs, which are downregulated by local and systemic IGF-1 and upregulated when IGF-1 binds to IGFBPs [161]. In the presence of GH, mesenchymal precursors favor chondrogenesis and osteoblastogenesis over adipogenesis [162]. IGF-1 regulation of chondrocyte differentiation in IGF-1 null mutants is abrogated as evidenced by impaired chondrocyte maturation and shortened femoral length [163]. Genetic, hormonal, and nutritional factors influence invasion of newly formed cartilage by blood vessels and bone cell precursors, which facilitates calcification into bone trabeculae, i.e., endochondral ossification [164]. As an adjunct to longitudinal bone growth, bone tissue also undergoes remodeling and modeling. Bone

TABLE 4.3 Effects of GH on Bone Function

Effects

GROWTH PLATE Chondrocyte replication

mm

Endochondral bone formation

mm

BONE REMODELING UNIT Osteoblastogenesis

m

Osteoblast proliferation

m

Function of mature osteoblasts

2m

Osteoprotegerin production

m

RANK-L production

2

Phosphate retention

m

2, no effect; m, minor stimulating effect; mm, major stimulating effect. From Giustina [169].

modeling occurs mostly during growth, whereas bone remodeling is a process of coordinated bone resorption and formation occurring in multicellular units throughout the lifespan [165]. During remodeling, multinucleated osteoclasts are attracted to specific sites to resorb bone, and osteoblasts are attracted to fill the cavity with newly synthesized matrix. GH stimulates proliferation of osteoblast cells and IGF-1 is required for selected anabolic effects of GH in osteoblasts [166]. GH also stimulates expression of bone morphogenetic proteins, which are important for osteoblast differentiation and bone formation [167]. In addition, GH stimulates mature osteoblast function, either directly or indirectly through IGF-1, and also stimulates carboxylation of osteocalcin, a marker of osteoblastic function [168] (Table 4.3, Fig. 4.8).

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FIGURE 4.8 Effects of GH and IGF-1 on bone growth. Skeletal effects of GH and IGF-1 are modulated by interactions between circulating and locally produced IGF-1 and IGFBPs. IGF-1 and IGF-2 are most abundant in skeletal tissue and are regulated by GH and PTH. GH may also exert direct effects on skeletal cells and induce IGF-1 action in bone. FGF, fibroblast growth factor; E2, estradiol; OPG, osteoprotegerin. Dotted line, no consistently demonstrated effect; thin solid line, minor stimulating effect; thick solid line, major stimulating effect. Source: From Giustina A, Mazziotti G, Canalis E. Growth hormone, insulin-like growth factors, and the skeleton. Endocr Rev 2008;29(5):535 59.

Low serum IGF-1 levels in GHR-mutated mice result in small growth plates, osteopenia, and reduced cortical bone with normal trabecular bone [170], suggesting a more pronounced effect of systemic IGF-1 on cortical bone than on trabecular bone. In contrast, mice with osteoblast-specific knockout of the IGF receptor gene exhibit decreased osteoblast number and function, causing reduced bone formation and trabecular volume [171], indicating a more significant role for skeletal IGF-1 in maintenance of trabecular bone. GH and IGF-1 influence bone metabolism throughout the lifespan [169]. During embryonic development, IGF-1 and IGF-2 are key determinants of bone growth, acting independent of GH, such that GH deficiency or insensitivity caused by GHR mutations or defects in GH signaling pathways, markedly impairs postnatal, but not prenatal growth [172]. Thus, GH plays a minor role in determining fetal growth. However, postnatally and during puberty, both GH and IGF-1 are critical in determining longitudinal skeletal growth [173] as well as skeletal maturation and acquisition of bone mass in the prepubertal period. Children with GH deficiency

manifest short stature, while GH excess in childhood causes gigantism. In contrast, an IGF-1 gene mutation causing IGF-1 deficiency [174] and IGF-1 resistance due to IGF receptor gene mutations [175] are associated with both prenatal and postnatal growth deficits. Anabolic effects of systemic and local skeletal GH and IGF-1 are important in the acquisition of bone mass and maintenance of skeletal architecture, particularly in the late adolescence and adulthood stages that are critical for peak bone mass achievement [169].

Bone Acquisition GH facilitates longitudinal bone growth and attainment of peak BMD during postnatal and pubertal growth, but is not required for intrauterine growth [154]. Childhood-onset GH deficiency is associated with increased fracture risk in adulthood and decreased BMD and is reversed by GH treatment [176]. Many of these GH effects are age-dependent; GH deficiency leads to a fourfold greater reduction in BMD

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during postpubertal growth than during prepubertal growth [177], possibly due to lack of GH activation of hepatic gene transcription prior to puberty [178]. IGF-1 mediates several GH effects on bone, as evidenced by GH insensitivity syndromes. Patients with Laron syndrome caused by GHR mutations, manifest with low/absent IGF-1 or IGFBP-3, decreased longitudinal bone growth; short body length at birth, and short stature [179], all of which are associated with reduced cortical bone parameters and endosteal and periosteal bone formation. In GH-insensitive patients, treatment with IGF-1 improves longitudinal bone growth, although it cannot fully restore all absent GH effects [179 181]. GH and IGF osteoblast activity are synergistic, and contribute to the stimulation of growth increase [182]. IGF-1 is required for growth throughout all stages of development [183], facilitating bone formation rates, and contributing to adequate BMD and femoral length via a combination of GH-dependent and GH-independent mechanisms [177,184]. IGF-1 effects in regulating bone formation vary in cortical versus trabecular bone and in appendicular versus axial skeletal regions, but both its independent effects, as well as its synergistic effects with GH, are essential for attainment of peak bone mass. IGF1 deficiency is associated with decreased BMD and growth retardation. GH thus acts independently as well as in synergy with IGF-1 to attain peak bone mass.

Bone Loss GH also increases bone resorption and turnover [185], independent of IGF-1, and patients with excess GH show an increased fracture risk [186]. Furthermore, the GH/IGF system may contribute to bone loss due to decline of GH secretion with increasing age, which contributes to age-associated bone loss. Serum IGF-1 and IGFBP levels [187] also decrease with age, and are associated with reduced stimulation of osteoprogenitor cells by both circulating serum and local bone IGF-1 [188]. However, the extent to which these age-related declines in the GH/IGF system contribute to age-related bone loss are unclear. Adults with GHD manifest low bone turnover, osteoporosis, and increased fracture risk, with decreased osteoid and mineralizing surfaces and a reduced rate of bone formation. Decreased osteocalcin and bone resorption markers reflect low bone turnover. Cortical loss is greater than trabecular bone loss [189], and bone loss is proportional to age of onset of GHD and duration and severity of the disease. Childhood-onset GHD is associated with more severely reduced vertebral bone mineral density than adult-onset patients, possibly due to failure to attain peak bone mass [190]. Nonvertebral fracture risk is increased threefold in

TABLE 4.4 Metabolic Effects of Growth Hormone EFFECTS ON CARBOHYDRATE METABOLISM Antagonism of insulin action EFFECTS ON LIPID METABOLISM Adipose tissue: increase lipolysis-increase free fatty acids Muscle/liver: increase lipoprotein lipase expression-triglyceride uptake EFFECTS ON PROTEIN METABOLISM Increase protein synthesis

untreated GHD patients, with fractures frequently localized to the radius [191].

GH AND METABOLISM GH continues to be secreted in adulthood after growth cessation, implying important metabolic functions in adult life. Metabolic actions may be acute and insulin-like or chronically antagonistic to insulin action, and may be direct or indirectly mediated by IGF-1. GH effects on carbohydrate metabolism are dominantly anti-insulin, with a net anabolic effect on protein metabolism (Table 4.4). GH-deficient children are mildly obese, with a decreased number of larger fat cells that have increased lipid content. GH replacement therapy leads to decreased body fat and, eventually, decreased size and lipid content of subcutaneous adipocytes. GHdeficient adults have altered body composition, with increased fat mass and decreased lean body mass (LBM). Initial acute effects of GH on lipid metabolism are antilipolytic (insulin-like) and subsequently, GH exerts a chronic lipolytic (anti-insulin) effect.

Lipids GH predominately stimulates adipocyte lipolysis, with increased circulating FFAs, and increased muscle and liver lipoprotein lipase expression with enhanced triglyceride uptake. GH increases lipolysis largely in visceral adipose tissue, and somewhat in subcutaneous adipose tissue, with release of circulatory FFAs [192]. GH activates hormone-sensitive lipase via enhanced agonist-induced stimulation of β-adrenergic receptors [193], resulting in increased hydrolysis of triglycerides to FFAs and glycerol. GH also facilitates differentiation of small preadipocytes into large, mature adipocytes, with increased capacity to store triglycerides and a higher lipolytic potential. Activation of STAT5 and possibly subsequent association with PPAR-γ may be associated with GH-induced adipogenesis [194].

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In the liver, however, effect of GH is opposite to that observed in adipose tissue. GH induces triglyceride uptake by increasing lipoprotein lipase and/or hepatic lipase expression and GH treatment increases hepatic triglyceride storage. In skeletal muscle, GH promotes lipid utilization by increasing lipoprotein lipase expression, which stimulates triglyceride uptake and subsequent storage as intramyocellular triglyceride, or stimulates lipid oxidation with energy release. GH-deficient adults have elevated total cholesterol, low-density lipoprotein cholesterol (LDL), triglycerides, and apolipoprotein B (ApoB) [195], with decreased highdensity lipoprotein (HDL). This lipid profile is associated with premature atherosclerosis and cardiovascular disease. GH replacement decreases total cholesterol [196], LDL cholesterol, and ApoB, and also increases HDL levels, although long-term surveillance is required to determine whether GH replacement therapy reverses premature atherosclerosis and reduces cardiovascular morbidity and mortality in GH-deficient adults. Lowdose GH replacement decreases total and visceral adipose tissue and reduces elevated levels of inflammatory markers, including highly sensitive c-reactive protein and interleukin-6 (IL-6) in women with hypopituitarism, with a relatively modest increase in IGF-1 levels and without worsening insulin resistance [197].

Body Composition Anabolic, lipolytic, and antinatriuretic GH actions impact body composition, affecting fat mass, LBM, and fluid volume in GH-deficient adults. LBM is reduced, and fat mass is increased in GH-deficient adults compared to predicted values for age-, sex-, and heightmatched normal controls. With GH deficiency, excess fat accumulates mostly in the visceral compartment in a central, mainly abdominal distribution and total body water is reduced. GH replacement therapy reverses these effects on body composition by increasing LBM. GH replacement also reduces fat mass by 4 6 kg in GH-deficient adults, with the most significant reduction in visceral fat. GH therapy increases total body water, especially extracellular water, within 3 5 days. Total blood volume increases after 3 months of treatment. GH and IGF-1 stimulate sodium reabsorption via epithelial sodium channels in the rat distal nephron [198], contributing to the antinatriuretic action of GH.

Carbohydrate Metabolism GH decreases glucose uptake in adipose tissue, and regulates glucose transporter-I in adipose tissuederived cell lines [199]. GH may antagonize adipocyte

insulin and lower serum leptin levels, but effects on adiponectin are unclear. In the liver, GH increases glycogenolysis, thereby increasing hepatic glucose production, possibly as a result of insulin antagonism. GH-deficient children have decreased fasting glucose levels [200], decreased insulin secretion [200], contradictory impairment of glucose tolerance [201], and increased insulin sensitivity due to increased glucose utilization and blunted hepatic glucose release. GH replacement increases fasting glucose levels [201], insulin levels [201], and hepatic glucose production. Endogenous GH secretion antagonizes insulin action. GH secretion increases 3 5 hours after oral glucose ingestion, and hyperinsulinemia occurs 2 hours after GH levels peak. Both intravenous and oral glucose tolerance tests are impaired if performed during periods of increased GH secretion, such as sleep onset. GH-deficient adults have elevated fasting insulin levels that correlate with fat mass and waist girth, suggesting the presence of insulin resistance. GH replacement increases insulin resistance in the first 1 6 weeks of therapy, but studies suggest unchanged insulin sensitivity over the long term [202].

Protein Metabolism Both insulin and IGF-1 have been implicated in the anabolic effects of GH on protein metabolism. GH causes urinary nitrogen retention and decreases plasma urea levels when administered to both normal and GH-deficient children.

Muscle Strength and Exercise Performance GH deficiency is associated with reduced muscle strength, due to altered body composition. Reduction in muscle cross-sectional area, as well as lack of conditioning and training, may contribute to weakness. Prolonged GH replacement therapy may increase muscle mass, but it may not result in improved strength.

GH AND REPRODUCTION The male and female reproductive systems are targets of GH action and also sites of GH synthesis, suggesting both autocrine and paracrine actions of GH within the reproductive system. The somatotrophic and gonadotrophic axes interact to signal the onset of puberty, sexual maturation, and accelerated pubertal growth. The GnRH pulse generator initiates puberty, while growth and nutritional factors influence timing and pace of puberty [203].

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In males, GH is expressed by the testis and accessory organs, and autocrine and paracrine actions promote seminiferous tubule differentiation and normal testicular growth [204], possibly mediated by IGF-1. GH stimulates androgen and/or estradiol production by Leydig cells in vitro [205] and promotes gametogenesis. Fertile GH-deficient males [206] receiving chronic GH therapy exhibit improved chorionic gonadotrophininduced testosterone production. In females, GH modulates steroidogenesis, folliculogenesis, and oocyte maturation, thus optimizing multiple processes that culminate in a viable embryo. Indeed, women with GHD have decreased fertility, and GH replacement improves spontaneous pregnancy rates in previously infertile GHD women [207]. While pituitary gonadotrophins are the prime regulators of ovarian steroidogenesis, GH also modulates progesterone and estradiol release [208] as well as gametogenesis, and may establish optimal conditions for nuclear maturation, perhaps by promoting follicular development [209]. GH is also proliferative and antiapoptotic in the corpus luteum [208]. The multiple actions of GH on the reproductive axis have led to the use of GH as an adjunct to assisted reproductive technology in women who are poor ovarian responders, but optimal use of this strategy remains somewhat unclear [210].

GH/IGF-1 AND CARDIOVASCULAR FUNCTION Data regarding blood pressure and peripheral resistance in untreated GH-deficient patients are unclear. Increased prevalence of hypertension [211], no change in blood pressure [212], and reduced blood pressure have been reported [213] in adult-onset GH-deficient patients. A stimulatory effect of GH on nitric oxide production [214] could explain reduced peripheral vascular resistance and diastolic blood pressure reported in some trials of GH replacement. Alternatively, GH replacement may reduce blood pressure in subgroups of GH-deficient adults with high baseline diastolic blood pressure, such as elderly patients or those with previous Cushing disease [215]. Atherosclerosis may be more prevalent in adults with GHD [212], who manifest increased carotid artery wall thickness, but early carotid artery atherosclerotic changes may be reversible with GH replacement [216]. In acromegaly, advanced cardiomyopathy is characterized by cardiomegaly, ventricular hypertrophy, replacement fibrosis, and cardiomyocyte degeneration [217]. Three stages of cardiovascular disease—early, intermediate, and late—have been identified in acromegaly [218]. Patients with acromegaly for a relatively short

duration display a “hyperkinetic” cardiovascular system with increased cardiac output and decreased total peripheral resistance. In contrast, untreated acromegaly and progression to more advanced stages is commonly associated with hypertension. The prevalence of hypertension is high in acromegaly patients (20 50%), due to expanded plasma volume, stimulation of smooth muscle cell growth leading to increased vascular resistance, and increased insulin resistance as a potential facilitator of increased blood pressure [219]. However, these patients do not have increased prevalence of coronary artery disease, carotid atherosclerosis, or carotid internal media thickness compared to normal subjects [220], possibly due to lower high-sensitive C-reactive protein [221].

Effects of GH and IGF-1 on Cardiac Structure and Function IGF-1 increases cardiomyocyte size [222] and protein synthesis [223]; there may also be a direct effect of GH, independent of IGF-1 [224]. IGF-1 promotes fibroblast collagen synthesis and GH increases cardiac collagen deposition [225], but collagen volume fraction is normal [226]. GH and IGF-1 may also modulate myocardial structure by preventing cardiomyocyte loss through apoptosis. IGF-1 acts as an inhibitor of ongoing apoptosis in the normal heart [227], and the antiapoptotic effect of GH/IGF-1 may confer myocardial protection during ischemic injury [228]. Cultured rat cardiac myocytes express GH and IGF-1 receptors, and IGF-1 induces cultured rat myocytes and delays apoptosis. At the same time, IGF-1 also sensitizes cultured rat myofilaments to calcium, thereby enhancing myocardial contractility. Moreover, locally produced IGF-1 promotes arterial cell growth, and paracrine IGF-1 contributes to inflammatory angiogenesis during atherosclerosis [229]. Untreated GH-deficient adults exhibit reduced left ventricular (LV) mass and cardiac output [230] and decreased exercise capacity [218], with more severe cardiac dysfunction in childhood-onset GH-deficient patients [229]. GH replacement therapy in adult GH-deficient patients has an anabolic effect on cardiac structure, resulting in improved diastolic and systolic function [231]. Increased cardiovascular morbidity and mortality are associated with both GH deficiency and GH excess [232]. Epidemiological studies suggest that lowernormal range IGF-1 levels in the general population may increase the risk of ischemic heart disease [233] and cardiac failure [234]. GH-deficient adults have several cardiovascular risk factors, including increased abdominal adiposity, insulin resistance, hypercoagulability, high total and LDL

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cholesterol, low HDL cholesterol, and decreased exercise performance and pulmonary capacity, all of which are negative cardiovascular risk factors for coronary artery disease [235]. Furthermore, GHD adults manifest less aortic distensibility and endothelial dysfunction, with higher fibrinogen, tissue plasminogen activator antigen and plasminogen activator inhibitor activity, and increased blood vessel intima-medial thickness. LV posterior wall and interventricular septal thickness is reduced, resulting in decreased LV mass index, and decreased LV internal diameter in adolescents with childhood-onset GHD. GH replacement improves peak exercise cardiac performance, and reduces carotid artery intima-medial thickness. GH replacement also has beneficial effects on lean body and fat mass, total and LDL cholesterol levels, and diastolic blood pressure [196], and may reduce the risk of premature cardiovascular mortality [195,236]. GH treatment has a beneficial effect on cardiovascular risk factors, including homocysteine and C-reactive protein [195], and may improve these cardiovascular risk factors and markers in hypopituitarism [237]. Body composition deteriorates in hypopituitary adults with increased body fat and decreased LBM. As extracellular water is decreased, which may result in reduced cardiac preload (Starling effect), decreased sweating, impaired thermoregulation, and increased risk for developing hyperthermia during exercise in hot environments [238] have been observed in GHdeficient adult patients. GH replacement therapy normalizes most cardiovascular risk factors observed in hypopituitary patients [237], and increased mortality in hypopituitary adults not treated with GH replacement is attributed to cardiovascular causes [239]. However, there are few data regarding the effects of long-term GH replacement therapy on cardiovascular morbidity and mortality.

GH Therapy in Congestive Heart Failure A meta-analysis suggests that GH treatment improves LV geometry, ejection fraction, and exercise parameters, and the improvement correlates with an increase in serum IGF-1 levels [240]. Discrepant data on IGF-1 levels in heart failure, suggest that low, normal, or even high IGF-1 levels might be attributable to variability in IGF-1 assays or inclusion of heterogeneous heart failure patients [241,242]. Using a GH provocative test to enroll only GH-deficient patients, heart failure patients treated with GH for 6 months in a randomized, single-blind study showed improved quality of life score, increased peak oxygen uptake, exercise duration, and flow-mediated vasodilation [243], with increased LV ejection fraction and reduced circulating

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N-terminal natriuretic peptide. These results suggest a potential therapeutic role for GH in patients with congestive heart failure.

GH as a Biomarker of CVD As GH levels and CV risk factors and outcomes have been linked [244], GH could be considered a biomarker for CVD [245]. GH and IGF-1 receptors are expressed in the heart and blood vessels, and the GH IGF-1 somatotrophic axis influences cardiac structure, function, and peripheral resistance via effects on vascular tone and central sympathetic outflow [244]. Ghrelin may also influence cardiovascular remodeling [246], thereby linking the pituitary gastric axis to cardiometabolic disease. Acromegaly patients have an increased risk of CVD and increased CVD-related and all-cause mortality [244], suggesting a link between excess GH and CVD and supporting the role of GH level as a CVD biomarker. A large Swedish study [244] found that increased fasting GH levels was associated with increased risk of coronary artery disease, stroke, heart failure, CVD-related mortality, and all-cause mortality, such that each standard deviation (SD) increment in GH levels increased the odds of early fatal myocardial infarction by 54%. Nevertheless, an effect on estimated 10-year CVD-related mortality was modest as assessed by multivariate analysis.

GH EFFECTS ON RENAL FUNCTION GH and IGF-1 regulate renal development, glomerular function, and tubular handling of sodium, calcium, phosphate, and glucose. Renal GHRs localize to epithelial cells in the proximal and distal tubules, mesangial cells, and podocytes in the proximal straight tubule and the medullary thick ascending limb of the loop of Henle, and, in both mice and humans, in the collecting duct and distal nephron [247]. Renal IGF-1 originates from circulating IGF-1, which is mainly synthesized in the liver, and acts in an endocrine manner on target tissues, as well as from IGF-1 synthesized locally in kidney, which acts as an autocrine/paracrine regulatory factor for renal cell metabolism [247]. Higher IGF-1 levels in renal venous blood compared to renal arterial blood also suggest renal IGF-1 biosynthesis [248], although relative growth contributions of circulating and locally produced GHdriven IGF-1 are poorly understood. The GH/IGF-1 axis acts on all the component cells of the glomerulus. GH and especially IGF-1 stimulate mesangial cell proliferation and migration and inhibit

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podocyte function, with increased permeability of the filtration barrier [249]. The GH-IGF-1 system is a significant hormonal modulator of renal tubular sodium and water reabsorption. GH stimulates sodium and water reabsorption in the kidney tubule, with GH and IGF-1 acting together to induce epithelial sodium channel (ENaC)dependent transepithelial sodium transport in the distal nephron [250]. GH and IGF-1 also facilitate increased phosphate requirements during growth, likely via increased renal tubular phosphate retention. GH and IGF-1 effects on renal structure and function are apparent in patients with acromegaly and GH deficiency. The sodium-retaining GH and IGF-1 action in the distal tubule, with enhanced ENaC-dependent sodium transport, is associated with extracellular volume expansion and contributes to soft-tissue swelling and arterial hypertension in acromegaly. Chronic renal exposure to the growth-promoting effects of GH/IGF-1 in acromegaly results in renal hypertrophy [251], increased glomerular filtration, and renal plasma flow [252]. GH enhances glomerular filtration through IGF-1-mediated decreased renal vascular resistance, leading in turn to increased glomerular perfusion [253]. This effect rapidly reverses if acromegaly treatment is undertaken prior to development of structural renal changes.

TESTS OF GH SECRETION Because of the pulsatile nature of pituitary GH secretion, a single random blood sample for GH measurement is not helpful in the diagnosis of GH hypersecretory or deficiency states, or GH neurosecretory disorders. Nonphysiologic provocative or suppression tests, or measurement of spontaneous GH secretion by 24-hour integrated serum GH concentration (IC-GH), are therefore employed to assess GH secretion.

Integrated 24-Hour GH Concentrations Pituitary GH secretion occurs episodically during waking hours, as well as during sleep, necessitating measurement over 24 hours [53] to accurately assess integrated GH secretion. Constant blood collection over a 24-hour period allows determination of a true mean or IC-GH, requiring a nonthrombogenic continuous withdrawal pump or patent indwelling catheter from subjects whose food intake and physical activity are not limited. Sampling intervals of 20 minutes are most widely used, but 5-minute and 30-second sampling frequencies detect significantly more pulses per hour. Samples from collection periods may be pooled,

producing a combined aliquot in which the IC-GH concentration is measured. The 24-hour IC-GH reflects the average GH concentration over a 24-hour period, eliminating peak or trough levels that might otherwise be obtained by single random sampling of GH.

Evaluation of GH Hypersecretion Increased serum IGF-1 levels are a consistent finding in acromegaly [254] and IC-GH levels show a log (dose) response correlation with serum IGF-1 levels [255]. The currently accepted diagnostic test of GH hypersecretion is failure of GH levels to be suppressed to less than 1 ng/mL within 2 hours following a 75-g oral glucose load using a two site immuno-radiometric assay or chemiluminescent assay [256]. In normal subjects receiving oral glucose loading, serum GH levels initially are suppressed and then subsequently increase as plasma glucose declines. However, in acromegaly, oral glucose fails to suppress GH to the normal range. GH levels may paradoxically increase in response to an oral glucose load, remain unchanged, or fall. As basal GH secretion is tonically elevated with minor bursts, a random GH value of less than 0.4 ng/mL invariably excludes the diagnosis of acromegaly [256].

Evaluation of GH Deficiency Single GH and IGF-1 Measurements Single GH measurements are not helpful for diagnosis of GH deficiency, as GH secretion is pulsatile and daytime levels are often low in normal subjects and also suppressed after meals. Low IGF-1 levels are suggestive of GH deficiency, but are also encountered in malnutrition, acute illness, celiac disease, poorly controlled diabetes mellitus, liver disease, and estrogen ingestion. Fifteen percent of children diagnosed as GHdeficient by stimulation tests may have normal IGF-1 levels [257]. IGF-1 levels are normally very low before 3 years of age and highest in adolescence. Normal and GH-deficient children may have IGF-1 levels that overlap with those observed in infancy [258]. Furthermore, both normal and low IGF-1 levels are encountered in children with growth delay and genetic short stature [259]. IGF-1 levels do not always correlate with GH levels after provocative GH stimulation and low IGFBP-3 levels are also encountered in children with GH deficiency. Importantly, normal IGF-1 levels occur in about 20% of patients with proven GHD. Provocative Tests Provocative testing for GHD should only be undertaken in the clinical context of probable GHD (childhood history of GHD or a clinical context predisposing

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to adult GHD). Dynamic testing of GH reserve involves stimulation of somatotrophs to elicit GH in response to a pharmacologic stimulus. Several GH stimulatory agents have been utilized, including insulin, clonidine, arginine, L-dopa, GHRH, propranolol, and glucagon. Diagnosis of adult GHD is established by demonstrating a subnormal rise in peak serum GH levels elicited in response to one or more dynamic stimulation tests. Insulin-Induced Hypoglycemia (Insulin Tolerance Test) This reliable stimulus for GH secretion is the historical standard provocative test [260]. Regular insulin 0.1 IU/kg is administered intravenously to decrease basal glucose levels by 50% to a value below 40 mg/dL. Maximal GH secretion peaks at 30 60 minutes. Patients may experience symptoms of hypoglycemia, including light-headedness, anxiety, tremulousness, sweating, tachycardia, seizures and, rarely, unconsciousness. Insulin-induced hypoglycemia is contraindicated in patients with a history of seizure disorder, coronary artery disease, or age over 55 years. The test should be performed under close supervision, and intravenous glucose (50%) should be readily at hand for rapid administration. The risk of inducing profound hypoglycemia is greater in GH-deficient patients because of increased insulin sensitivity. A potential advantage of the ITT is the ability to simultaneously assess the hypothalamic pituitary adrenal axis for adrenal insufficiency. Clonidine This α-adrenergic agonist stimulates GH release via a central action. Clonidine (0.15 mg/m2) is administered orally, with a maximum GH secretory peak occurring after 60 90 minutes. Patients may experience drowsiness, with decreased systolic blood pressure in sodium-depleted GH-deficient adults at doses required to release GH (0.25 0.30 mg orally). Clonidine, although used as a stimulus for GH release in children, is not reliable to assess GHD in adults. L-Dopa/Propranolol L-Dopa induces GH release by stimulating hypothalamic dopaminergic receptors. Adrenergic blockade using propranolol enhances GH response to L-dopa. Ldopa is administered orally according to the patient’s weight (125 mg if weighing ,30 kg; 250 mg if 10 30 kg; and 500 mg if .30 kg) together with propranolol 0.75 mg/kg (maximum dose 40 mg) after an overnight fast. Maximum GH secretion is elicited after 60 90 minutes. L-dopa is effective in stimulating GH release and rarely results in adverse effects.

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Arginine/GHRH Arginine potentiates maximal somatotroph responsiveness to GHRH by inhibiting release of somatostatin from the hypothalamus. GHRH directly elicits GH secretion from pituitary somatotroph cells, potentiated by arginine. After an overnight fast, GHRH (1 μg/kg) is administered as an intravenous bolus at 0 minutes with arginine (30 g) in 100 mL infused from 0 to 30 minutes, with subsequent blood sampling for GH performed every 15 minutes for 90 minutes. Combined arginine/GHRH responses are age-independent. This highly reproducible GH provocative test [261] is at least as sensitive as insulin-induced hypoglycemia [262,263]. The arginine/GHRH test has been validated as a reliable alternative test when the ITT is contraindicated or impractical [262], and is endorsed by the Endocrine Society [260], the American Association of Clinical Endocrinologists [264], and the GH Research Society [265]. Ghrelin Mimetics GHRPs are synthetic secretagogues that elicit dosedependent and specific GH release by binding to GH5R, for which ghrelin has been shown to be the natural ligand [25]. GHRPs can be administered alone or in combination with GHRH. Combined administration of GHRP-6 and GHRH is the most potent stimulus to GH release, with excellent reproducibility and no serious side effects [23]. GHRH/GHRP-6 is highly specific, but is less sensitive than ITT. It is a viable alternative to the ITT in patients with organic pituitary disease, but overlap has been reported between GH levels attained in the control group and severely GH-deficient patients. Since GHRH and GHRP act directly on the pituitary, coadministration restores GH secretion in patients with hypothalamic disease [266]. GHRP-2 administration has different diagnostic cut-off points in adult GHD compared to ITT, and is highly reproducible [267]. A multicenter study comparing the oral GH secretagogue macimorelin with arginine/GHRH found it to be safe, convenient, and of comparable efficacy (82% sensitivity, 92% specificity, and 87% accuracy in diagnosing adult GHD), with a GH cut-off point of 6.8 μg/L for patients with a body mass index (BMI) ,30 kg/m2 and 2.7 μg/L for patients with a BMI .30 kg/m2 [268]. GHRPs are not currently commercially available in the United States. Glucagon Glucagon elicits GH secretory potency similar to or only slightly lower than the ITT for differentiating GHdeficient patients from normal subjects [269]. The

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mechanism of glucagon-induced GH release is not fully understood. After fasting for at least 8 hours, 1 mg glucagon (1.5 mg if patient weight .90 kg) is administered intramuscularly, with serum GH and capillary blood glucose levels measured every 30 minutes for 4 hours. Glucagon stimulation is contraindicated in malnourished patients and in patients who are fasting for 48 hours. Side effects include nausea and late hypoglycemia. A normal response is defined as a GH peak above 3 μg/L; in adults with GHD, GH levels do not rise above 3 μg/L. With the unavailability of GHRH in the United States, glucagon stimulation has been increasingly used as an alternative to the ITT because of its availability, reproducibility, safety, and lack of influence by gender and hypothalamic cause of GHD [270].

Approach to Provocative GH Testing As GH reserve testing is expensive and also fraught with high rates of false-positive results, patients should fulfill at least one of these rigorous preexisting criteria [271]: (1) young adults transitioning from adolescence who required GH therapy for short stature and had demonstrated anatomic genetic or acquired cause of short stature; (2) evidence for a pituitary lesion or damage including surgery or irradiation; (3) MRI evidence for a sellar lesion, pituitary hypoplasia, hypophysitis, or infiltration; (4) history of significant head trauma. Multiple sampling of GH levels most accurately reflects GH secretion. However, it is not practical in clinical practice as GH secretion is influenced by age, nutritional status, exercise, and BMI [262]. Provocative tests of GH secretion are employed when patients suspected of having GHD require confirmation of the diagnosis [260]. Serum IGF-1 levels below the age-adjusted normal range, in the absence of liver dysfunction and catabolic disorders, usually indicate GH deficiency [265]. However, the finding of normal IGF-1 levels does not exclude the diagnosis of GHD [262] and GH provocative testing is still required for diagnosis in the appropriate clinical setting [260]. A single GH stimulation test is sufficient to confirm the diagnosis of adult GHD [265], and provocative GH testing is not required for hypopituitary patients, those with serum IGF-1 levels below the reference range, and those exhibiting three or more other pituitary hormones deficits, as these patients have a .97% chance of having GHD [272]. Historically, the ITT has been the “gold standard” GH provocative test, but is contraindicated in patients with seizure disorders or cardiovascular disease and requires intensive monitoring. Combined arginine/

GHRH testing is considered a reliable alternative, with 95% sensitivity and 91% specificity at a GH cutoff of 4.1 ng/mL, compared to 96% sensitivity and 92% specificity for the ITT with an optimal GH cutoff of 5.1 ng/mL [262]. A caveat for the arginine/GHRH test is the falsely normal GH response in patients with GHD due to hypothalamic disease, in whom GHRH directly stimulates the pituitary gland [273]. The relative performance of ITT and arginine/GHRH stimulation is comparable; however arginine alone, clonidine, levodopa, and the combination of arginine plus levodopa are less robust tests for the diagnosis of adult GHD [262]. The GH cutoff for diagnosis of GHD varies with the test used. A peak GH response of ,3 ng/mL during ITT and glucagon test confirms the diagnosis of GHD. Relative adiposity in the abdominal region blunts GH responses to stimulation [274], and thus cutoffs for arginine/GHRH testing have been validated by BMI: validated GH cutoff levels are defined as peak GH ,11 ng/mL for patients with BMI ,25 kg/m2, peak GH ,8 ng/mL for BMI 25 30 kg/m2, and peak GH ,4 ng/mL for BMI .30 kg/m2 [275]. Lack of age- and gender-adjusted normative data, as well as assay variability influence definitions of GH cutoff diagnostic criteria. GH levels between 3 and 5 ng/mL were previously defined using polyclonal radioimmunoassays. Cutoff values for newer, more sensitive, two-site assays have not been rigorously defined [265]. However, GH values of 5.1 ng/mL and 4.1 ng/mL have been proposed using ITT and arginine/GHRH respectively using immunochemiluminescent two-site assays [262]. Pediatric patients with idiopathic GHD (either isolated or with one additional hormone deficit) should be retested for GHD after completion of puberty [276] after discontinuing GH treatment for at least a month [260]. Patients with a high likelihood of permanent GHD and who may not require retesting after puberty include those with radiologically confirmed sellar/ suprasellar abnormality, a transcription factor mutation, or acquired hypothalamic pituitary disease, as well as those who have had hypothalamic pituitary surgery or who have undergone hypothalamic pituitary radiation. Lack of international assay standardization further hinders the definition of GH cutoff values. Analytic methods used in individual assays influence GH results and ideally assay-specific cutoff values should be defined for each provocative test [265]. The calibrant used in the assay, GH isoform detected, as well as the presence or absence of GHBP all influence GH assay results. Adoption of a universal GH calibration standard would be valuable in the international harmonization and standardization of GH provocative test results.

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TESTS OF GH SECRETION

Variability of GH Assays The comparison of results of various GH assays obtained in different laboratories is difficult because of differences between several aspects of the immunoassays. Older assay methods employed polyclonal competitive techniques and were relatively insensitive. Newer sensitive noncompetitive sandwich-type GH immunoassays employ antibodies directed against different epitopes on the surface of the GH molecule. One antibody captures the GH molecules, whereas the second labeled antibody generates a signal proportional to the amount of GH in the sample. Also, older radioimmunoassays used radiolabelled GH, whereas newer nonradioactive sandwich-type assays employ various labels, including enzyme-linked, fluorescence, and chemiluminescence. Different circulating forms of GH are not all recognized in GH assays. Because monomeric 22k is the only GH form available as a standard in sufficient purity and quantity, and because monomeric 22k is also the most abundant circulating form, it is used as the basis for GH measurement. Other GH forms are recognized to varying and largely unknown degrees. Thus, different antibodies or assay protocols yield different results. Polyclonal antibodies used in the early radioimmunoassays recognized several molecular forms of GH, thus inducing higher estimates of GH compared to newer immunometric assays employing highly specific monoclonal antibodies. GH standards also affect comparison of GH values in different laboratories. In 1994, the first international standard for somatotrophin, IRP 88/624, was prepared by the World Health Organization (WHO) using recombinant technology in contrast to the previous standards prepared from pituitary extracts. Use of an international standard enables uniformity of calibration between different GH kits, and provides an opportunity for international use of a single calibrant for GH assays. The recent recombinant International

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Standard (IS) preparation, WHO IS 98/574, is a recombinant 22-kDa GH of .95% purity. GHBPs may influence GH estimates by interfering in some GH assays, as approximately 50% of circulating GH is complexed to GHBP. GHBP present in high concentrations in serum samples can block epitope accessibility of respective antibodies used in GH assays, and lead to underestimation of GH concentrations. However, as GHBP has a greater affinity for the 20-kDa GH molecule, it presumably does not interfere in GH estimates in the new GH assays, specific for the 22-kDa GH molecule [277]. GH immunoassay heterogeneity thus poses a major challenge in the definition of standards for the diagnosis of GHD. Different conversion factors are used to report GH assay results in mass units, a further cause for assay variability. In one series, a borderline GH value obtained from a patient with suspected acromegaly, was sent to 104 laboratories for analysis [278] (Fig. 4.9). The median GH was 2.6 mU/L (range 1.04 3.5 mU/L). When a conversion factor of 3.0 (1 μg/L 5 3 mU/L) was used, 11% of result values were consistent with acromegaly; with a conversion factor of 2.6, 55% diagnosed acromegaly, whereas using a conversion factor of 2.0, 86% diagnosed acromegaly. Reliable and harmonious GH assays with robust reference standards still need to be developed [279].

Variability of IGF Assays Serum IGF-1 levels are regulated by GH, as well as nutrient intake, estrogen, thyroid, cortisol levels, and IGFBPs. Testosterone, age, gender, ethnicity, and BMI also influence IGF-1 levels [280]. IGF-1 levels increase until puberty and then decline (Fig. 4.10), necessitating adequate age-adjusted ranges with large numbers of healthy male and female control subjects within each age range [281].

FIGURE 4.9 The impact of conversion factors (CFs) on GH results. 1, Immunotech IRMA; 2, Wallac DELFIA; 3, NETRIA IRMA; 4, Nichols Allegro IRMA; 5, Tosoh AIA; 6, Nichols Advantage; 7, Nichols ICMA; 8, Beckman Access; 9, DSL ELISA; 10, DPC Immulite; 11, DPC Immulite 2000; 12, in-house ELISA; 13, DiaSorin IRMA; 14, in-house IRMA. The lines present the Cortina Consensus cutoff value transformed into mU/L using various CFs: solid line, CF 3, 11% of results consistent with active acromegaly; wide dashed line, CF 2.6, 55% acromegaly; narrow dashed line, CF 2, 86% active acromegaly. Source: From Pokrajac A, Wark G, Ellis AR, Wear J, Wieringa GE, Trainer PJ. Variation in GH and IGF-I assays limits the applicability of international consensus criteria to local practice. Clin Endocrinol (Oxf) 2007;67(1):65 70.

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FIGURE 4.10 Left, 24-h integrated GH levels in 173 nonobese subjects, aged 7 65 years, stratified by age decades. From Zadik [96]. Right, age- and gender-specific IGF-1 levels in 3900 healthy subjects. Serum IGF-1 levels measured by Nichols Advantage Assay. Source: From Brabant G, von zur MA, Wuster C, et al. Serum insulin-like growth factor I reference values for an automated chemiluminescence immunoassay system: results from a multicenter study. Horm Res 2003;60(2):53 60.

Commonly used commercial IGF-1 assays are mostly calibrated against the older standard preparation WHO 87/518, and have similar sensitivities and coefficients of variation, but exhibit marked nonlinear differences in comparative studies [282]. Newer assays are calibrated against the 02/254 standard [283]. Reliable assays require validation of recovery of exogenous IGF-1, crossreactivity with IGF-2, and assay reproducibility, as well as comparison of sample types, in order to ensure accurate data. Validation of reported results should be published in the kit inserts of commercial assays [284]. Meaningful interpretation of IGF-1 concentrations requires rigorous assay- and age-specific reference ranges. High-quality, method-specific reference ranges and a high degree of IGF-1 assay methodological consistency are essential for reliable comparison of results across studies and for long-term monitoring acromegaly therapy and GH replacement therapy in GHdeficient patients. IGF-1 immunoassays, in which antibodies competitively or noncompetitively bind to IGF-1, are commonly used to measure circulating IGF-1 levels. No universally accepted assay has clearly emerged. Available immunoassays employ different methodologies, laboratory protocols, and reference ranges [283,284], resulting in a well-documented lack of consistency in IGF-1 measurement. Wide variations in IGF-1 measurement are encountered when submitting the same patient samples to multiple laboratories using the same assay [278]. Furthermore, lot-to-lot variation with current immunoassays resulted in a nearly twofold range of results, observed in two laboratories using the same reagents, over a 5-year period [285].

Several factors influence the results of IGF-1 immunoassays [279]. Under physiologic conditions, approximately 99% of IGF-1 is bound to IGFBPs, predominantly IGFBP-3 [286], and improper extraction techniques dissociating the IGF-1/IGFBP complex may undermine assay performance [279]. Antibody specificity for IGF-1 also varies between assays, which affects measurement of IGF-1 levels. Reference ranges for many of the currently used immunoassays were developed based on a small number of samples and/or a short age span, so reported “normal” and “abnormal” values may not be reflective of all adult patients. Comparing results obtained from IGF-1 assays calibrated to the older WHO 87/518 standard against those from assays calibrated to the new 02/254 standard can be challenging [279,284,287]. The chemiluminescent IGF-1 immunoassay IDSiSYS (Immunodiagnostic Systems; Tyne & Wear, United Kingdom) was developed in an attempt to avoid pitfalls of commonly used immunoassays [283]. IDS-iSYS immunoassay reference ranges are robust, ensuring that they are representative of the general population. The immunoassay was calibrated to the new 02/254 standard, and specificity of the two mouse monoclonal antibodies used was validated against recombinant human IGF-1, exhibiting adequate dissociation of IGF-1/IGFBP complexes and preventing IGFBP interference [283]. Liquid chromatography mass spectrometry (LC-MS) has emerged as an alternative to immunoassay. Unlike immunoassays, which introduce antibodies to bind and separate target antigens, LC-MS selects the target analyte based on mass, and quantifies it based on a unique mass: charge ratio after passing through an electric ion

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CLINICAL USE OF GH

field [288 290]. The narrow-mass-extraction, highresolution LC-MS (Quest Diagnostics, San Juan Capistrano, California, USA) selects IGF-1 from the serum sample using a one-step extraction for dissociation of IGF-1/IGFBP complexes, and does not employ antibodies to isolate serum IGF-1 molecules, mitigating concerns about antibody specificity or interference at IGF-1/IGFBP binding sites [291]. This assay has a robust set of sex- and age-specific reference ranges, and is calibrated to the 02/254 WHO IGF-1 standard. Although mass spectrometry technology is not limited by the pitfalls of immunoassay technology, it is susceptible to interlaboratory variability [292]. In the absence of a standardized kit, there is concern for both technical and human laboratory error, and discrepancies between laboratories using the same technology have been reported [289].

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TABLE 4.5 Indications for GH Therapy APPROVED USE Adults GH deficiency AIDS-associated muscle wasting Children GH deficiency Idiopathic short stature Turner syndrome Born small for gestational age Chronic renal insufficiency SHOX gene deficiency INVESTIGATIONAL Frailty

CLINICAL USE OF GH

Osteoporosis Catabolic states, cachexia

GH Therapy in Childhood

Burns

Recombinant hGH is administered to promote linear growth in short children. In the United States, the FDA has approved GH treatment for the following conditions: GH deficiency, idiopathic short stature (ISS), chronic kidney disease, Turner syndrome, Prader Willi syndrome, SHOX gene haploinsufficiency, Noonan syndrome, and small for gestational age (SGA) age infants (Table 4.5). The efficacy of GH treatment in children with non-GHD growth disorders is also well-established. However, individual responses are variable, and prediction of adult height is guarded. Long-term follow-up studies do not confirm a higher incidence of neoplasia in children or adults who received GH therapy in childhood. However, in light of high GH doses employed, careful monitoring of IGF-1 and IGFBP-3 is recommended. Isolated GH deficiency may be due to congenital or acquired causes and is most commonly idiopathic. About 10% of patients with sporadic GH deficiency exhibit identifiable mutations [293], while up to 30% exhibit familial patterns of inheritance. Hereditary GH deficiency (Table 4.6) may be due to mutations occurring at each level of the hypothalamic pituitary/ GH-IGF-1 axis. Mutations of the GHRH receptor, transcription factors determining GH synthesis, the GH molecule itself, or peripheral GHR may all lead to short stature [294,295]. True familial isolated GHD may occur as four distinct syndromes (Table 4.7). Childhood GH deficiency ranges from complete absence of GH associated with severe growth retardation to partial GH deficiency resulting in short stature. Diagnosis is based on decreased height ( .2.5 SDs below the mean for age-matched normal children),

Postoperative recovery Wound healing Parenteral nutrition Ovulation induction Immune deficiency

poor growth velocity (,25th percentile), delayed bone age, and a predicted adult height below mean parental height [296]. GHD is usually confirmed by inadequate pituitary GH responses to standard provocative stimuli. Combined clinical evaluation and provocative testing are used in assessment and concomitant endocrine deficiencies, especially hypothyroidism, should be corrected to maximize growth-promoting benefits of hGH. GH replacement should be started as early as possible before height drops below the third percentile, as total height gain is inversely proportional to the pretreatment chronologic and bone age, as well as severity of GH deficiency. The most pronounced acceleration in linear growth rate occurs during the first 2 years of treatment. Dose and frequency of administration of hGH both influence height velocity.

Idiopathic Short Stature ISS describes otherwise normal children who are at or below the 5th percentile for height, with normal GH responses to provocative stimuli. This group of

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TABLE 4.6 Etiology of Inherited GH Deficiency STRUCTURAL Pituitary aplasia Pituitary hypoplasia CNS masses RECEPTOR MUTATION GHRH mutation-GH deficit TRANSCRIPTION FACTOR MUTATION Gene

Chromosome Deficiency

Pituitary

Associated malformations

Inheritance

POU1F1 3p11

GH, PRL, 6 TSH

Normal or hypoplastic

Recessive, dominant

PROP1

5q35

GH, PRL, TSH, LH, FSH

Normal, hypoplastic, hyperplastic, or cystic

Recessive

HESX1

3p21

GH, PRL, TSH, LH, FSH, ACTH; posterior defects

Hypoplastic or hyperplastic; normal or ectopic posterior

PITX2

4q25

GH, PRL, TSH, FSH, LH

LHX3

9q34

GH, PRL, TSH, LH, FSH

Hypoplastic or hyperplastic

LHX4

1q25

GH, TSH, ACTH

Hypoplastic

GH, PRL, TSH, LH, FSH, ACTH

Hypoplastic

Eye malformations Anophthalmia, esophageal atresia

OTX2 SOX2

3q26

GH, FSH, LH

Hypoplasia, mid-brain defects

SOX3

Xq27

GH, TSH, ACTH, FSH, LH

Hypoplasia, ectopic posterior pituitary

IGSF1

Xq25

GH, PRL, TSH

Septo-optic dysplasia

Recessive

Rieger syndrome

Dominant

Stubby neck with rigid cervical spine

Recessive Dominant Dominant/ negative

X-linked recessive Testicular enlargement

X-linked recessive

HORMONE MUTATION GH1-GH deficiency Bioinactive GH-GH deficiency From Kaiser U, Ho KKY. Pituitary physiology and diagnostic evaluation. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM, editors. Williams Textbook of Endocrinology, 13th edition. Elsevier, Philadelphia; 2016. p. 176 231.

TABLE 4.7 Genetic Forms of Isolated GH Deficiency Type Inheritance

Phenotype

Gene

Mutations

IA

Autosomal recessive

Severe short stature; serum GH undetectable; anti-GH antibodies on treatment

GH1

Deletions, frameshift, nonsense

IB

Autosomal recessive

Less severe short stature; serum GH low but detectable; no anti-GH antibodies on treatment

GH1 Splice site, frameshift, GHRHR missense, nonsense

II

Autosomal dominant

Variable height (severe short stature to normal); normal or hypoplastic anterior pituitary; other pituitary hormone deficiencies

GH1

III

X-linked

GH deficiency with agammaglobulinemia with or without mental retardation; ectopic posterior pituitary on MRI

SOX3 Deletions, expansions, Others? others

Splice site, missense, splice enhancer; intronic deletions

From Alatzoglou KS, Dattani MT. Genetic causes and treatment of isolated growth hormone deficiency-an update. Nat Rev Endocrinol 2010;6(10):562 76.

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short-stature children may also harbor as yet unidentified mutations. Children with ISS are normal size at birth, but grow slowly during early childhood so that the average height falls below 2.0 SD by school-age, maintaining a height velocity within the lower normal range, growing below but parallel to the normal centile channels. Untreated adult height is below the normal range and below mid-parental height by about 1 SD. Increasing numbers of genetic defects in genes associated with GH/IGF-1 secretion/growth, resulting in short stature, and previously labeled as ISS, have been described, including pituitary gene defects (GHSR and GH1 locus), defects in the GHR and intracellular signaling, and defects in GHR extracellular domain (Sta5b and SH2) and growth plate (SHOX transcription factor deficiency) [297]. GH administered to children with ISS induces an adult height gain of between 3 and 7 cm, depending on the duration of treatment [181]. Rates of adverse events associated with GH therapy in children with ISS are lower compared to side-effect profiles observed in other GH-treated disorders, as these children are generally otherwise healthy.

Turner Syndrome Patients with Turner syndrome manifest dysmorphic body features, ovarian failure, and reduced growth rate, starting during intrauterine life and continuing through childhood and puberty, resulting in reduced final adult height. GH therapy in girls with Turner syndrome increases predicted height, with a greater increase in height when treatment is started early and when estrogen replacement is postponed until at least age 14 years. In a randomized controlled study, mean adult height was 7 cm greater than the untreated group after 6 years [298]. However, GHtreated Turner syndrome patients, may manifest increased incidence of type 2 diabetes mellitus.

Children Born Small for Gestational Age Children with a birth length at least 2 SD below the mean are defined as SGA. Poor fetal growth may be idiopathic, or due to maternal toxins or associated with defined syndromes. Most SGA infants experience catchup growth within the first or second year of life; the remaining 10 15% increase adult height by approximately 1.0 1.4 SD with long-term GH treatment [299].

malnutrition, acid base disturbances, hyperparathyroidism, or GH insensitivity manifest by elevated GH levels and reduced IGF-1:IGFBP ratios with decreased free IGF-1 concentrations. Decreased renal GH clearance is also present, with consequent high basal and elicited GH levels. Approximately one-third of children with chronic renal insufficiency have heights below the third centile [300]. Growth patterns vary depending on age of onset of renal insufficiency. Despite high GH levels, children with renal failure are short. Following renal transplantation, return to normal growth is variable. A meta-analysis of randomized controlled trials concluded that catch-up growth occurred in the first year of treatment and continued GH treatment likely prevents progressive growth failure [301]. The indication for GH treatment in chronic renal insufficiency is growth failure (subnormal height velocity) rather than short stature. GH treatment elicits a doubling of pretreatment height velocity in the first year of treatment [302]. Children with chronic renal insufficiency receiving GH treatment should be carefully monitored for impaired glucose tolerance, as they have relative glucose intolerance, even in the absence of GH treatment.

SHOX Gene Deficiency The SHOX gene, at the distal ends of the X and Y chromosomes, encodes a homeodomain transcription factor responsible for a significant proportion of long bone growth. Deficiency leads to atypical proliferation and differentiation of chondrocytes, with delayed bone growth in intrauterine and postnatal growth. The SHOX gene plays a role in the short stature of Turner syndrome, Leri Weill syndrome, and some cases of ISS. A recent study showed that 57% of patients with SHOX gene deficiency and 32% of Turner syndrome patients treated with GH for a mean of 6 7 years reached a final height greater than 2 SD, with no effect in pubertal maturation [303].

GH THERAPY IN ADULTS In adults, GH is indicated for GH deficiency, muscle wasting due to HIV/AIDS, and short bowel syndrome.

Adult GHD Syndrome Etiology

Chronic Renal Insufficiency Chronic renal insufficiency is frequently associated with growth failure, which may be due to protein-calorie

The diagnosis of adult GHD (AGHD) should be suspected in patients with hypothalamic or pituitary disease, or in those with a history of having received

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cranial irradiation or pituitary adenoma treatment, or prior traumatic brain injury or subarachnoid hemorrhage. AGHD may be isolated or can occur in association with several other pituitary hormone deficiencies (panhypopituitarism). Childhood-onset GHD is most commonly idiopathic, but may be genetic, or associated with congenital anatomical malformations in the brain or sella turcica region (Table 4.8). AGHD may follow childhood-onset GHD, which persists into adulthood or can be acquired in adulthood secondary to structural sella and parasellar lesions or may be secondary to head trauma. In the United States, the incidence of AGHD is about 6000 new cases annually, with a population prevalence of about 50,000 cases [304]. In recent years, with the availability of recombinant GH, the pattern of AGHD diagnosis has changed, with an increase in idiopathic etiologies accounting for about 17% of patients in the HypoCSS surveillance database [305]. Pitfalls of diagnosing isolated adult GDH include inappropriate testing and less rigorous diagnostic criteria [271]. The most common cause of AGHD is a pituitary macroadenoma (30 60% of which are associated with single or multiple pituitary hormone deficiencies), or pituitary adenoma treatment (surgery or radiotherapy). Up to 20% of patients who sustain traumatic brain injury subsequently develop GHD with varying degrees of concomitant hypopituitarism [306]. GHD is usually the first hormone deficiency to develop when pituitary damage occurs; thus in patients diagnosed with multiple pituitary hormone deficits, the likelihood of GHD is high. The incidence of hypopituitarism associated with pituitary irradiation increases over time, with 50% of patients diagnosed with varying degrees of hypopituitarism 10 years after having received conventional radiotherapy. Diagnosis GHD adults have altered body composition with increased fat mass and decreased muscle volume and strength, decreased bone mineral density, altered glucose and lipid metabolism, lower psychosocial achievement, and possibly increased mortality due to cardiovascular disease (Table 4.9). These patients have a lower employment rate, are more often on sick leave or disability, and either live alone or with parents [308]. IGF-1 is a robust screening test in lean, younger patients (,40 years) suspected of having GHD. However, at any age, screening IGF-1 levels in hypopituitary adults may be normal in the presence of severe GHD. Other causes of low IGF-1 levels include liver disease and malnutrition. Single GH serum measurements are not informative. The diagnosis of AGHD is confirmed by

TABLE 4.8 Causes of GH Deficiency PRESENTING IN CHILDHOOD Congenital Idiopathic Embryologic defects (structural) Agenesis of corpus callosum Hydrocephalus Septo-optic dysplasia Arachnoid cyst Empty sella syndrome Genetic Transcription factor defect GHRH receptor defect GH gene defect GH receptor/postreceptor defect GH resistance Laron dwarfism Pygmy Neurosecretory defects Radiation for brain tumors, leukemia Head trauma Perinatal birth injury Child abuse Accidental Inflammatory diseases Viral encephalitis Meningitis, bacterial, fungal, tuberculosis ACQUIRED IN ADULTHOOD Pituitary/hypothalamic/tumors Pituitary adenoma Craniopharyngioma Rathke’s cleft cyst Metastasis Parasellar tumors Germinoma Astrocytoma Postpituitary surgery Head trauma Hemochromatosis Sickle cell disease Thalassemia Lymphocytic hypophysitis Cranial irradiation Infiltrative/granulomatous/infectious disease Histiocytosis Sarcoidosis Idiopathic Tuberculosis Syphilis Vascular Acromegaly treatment

provocative testing of GH secretion after other hormonal deficits have been adequately replaced (Table 4.10). A single stimulation test is adequate for the diagnosis of AGHD, but not all patients suspected of having GHD require a GH stimulation test for diagnosis. Adult patients with three or four pituitary hormone deficits and a low IGF-1 level, do not require GH stimulation testing to establish the diagnosis [272].

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The ITT remains the test of reference despite concerns about reproducibility, safety, and specificity. ITT is contraindicated in adults with ischemic heart disease and seizure disorders, and is a potential risk in elderly patients as occult vascular disease increases with age. ITT requires close clinical monitoring to attain adequate hypoglycemia, with prompt reversal of severe insulininduced hypoglycemia to avoid neuroglycopenia. Sensitive and reliable alternative GH stimulants have been evaluated. The GHRH-arginine test, with 95% sensitivity, and 91% specificity, at a GH cutoff of 4.1 μg/L compares very favorably to the ITT, with a GH cutoff of 5.1 μg/L (96% sensitivity and 92% specificity) [262]. Arginine alone, clonidine, levodopa, and arginine plus levodopa are not reliable alternatives to the ITT. GHRH-arginine is well-tolerated and requires less monitoring than the ITT. However, two caveats should be considered when interpreting results of GHRHarginine stimulation testing: the impact of increased BMI on GH secretion, and whether the GHD is due to hypothalamic or pituitary damage. TABLE 4.9 Physical Findings in the Adult Growth Hormone Deficiency Syndrome Truncal adiposity Increased waist/hip ratio Thin, dry, cool skin Reduced exercise performance Reduced muscle strength Reduced bone mineral density Depressed mood Psychosocial impairment From Carroll [307].

TABLE 4.10

Obese subjects have reduced spontaneous and stimulated GH secretion; negatively associated with BMI [274]. Diagnostic GH cutoff values have been evaluated for lean (BMI , 25 kg/m2), overweight (BMI .25 but ,30 kg/m2), and obese (BMI .30 kg/m2) subjects, with high sensitivity and specificity for GH deficiency. In lean subjects, a peak GH cutoff point of 11.5 μg/L had the highest sensitivity and specificity using receiver operator characteristics (ROC) curve analysis; in the overweight and obese population lower cutoff points were determined, at 8 4.2 μg/L [275]. To avoid false-positive responses in overweight and obese subjects, and false-negative results in lean subjects, BMI must be considered in the interpretation of GH responses to GHRH-arginine provocative stimulation, and approximate GH cutoff points must be considered. GHRH stimulates the pituitary directly, and thus falsely “normal” responses can be elicited in patients with hypothalamic GHD, because exogenously administered GHRH directly stimulates pituitary somatotroph cells. Therefore, in patients with suspected hypothalamic damage (e.g., after cranial irradiation), the peak GH response to GHRH and arginine may be normal, whereas the ITT may reveal an abnormal response [273]. GHRH-arginine is accepted as a reliable alternative to the ITT [260,265], however, since 2008, GHRH has been difficult to obtain in the United States. Glucagon stimulation testing is a well-tolerated alternative GH provocative test to GHRH-arginine. Glucagon is relatively inexpensive and widely available for treating hypoglycemia in patients with diabetes mellitus. Glucagon is contraindicated in patients who have fasted for more than 48 hours. Side-effects may include nausea and late hypoglycemia, which can be prevented by eating small frequent meals after completing

Diagnostic Tests for Adult GHD

Test

Procedure

Interpretation/expected normal response

Insulin tolerance

• Administer insulin 0.05 0.15 U/kg IV • Sample blood at 30, 0, 30, 60, and 120 min for GH and glucose

• Glucose should drop ,40 mg/dL (2.2 mmol/L) • GH should be .3 5 μg/L • Cutoffs for GH response are BMI-related

GHRHarginine

• Administer GHRH 1 μg/kg (max 100 μg) IV followed by arginine infusions 0.5 g/kg (max 35 g) over 30 min • Sample blood at 0, 30, 45, 60, 75, 90, 105, and 120 min for GH

• Can give false-normal GH response if GHD is due to hypothalamic damage (e.g., following radiation) • Cutoffs for GH response should be correlated to BMI (obesity may blunt GH response to stimulation)

Glucagon

• Administer glucagon 1 mg (1.5 mg if weight .90 kg) IM • Sample blood at 0, 30, 60, 90, 120, 150, 180, 210, and 240 min for GH and glucose

From Molitch ME, Clemmons DR, Malozowski S, Merriam GR, Vance ML, Endocrine S. Evaluation and treatment of adult growth hormone deficiency: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2011;96(6):1587 609.

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the test. Glucagon is administered intramuscularly and GH is measured half-hourly for 4 hours. In adults with GHD, GH levels do not rise above 3 μg/L. Using ROC analysis, a GH cutoff value of 3 μg/L provides the best pair of sensitivity (100% and 97%, respectively) and specificity (100% and 88%, respectively). Unlike the GHRH-arginine test, there is no inverse correlation between BMI and peak GH response to glucagon [270].

TABLE 4.11

Effects of GH Replacement in Adults

Clinical consequence

Effect of GH replacement

BODY COMPOSITION General and central adiposity

Decrease

Reduced lean mass

Increase

Reduced bone mass

Increase

FUNCTION

GH Replacement Therapy GH secretion continues into adulthood, and GH influences many metabolic systems other than growth. The goal of GH replacement therapy in adulthood is to correct the metabolic, functional and psychological deficiencies associated with rigorously diagnosed adult GHD (Table 4.11). GH replacement may also reduce mortality associated with pituitary failure from a standardized mortality ratio (SMR) of 2.4 (95% CI, 1.46 3.34) to 1.99 (95% CI, 1.21 2.76). This effect is more pronounced in men [309]. GH replacement in GH-deficient adults is associated with increased energy levels, improved mood, vitality and emotional reactions, and less feeling of social isolation. GH replacement therapy is associated with significantly improved quality of life scores [310]. GH-deficient adults demonstrate reduced VO2 max (maximum capacity to take in and use oxygen) with impaired exercise capacity. A meta-analysis of 268 GHD patients treated with 3.3 15.7 mg/week GH for 6 18 months in 11 randomized placebo-controlled studies, demonstrated significant improvement in exercise capacity evaluated by maximally increased work rate and VO2 max [311]. Lipolytic effects of GH increase availability of circulating FFAs to muscle during prolonged exercise [312], with potential conservation of glycogen stores. GH-enhanced increase in the cardiac LV ejection fraction also may contribute to improved oxygen delivery to exercising muscle [313]. GHD adults manifest reduced skeletal muscle mass, with reduced isometric muscle strength and possibly reduced isokinetic strength. GH and IGF-1 exert anabolic effects on skeletal muscle [314], with increased protein synthesis and reduced protein oxidation, an effect which is enhanced with concurrent administration of testosterone [315]. GH replacement in GHD adults rescues isometric and isokinetic strength, especially in those patients with the most compromised baseline muscle strength; an effect which is sustained for 5 years [316]. GH replacement in GHD adults also improves body composition and thermoregulation, with increased sweat secretion rates during heat exposure and exercise in GHD adults.

Reduced exercise capacity

Improve

Muscle weakness

Improve

Impaired cardiac function

Improve

Hypohydrosis

Improve

QUALITY OF LIFE Low mood

Improve

Fatigue

Improve

Low motivation

Improve

Reduced satisfaction

Improve

CARDIOVASCULAR RISK PROFILE Abnormal lipid profile

Improve

Insulin resistance

Improves in long term

Inflammatory markers

Decrease

Intimal media thickening

Decrease

Cardiovascular and cerebrovascular events

Unknown

LABORATORY Blunted peak GH to stimulation Low IGF-1

Increase

Hyperinsulinemia

Increase

High LDL and low HDL cholesterol

Improve

Longevity

Unknown

From Melmed [271].

Within a year of GH replacement, visceral adipose tissue mass decreases by 9% [197], while LBM improves by up to 7% [317]. GH antagonizes insulin action and lipoprotein profiles improve, with reduced total and LDL cholesterol, and increased HDL cholesterol, triglycerides, and ApoB 100 levels [196]. Although LBM, cardiac stroke volume, and LV mass are increased [196], reports of cardiovascular risk profile improvement have been inconsistent [318]. Effects of GH replacement on bone mineral density are more beneficial in GH-deficient men [319] and bone fracture development is slowed in patients with no prior history of osteoporosis [320]. In postmenopausal women

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GH THERAPY IN ADULTS

113

FIGURE 4.11 Algorithm for management of adult GH deficiency.

followed for 10 years, GH treatment improved fracture outcomes [321]. GH doses in adulthood are adjusted to individual needs (Fig. 4.11). As more GH is secreted in younger, lean individuals and in females, elderly, male, or obese individuals require lower GH replacement doses. The use of oral estrogen replacement affects the GH replacement dose. Premenopausal women or postmenopausal women using transdermal estrogen replacement require lower GH doses than postmenopausal women receiving oral estrogen replacement [322]. Oral, but not transdermal estrogens antagonize GH actions and reduce IGF-1 levels (Fig. 4.12). Historically, GH treatment regimens were weight-based, resulting in a higher incidence of side-effects as well as higher maintenance doses than in currently used individualized dose-titration GH replacement regimens. Current dosing recommendations suggest a starting GH dose of 0.2 0.4 mg/day in young patients and 0.1 0.2 mg/ day in those over age 60 years [323]. GH is selfadministered as a single subcutaneous evening injection in an attempt to recapitulate normal physiological nocturnal GH secretion. Daily doses are titrated by 100 200 μg/day every 6 weeks according to clinical responses, side-effects, and IGF-1 levels [260]. After maintenance doses have been established, patients can be monitored at 6 12-monthly intervals, for clinical evaluation, side-effects, and serum IGF-1 levels

FIGURE 4.12 Time course of GH dose and serum IGF-1 concentration in a representative patient (38-year-old woman) who was switched from oral to transdermal estrogen therapy during the course of GH replacement. Source: From Cook DM, Ludlam WH, Cook MB. Route of estrogen administration helps to determine growth hormone (GH) replacement dose in GH-deficient adults. J Clin Endocrinol Metab 1999;84(11):3956 60.

(Table 4.12). GH doses are adjusted until IGF-1 levels reach mid-normal range for age and sex (see Fig. 4.10). Lipid profile and fasting blood glucose levels should be evaluated annually. If the pretreatment bone DEXA scan is abnormal, follow-up DEXA scan is evaluated at 1 2-year intervals. Hypopituitary patients may require

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4. GROWTH HORMONE

TABLE 4.12 Patient Monitoring After Initiating GH Replacement in Adults

TABLE 4.13 Replacement

1 Assess body weight, blood pressure, waist circumference, and BMI at initiation and every 6 months

Patient age and gender

2 Measure IGF-1 6 weeks after initiating GH replacement, after dose escalations, and every 6 months thereafter 3 Assess thyroid and adrenal function, and replace or adjust replacement doses as indicated 4 Assess metabolic profile including blood sugar and lipids every 6 months 5 Assess BMD by DEXA annually

Factors Determining Side Effects of GH

Enhanced IGF-1 response Greater body weight and body mass index Adult onset versus childhood onset of GH deficiency

TABLE 4.14

Side Effects of GH Replacement Therapy

ADULTS

6 Periodically assess residual pituitary mass with a pituitary MRI 7 Assess quality of life

Salt and fluid retention; peripheral edema Glucose homeostasis

From Fleseriu [324].

Unmasking of thyroid dysfunction Arthralgias and myalgias

evaluation of thyroid and adrenal axes after initiation of GH therapy [323]. Duration of GH therapy depends on benefits of treatment. Discontinuing GH therapy may be appropriate, if objective benefits are not apparent after at least 1 year of treatment; however, if objective clinical benefits are obtained from GH replacement, treatment is continued. Side-effects of GH replacement are reported in about 20% of patients (Tables 4.13 and 4.14). They are usually transient, and include arthralgias, edema, and carpal tunnel syndrome due to fluid retention; dose reduction may be required to alleviate these effects. As GH may reduce insulin sensitivity, glycemic control should be monitored, especially in patients receiving higher GH doses. In a placebo-controlled study, GH therapy induced impaired glucose tolerance in 13% of patients, and diabetes mellitus in 4%, with a significant number of patients developing worsening of glucose tolerance in the GH-treated group [317]. New-onset diabetes mellitus occurs in ,5% of patients. GH replacement for up to 9.6 years in males with nonfunctioning adenomas did not increase all-cause mortality [325]. Associated pituitary dysfunctions were likely independent causes of increased mortality. GH replacement therapy is contraindicated in patients with an active malignancy, benign intracranial hypertension, and proliferative diabetic retinopathy. There is concern that the growth-promoting and mitogenic effects of GH and IGF-1 could potentially increase cancer risk and promote tumor regrowth. However, significant increases in the occurrence of intrasellar, intracranial, or extracranial tumors has not been reported in adult GHD patient on long-term GH replacement therapy [326,327]. Increased rates of regrowth of postoperative craniopharyngioma [328], other childhood brain tumors [329], or adult pituitary macroadenomas have

Carpal tunnel syndrome Sleep apnea Headache Iatrogenic acromegaly CHILDREN Slipped capital femoral epiphysis Benign intracranial hypertension

not been reported following GH replacement. GH administered to patients after pituitary tumor resection does not induce adenoma regrowth [330]. In healthy men, serum IGF-1 levels in the upper normal range may be of predictive value for risk of developing prostate cancer [331], breast cancer in healthy premenopausal females [332], and colorectal cancer [333]. Furthermore, cancer risk was inversely correlated with serum IGFBP-3 levels. These studies which have not been uniformly reproduced, provide a strong rationale for maintaining IGF-1 in the mid-normal age-adjusted range, in AGHD patients on GH replacement therapy. Effects of GH Replacement Therapy HYPOPITUITARISM

Complex hormonal interactions occur between GH and other pituitary hormones, which impact diagnosis as well as optimal hormone replacement therapy. Thyroid, adrenal, and sex steroid replacement must be optimized for at least 3 months prior to testing for GHD. In addition, GH replacement may unmask incipient adrenal and thyroid insufficiency, necessitating monitoring of these hormonal interactions in order to achieve optimal hormone replacement [324].

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GH THERAPY IN ADULTS

THYROID HORMONE

GH replacement increases conversion of T4 to T3 and decreases conversion of T4 to reverse T3 [334]. Initiation of GH replacement therapy in eurthyroid patients may therefore be associated with a fall in serum T4 levels, unmasking pre-existing central hypothyroidism [334]. GH stimulation performed in untreated central hypothyroidism may lead to an inaccurate GHD diagnosis. Careful monitoring of thyroid function is important in patients taking thyroid replacement, who are initiated on GH replacement therapy, as thyroid hormone dose increases may be required to maintain normal T4 [324]. GONADAL STEROIDS

Female patients taking oral estrogens require at least twofold higher doses of GH replacement [322], as estrogen administered orally impairs GH action. Therefore, to reduce GH requirements, a nonoral route (such as a transdermal patch) for estrogen replacement in hypopituitary women should be considered. Recommendations for sex steroid replacement in hypopituitary patients after menopause should follow guidelines for the general population. If adjustments are made in the dose of oral estrogens in hypopituitary female patients, the GH replacement dose should be reevaluated, as it may need to be changed. Changes in dose or route of androgen replacement therapy do not require reevaluation of GH dosage. GH replacement during pregnancy is not recommended as there is as yet no clear-cut evidence for efficacy or safety. Furthermore, the placenta is an abundant source of GH [335]. Nevertheless, an observational study of 201 pregnant women reported that, in more than 50% who continued GH therapy, pregnancy outcomes were unchanged [335]. GLUCOCORTICOIDS

GH or IGF-1 decrease 11 β-hydroxysteroid dehydrogenase type I activity, resulting in reduced conversion of inactive cortisone to active cortisol, and thus may unmask secondary hypoadrenalism. The hypothalamic pituitary adrenal axis should therefore be reevaluated during initiation of GH therapy as increased glucocorticoid replacement therapy or initiation of steroid replacement therapy may be required [336]. GH Replacement in Acromegaly In cured acromegaly patients, GHD may sometimes be documented [337]. Determinants of postoperative GHD, seen in about 10% of patients, include immediate (72 hours) postoperative GH levels as well as bilaterality of intrasellar tumor [338]. A retrospective study found that cardiovascular mortality was higher

115

in patients with documented GHD associated with treated acromegaly as compared to mortality occurring after resection of nonfunctioning pituitary adenomas (SMR 3.03, p , 0.02) [339]. In 42 acromegaly patients receiving GH replacement, quality of life was improved and body composition and lipid profiles were controlled without development of glucose intolerance, a hallmark of acromegaly [340].

GH in the Healthy Elderly In animal models, mutations resulting in suppressed GH/IGF-1 axis with reduced GH/IGF-1 signaling actually increase life span [341]. Snell and Ames dwarf mice with defects in anterior pituitary function due to Pit-1 and PROP-1 mutations, respectively, exhibit severely reduced insulin, IGF-1, glucose, and thyroid hormone levels, female infertility, and increased longevity [342]. Lit/Lit drwarf mice with mutations in the extracellular domain of the GHRH receptor had reduced serum IGF-1 levels, increased adiposity, and B25% increased longevity. Heterozygous IGF-1 receptor gene disrupted mice have a 50% reduction in receptor levels, and a 33% increased life-span in females, who are not dwarf [343]. Caloric restriction, another mechanism of decreasing circulating IGF-1 levels, also prolongs the life-span in several species [344]. Serum GH and IGF-1 levels decline progressively with age, a phenomenon referred to as “somatopause.” Increased adiposity and decreased LBM observed in the adult GHD syndrome, also occur with aging. The purported rationale for GH use as an antiaging therapy is the potential for improvement in body composition, bone density, and cholesterol levels observed in GH-deficient adults treated with GH replacement therapy. GH is not approved for use as an antiaging hormone by the FDA, but abuse of GH for this purpose continues to escalate [345]. Randomized controlled studies evaluating safety and efficacy of GH in the healthy elderly are limited [345]. The scant data suggest small but clinically nonsignificant improvements in body composition with adverse events including impaired fasting glucose, onset of diabetes mellitus, carpal tunnel syndrome, edema, arthralgias, and no beneficial effect on strength or physical function. Thus, available evidence does not validate physiological benefits from augmenting the declining GH levels in the normal aging process [346]. Sarcopenia (loss of muscle mass) increases with age and contributes to frailty. Clinical trials using GH in healthy elderly have not been proven to enhance muscle strength or quality of life. Ghrelin may prove to be beneficial in catabolic states, and increases appetite and LBM in healthy older men [347].

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4. GROWTH HORMONE

A study evaluating the contribution of physiological supplementation with GH and testosterone for 16 weeks in elderly community-dwelling males showed improved LBM, muscle strength, and performance with reduced total body and truncal fat [348]. However, as there are no prospective controlled studies, and as inappropriate GH replacement may cause adverse events, GH should not be administered to elderly adults with low IGF-1 and no history of hypothalamic pituitary disease [260,324,346].

endurance. Maximum oxygen consumption was also unchanged [356]. In a single study of a highly select group of abstinent dependent users of anabolic androgenic steroids, hGH (19 μg/kg per day) for 1 week improved strength, peak power output, and IGF-1 levels [357]. Given the published evidence, GH administered to enhance athletic performance has unsubstantiated efficacy and scientific or ethical justification, and the practice is illegal [324,346]. Testing for GH Doping in Athletes

GH Abuse by Athletes Exercise is a potent stimulus of GH secretion and GH levels increase within 10 20 minutes of the onset of exercise, and are sustained for up to 2 hours following exercise. Furthermore, healthy subjects who exercise regularly demonstrate increased 24-hour GH secretion rates. Age, gender, BMI, physical fitness, and duration and intensity of exercise influence the magnitude of the GH response to exercise [349]. Beneficial effects of GH replacement therapy on exercise capacity in truly GHD adults have encouraged unapproved use of GH by athletes [350], and inappropriate use of rhGH by athletes (“doping”) increased from 6% in 2001 to 24% in 2006 [351]. Supraphysiological GH doses to pituitary-replete athletes increases FFA availability, with no effect on fat oxidation [352]. Oxidative protein loss at rest, during, and following exercise is reduced [353] and LBM is increased [354]. However, effects on performance are limited to anaerobic exercise capacity [354]. A systematic review [355] of 56 studies reported on 303 young recreational athletes, average age 27 years, who had received GH for an average of 20 days, many of whom only received one hGH injection. The average GH dose was 36 μg/kg per day (approximately 5 10-fold the replacement dose used in GHdeficient adults). LBM increased in the treatment group, compared to those not treated, with a statistically insignificant decrease in fat mass. There was no improvement in muscle strength after 24 and 84 days of GH administration in two studies. This analysis revealed little beneficial effect of GH in recreational athletes and failed to document improved performance. In a double-blind, randomized, placebo-controlled trial of 96 recreationally trained healthy athletes [356], GH supplementation (2 mg/day, or about 30 μg/kg for a 70-kg person) significantly reduced fat mass and increased LBM, and the addition of testosterone enhanced these effects. The only measure of enhanced physical performance was sprint capacity; no changes were seen in measures of strength, power, or

GH is not FDA-approved for enhancement of athletic performance, and the International Olympic Committee has prohibited GH doping. The GH-2000 project, comprising endocrinologists from four European countries, proposed a test based on the measurement of two GH-sensitive markers, IGF-1 and type III procollagen, which was first used at the Olympic Games in Athens in 2004 [358,359]. Pituitary GH contains several different GH isoforms, while recombinant human GH comprises only the 22-kDa isoform [360,361]. Administration of rhGH suppresses endogenous GH secretion, with increased 22-kDa GH to total GH ratios. To detect the 22-kDa GH isoform, the test must be performed within 24 hours of GH administration; discontinuation of GH the day before the test will result in a false-negative result [361]. The GH-2004 project demonstrated minor ethnic differences in IGF-1 and procollagen III peptide levels in athletes, which did not affect test performance [362]. However, a large cross-sectional study in over 1000 athletes from 12 countries, representing four major ethnic groups and 10 major sport types showed that age, gender, BMI, ethnicity, and sport type contributed to 56% of the variability of IGF-1 axis markers (IGF-1, IGFBP-3, and ALS) and collagen markers (type I procollagen, cterminal telopeptide of type I collagen, and N-terminal propeptide of type III procollagen) [363]. Thus, demographic factors should be taken into account in interpretation of tests using IGF-1 and collagen markers to detect GH doping. Gene expression analysis of peripheral blood leukocytes was attempted as a method to detect GH doping. However, this approach was not found to be clinically valuable for widespread screening [364].

Complications of GH Treatment Adverse reactions to adult GH replacement include peripheral edema, glucose intolerance, arthralgias, myalgias, backache, parasthesias, carpal tunnel syndrome, headache, hypertension, and rhinitis. These are frequent, often transient or disappear with

I. HYPOTHALAMIC PITUITARY FUNCTION

REFERENCES

lowering of the GH dose, and are more common in adult-onset than childhood-onset GH deficiency. Side-effects of GH treatment have been described in GH-deficient patients inappropriately treated with GH (see Table 4.14). Non-GH-deficient adult patients receiving excess GH doses can be likened to acromegaly patients, with increased GH levels. In a metaanalysis of seven studies with 22,654 patients, there was no association of GH replacement therapy with pituitary tumor recurrence (RR 0.87; 95% CI, 0.56 1.33) or the risk of secondary malignancies (RR 1.24; 95% CI, 0.65 2.33) [365]. Prospective surveillance for a mean of 2.3 years of 1988 GH-replaced adult GHD patients compared to controls showed no significant differences in mortality, cancer, diabetes, pituitary tumor growth, or cardiovascular events [366]. Insomnia and sleep apnea are observed to occur at higher frequencies. Elevated (but still within normal range) endogenous IGF-1 concentrations have been epidemiologically correlated with prostate, breast, colon, and lung cancer risk [331,332].

Decreased IGF-1 Levels Protein Calorie Malnutrition, Starvation, Anorexia Nervosa Short-term fasting and protein calorie malnutrition result in elevated basal GH levels and low IGF-1 levels [367], reflecting an uncoupling of the IGF-1 feedback regulation of GH secretion. In patients with anorexia nervosa, basal GH levels are also elevated [368]. Diabetes Mellitus Poorly controlled diabetes mellitus is associated with elevated basal GH levels and increased GH response to exercise. Elevated GH levels return to normal with improved diabetic control after insulin administration. IGF-1 levels are low in children with poorly controlled insulin-dependent diabetes, including those entering puberty, suggesting insulin resistance exacerbated by GH. Laron Syndrome Laron syndrome, an autosomal recessive disorder, is a condition of peripheral unresponsiveness to GH due to inactivating mutations of the GHR [369]. Serum GH levels are normal or elevated, circulating IGF-1 is absent, and there is no IGF-1 response to exogenously administered GH [118]. IGF-1 therapy increases height velocity, with improved body composition as evidenced by loss of fat mass [370].

117

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receiving long-term GH replacement therapy. J Clin Endocrinol Metab 2001;86(11):5172 5. Child CJ, Conroy D, Zimmermann AG, Woodmansee WW, Erfurth EM, Robison LL. Incidence of primary cancers and intracranial tumour recurrences in GH-treated and untreated adult hypopituitary patients: analyses from the Hypopituitary Control and Complications Study. Eur J Endocrinol 2015;172 (6):779 90. Karavitaki N, Warner JT, Marland A, et al. GH replacement does not increase the risk of recurrence in patients with craniopharyngioma. Clin Endocrinol (Oxf) 2006;64(5):556 60. Jostel A, Mukherjee A, Hulse PA, Shalet SM. Adult growth hormone replacement therapy and neuroimaging surveillance in brain tumour survivors. Clin Endocrinol (Oxf) 2005;62 (6):698 705. van Varsseveld NC, van Bunderen CC, Franken AA, Koppeschaar HP, van der Lely AJ, Drent ML. Tumor recurrence or regrowth in adults with nonfunctioning pituitary adenomas using GH replacement therapy. J Clin Endocrinol Metab 2015;100(8):3132 9. Chan JM, Stampfer MJ, Giovannucci E, et al. Plasma insulinlike growth factor-I and prostate cancer risk: a prospective study. Science 1998;279(5350):563 6. Hankinson SE, Willett WC, Colditz GA, et al. Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 1998;351(9113):1393 6. Ma J, Pollak MN, Giovannucci E, et al. Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J Natl Cancer Inst 1999;91(7):620 5. Losa M, Scavini M, Gatti E, et al. Long-term effects of growth hormone replacement therapy on thyroid function in adults with growth hormone deficiency. Thyroid 2008;18 (12):1249 54. Vila G, Akerblad AC, Mattsson AF, et al. Pregnancy outcomes in women with growth hormone deficiency. Fertil Steril 2015;104(5) 1210-7 e1 Mazziotti G, Giustina A. Glucocorticoids and the regulation of growth hormone secretion. Nat Rev Endocrinol 2013;9 (5):265 76. Mazziotti G, Marzullo P, Doga M, Aimaretti G, Giustina A. Growth hormone deficiency in treated acromegaly. Trends Endocrinol Metab 2015;26(1):11 21. Ku CR, Hong JW, Kim EH, Kim SH, Lee EJ. Clinical predictors of GH deficiency in surgically cured acromegalic patients. Eur J Endocrinol 2014;171(3):379 87. Tritos NA, Johannsson G, Korbonits M, et al. Effects of long-term growth hormone replacement in adults with growth hormone deficiency following cure of acromegaly: a KIMS analysis. J Clin Endocrinol Metab 2014;99(6):2018 29. Giavoli C, Profka E, Verrua E, et al. GH replacement improves quality of life and metabolic parameters in cured acromegalic patients with growth hormone deficiency. J Clin Endocrinol Metab 2012;97(11):3983 8. Berryman DE, Christiansen JS, Johannsson G, Thorner MO, Kopchick JJ. Role of the GH/IGF-1 axis in lifespan and healthspan: lessons from animal models. Growth Horm IGF Res 2008;18(6):455 71. Bartke A, Wright JC, Mattison JA, Ingram DK, Miller RA, Roth GS. Extending the lifespan of long-lived mice. Nature 2001;414 (6862):412. Flurkey K, Papaconstantinou J, Miller RA, Harrison DE. Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc Natl Acad Sci USA 2001;98(12):6736 41.

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[344] Barzilai N, Bartke A. Biological approaches to mechanistically understand the healthy life span extension achieved by calorie restriction and modulation of hormones. J Gerontol A Biol Sci Med Sci 2009;64(2):187 91. [345] Liu H, Bravata DM, Olkin I, et al. Systematic review: the safety and efficacy of growth hormone in the healthy elderly. Ann Intern Med 2007;146(2):104 15. [346] Clemmons DR, Molitch M, Hoffman AR, et al. Growth hormone should be used only for approved indications. J Clin Endocrinol Metab 2014;99(2):409 11. [347] Nass R, Pezzoli SS, Oliveri MC, et al. Effects of an oral ghrelin mimetic on body composition and clinical outcomes in healthy older adults: a randomized trial. Ann Intern Med 2008;149 (9):601 11. [348] Sattler FR, Castaneda-Sceppa C, Binder EF, et al. Testosterone and growth hormone improve body composition and muscle performance in older men. J Clin Endocrinol Metab 2009;94 (6):1991 2001. [349] Holt RI, Webb E, Pentecost C, Sonksen PH. Aging and physical fitness are more important than obesity in determining exercise-induced generation of GH. J Clin Endocrinol Metab 2001;86(12):5715 20. [350] Holt RI, Erotokritou-Mulligan I, Sonksen PH. The history of doping and growth hormone abuse in sport. Growth Horm IGF Res 2009;19(4):320 6. [351] Baker JS, Graham MR, Davies B. Steroid and prescription medicine abuse in the health and fitness community: a regional study. Eur J Intern Med 2006;17(7):479 84. [352] Irving BA, Patrie JT, Anderson SM, et al. The effects of time following acute growth hormone administration on metabolic and power output measures during acute exercise. J Clin Endocrinol Metab 2004;89(9):4298 305. [353] Healy ML, Gibney J, Russell-Jones DL, et al. High dose growth hormone exerts an anabolic effect at rest and during exercise in endurance-trained athletes. J Clin Endocrinol Metab 2003;88 (11):5221 6. [354] Birzniece V, Nelson AE, Ho KK. Growth hormone and physical performance. Trends Endocrinol Metab 2011;22(5):171 8. [355] Liu H, Bravata DM, Olkin I, et al. Systematic review: the effects of growth hormone on athletic performance. Ann Intern Med 2008;148(10):747 58. [356] Meinhardt U, Nelson AE, Hansen JL, et al. The effects of growth hormone on body composition and physical performance in recreational athletes: a randomized trial. Ann Intern Med 2010;152(9):568 77. [357] Graham MR, Baker JS, Evans P, et al. Physical effects of shortterm recombinant human growth hormone administration in abstinent steroid dependency. Horm Res 2008;69(6):343 54.

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[358] Powrie JK, Bassett EE, Rosen T, et al. Detection of growth hormone abuse in sport. Growth Horm IGF Res 2007;17 (3):220 6. [359] Erotokritou-Mulligan I, Guha N, Stow M, et al. The development of decision limits for the implementation of the GH2000 detection methodology using current commercial insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays. Growth Horm IGF Res 2012;22 (2):53 8. [360] Wallace JD, Cuneo RC, Bidlingmaier M, et al. Changes in non-22-kilodalton (kDa) isoforms of growth hormone (GH) after administration of 22-kDa recombinant human GH in trained adult males. J Clin Endocrinol Metab 2001;86 (4):1731 7. [361] Baumann GP. Growth hormone doping in sports: a critical review of use and detection strategies. Endocr Rev 2012;33 (2):155 86. [362] Erotokritou-Mulligan I, Bassett EE, Cowan DA, et al. Influence of ethnicity on IGF-I and procollagen III peptide (P-III-P) in elite athletes and its effect on the ability to detect GH abuse. Clin Endocrinol (Oxf) 2009;70(1):161 8. [363] Nelson AE, Howe CJ, Nguyen TV, et al. Influence of demographic factors and sport type on growth hormone-responsive markers in elite athletes. J Clin Endocrinol Metab 2006;91 (11):4424 32. [364] Mitchell CJ, Nelson AE, Cowley MJ, et al. Detection of growth hormone doping by gene expression profiling of peripheral blood. J Clin Endocrinol Metab 2009;94(12):4703 9. [365] Jasim S, Alahdab F, Ahmed AT, et al. Mortality in adults with hypopituitarism: a systematic review and meta-analysis. Endocrine 2016; [Epub ahead of print] [366] Hartman ML, Xu R, Crowe BJ, et al. Prospective safety surveillance of GH-deficient adults: comparison of GH-treated vs untreated patients. J Clin Endocrinol Metab 2013;98(3):980 8. [367] Cahill Jr. GF, Herrera MG, Morgan AP, et al. Hormone-fuel interrelationships during fasting. J Clin Invest 1966;45 (11):1751 69. [368] Isley WL, Underwood LE, Clemmons DR. Dietary components that regulate serum somatomedin-C concentrations in humans. J Clin Invest 1983;71(2):175 82. [369] Backeljauw PF, Underwood LE. Therapy for 6.5-7.5 years with recombinant insulin-like growth factor I in children with growth hormone insensitivity syndrome: a clinical research center study. J Clin Endocrinol Metab 2001;86 (4):1504 10. [370] Laron Z, Klinger B. Comparison of the growth-promoting effects of insulin-like growth factor I and growth hormone in the early years of life. Acta Paediatr 2000;89(1):38 41.

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C H A P T E R

5 Prolactin Nadine Binart

INTRODUCTION Prolactin (PRL) is mainly produced by pituitary lactotrophs and is tonically inhibited by the hypothalamus by the neurotransmitter dopamine. The discovery of multiple extrapituitary sites of PRL secretion also increases the range of known functions of this hormone. Its primary function is to enable breast milk production, although PRL receptors (PRLRs) are found in many other tissues. The major isoform, 23-kDa PRL, acts via a membrane receptor, the prolactin receptor (PRLR), a member of the hematopoietic cytokine superfamily, and for which the mechanism of activation has been elucidated. High levels of PRL in humans may interfere with reproductive function mainly by actions at the hypothalamus.

HISTORICAL OVERVIEW In the late 1920s, it was found that pituitary extracts induce milk secretion. Riddle and coworkers found that this substance, which they named prolactin (PRL), could be differentiated from the known growth- and gonad-stimulating substances [1]. In these experiments, they showed that PRL stimulated milk production by guinea pig mammary glands and a milk-like substance from the crop sacs of pigeons and doves, giving rise to the pigeon crop sac bioassay for PRL. Over the ensuing years, PRL was characterized, sequenced and specific radioimmunoassays (RIAs) developed for PRL from a number of species. Because of the high lactogenic activity of even very highly purified preparations of human growth hormone (GH), however, it was impossible to separate human PRL from GH using the relatively crude pigeon crop assay. However, several human disease states provided strong evidence that these two hormones were separate. For example, it was observed that most patients with

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00005-2

pituitary tumors in whom galactorrhea and amenorrhea were the cardinal clinical features did not have acromegalic features and patients who were known to have isolated, congenital GH deficiency were able to undergo postpartum lactation. Finally, in 1970, Frantz and Kleinberg developed a sensitive in vitro bioassay which involved staining milk produced by cultured, lactating mouse mammary tissue in response to PRL that was capable of measuring PRL levels as low as 5 ng/mL. In this assay they added excess antibody to GH to neutralize any potential lactogenic effects it had and, for the first time, were able to demonstrate measurable PRL levels in women with puerperal and nonpuerperal galactorrhea but not in most normal men and women. Shortly thereafter, an RIA for human PRL was developed which could finally measure PRL levels in the sera of normal individuals, permitting the entire amino acid sequencing of human PRL [2] and determination of its cDNA sequence.

CELL OF ORIGIN PRL is made by the pituitary lactotrophs (also known as mammotrophs). In the normal human pituitary, the lactotrophs comprise about 15 25% of the total number of cells, are similar in number in both sexes, and do not change significantly with age. During pregnancy and subsequent lactation, however, lactotroph hyperplasia may be observed, presumably as a result of lactotroph proliferation, transdifferentiation of somatotrophs, and/ or expansion from a stem cell population [3]. The hyperplastic process involutes within several months after delivery, although breastfeeding retards this process. This stimulatory effect of pregnancy on the lactotrophs also holds true for prolactinomas, which may be subject to significant pregnancy-induced tumor enlargement (see chapter: Prolactinoma).

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Lactotroph Ontogeny Continuous hypothalamic pituitary interaction takes place during embryologic development. During the formation of Rathke’s pouch, the primordium of the anterior pituitary, the ectodermal primordial cells of the anterior and intermediate lobes of the pituitary make contact with the neuroectoderm of the floor of the diencephalon and experimental studies have shown inductive interactions between these tissues that are necessary for their subsequent interdependent development. Elegant studies using a series of targeted mutations have shown that there are a number of transcription factors (Six-3, Hesx1, Lhx3, Lhx4, Sox2, Sox 3, Pitx2, Otx2, Bmp2, Bmp4, and Gli2) that are sequentially expressed in the developing hypothalamus and pituitary that lead to the final determination of the five mature pituitary cell types and the functional integration of the hypothalamic pituitary system [4]. Mutations in most of the genes for these factors have been shown in humans to result in disordered development of hypothalamic, pituitary, and other brain structures with varying degrees of hypopituitarism. The POU homeodomain transcription factor Pou1f1 (also called Pit-1) gene becomes activated relatively late in development and is necessary for the activation of the PRL, GH, growth hormone-releasing hormone (GHRH) receptor, and TSHβ genes as well as being necessary for the differentiation and proliferation of these cell lineages. A point mutation in the POU homeodomain of Pou1f1 has been found to be the cause of the GH, PRL, and TSH deficiencies found in the Snell dwarf mouse, with absence of somatotroph, lactotroph, and thyrotroph cells. Similar mutations in Pou1f1 have now been found to cause a similar deficiency of GH, PRL, and TSH in humans [4]. A second paired-like homeodomain factor, known as Prophet of Pit-1 (Prop-1) has also been found to be necessary for the expression of Pit-1 and mutations of the Prop-1 gene cause the dwarfism in mice known as the Ames mouse (defects of somatotrophs, lactotrophs, and thyrotrophs) and similar mutations in human cause variable deficiencies of GH, PRL, TSH, LH, and FSH [4]. Many lactotrophs arise from cells that at least at some point expressed the GH gene. However, at least in mice, it appears that many, if not most, lactotrophs may derive from an earlier precursor [5]. Subsequent lactotroph proliferation occurs once estrogen receptors (ERs) appear. Estrogen stimulates PRL gene transcription (see below) only if Pou1f1 is bound to the PRL promoter. Stimulation by Prop-1 is necessary for the subsequent development of all noncorticotroph cells. Pou1f1 is then necessary for the development and proliferation of thyrotrophs and somatomammotrophs, but the separation of these two cell lines occurs before the appearance of the ability to

synthesize α-subunits. The final differentiation of somatomammotrophs occurs at least in part in relationship to estrogen stimulation.

PROLACTIN GENE PRL belongs to the somatotrophin/PRL family, a large family of proteins that includes GH, placental lactogens (PLs), PRL-like and PRL-related proteins, proliferins, and proliferin-related proteins. The human PRL gene is located on chromosome 6p22.2-p21.3 and consists of five coding exons, one noncoding exon, and four introns. It is believed that PRL, GH, and PL arose from duplication of a common ancestral gene B400 million years ago. The entire PRL locus in both humans and rats spans a region of B10 kb. The human PRL cDNA is 914 nucleotides long and contains a 681-nucleotide open reading frame encoding mRNA for a prohormone (preprolactin) of 227 amino acids. During PRL processing, the 28-amino-acid signal peptide is proteolytically cleaved, resulting in a mature 199-amino-acid PRL polypeptide with a molecular weight of 23 kDa. Genes for the related hormones GH and PL (or chorionic somatomammotrophin) are clustered on chromosome 17. Much of the knowledge regarding regulation of PRL gene expression has been derived from studies utilizing the rat pituitary PRL promoter. Less information is available on regulation of hPRL and mouse PRL. Although attempts have been made to investigate hPRL regulation by transfecting the hPRL promoter into rat GH3 cells, it is uncertain how reliably this type of experimental system recapitulates hPRL promoter activity, since transformed rat and human lactotrophs may not share the same repertoire of endogenous transcriptional and/or epigenetic regulators. The rprl gene is controlled by a proximal promoter and a distal enhancer, located 2433/ 220 bp and 1800/ 21500 bp, respectively, relative to the pituitary transcriptional start site. In rats, 3 kb of 5’-flanking region is sufficient to direct lactotroph-specific transgene expression and a synergistic interaction between the distal enhancer and proximal promoter region is required for high levels of expression. Sequences flanking the enhancer restrict prl expression to pituitary lactotrophs. Organization of the hPRL gene is more complex, and its transcription is regulated by two main independent promoter regions (Fig. 5.1). The proximal region directs pituitary-specific expression, while a more upstream promoter region, designated the “superdistal PRL promoter,” is responsible for directing extrapituitary expression [6]. Although most of the circulating PRL in serum is produced by pituitary lactotrophs, PRL is also expressed in several extrapituitary tissues, including uterine decidualized

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FIGURE 5.1 Schematic diagram of the human PRL 5’ gene regulatory region, gene, and mRNA transcript. The PRL gene consists of five

exons, designated by the numbers (1 5). The region from 25800 base pairs (bp) to 0 indicates the region known to regulate pituitary PRL synthesis whereas the region from 28789 to 25800 is referred to as the “superdistal” or “extrapituitary” promoter region. Thirteen Pit-1 binding sites (blue) are present in the regulatory region. A single degenerate estrogen response element (ERE) (red) has been identified at 21189 bp. The extrapituitary PRL mRNA is B150 bp longer than the pituitary transcript, and has a different 5’ UTR, but transcription from either promoter produces identical protein-coding sequences.

endometrium, breast, brain, ovary, prostate, endothelial cells, lymphocytes, skin, adipose tissue, and cochlea [6]. PRL expression at these sites is cell typespecific and Pit-1-independent. In human extrapituitary tissues, PRL mRNA transcription is driven by the alternative “superdistal PRL promoter” located 5.8 kb upstream of the pituitary transcription start site, resulting in transcription of an extra exon, designated exon 1a. This alternative exon 1a of hPRL gene is noncoding, and transcription from either promoter produces mRNAs with differing 5’ untranslated regions (UTRs) but identical protein-coding sequences. Although the purpose of the alternative promoter is not known for certain, it appears to confer tissue-specific expression, and it is likely that the variant 5’ UTRs may influence the stability or translational efficiency of the disparate PRL transcripts [7]. Characterization and analysis of the far upstream elements dictating extrapituitary hPRL expression are incomplete but evolving. In silico analysis of the extrapituitary hPRL promoter has revealed that the 5’ proximal region of the extrapituitary hPRL promoter, as well as exon 1a and a portion of intronic

sequence, occur within a long terminal repeat-like transposable element (TE) of the medium reiteration frequency (MER) family. The LTR sequence, named MER39, appears to have been inserted B30 million years ago before the divergence of monkeys from higher apes. In addition, an older TE, MER20, provides an additional 198 bp of the 5’ flanking region of exon 1a. TE-derived sequences typically regulate nearby human genes, and in most cases, the LTR acts as an alternative promoter, but does not alter the coding sequence. Some of the transcription factor binding sites that mediate extrapituitary PRL expression are located within these TE sites. Thus, the evolutionary acquisition of these two TEs may underlie the ability of mammals to express PRL in extrapituitary tissues.

Pit-1 Transcriptional regulators that control the development of anterior pituitary lactotrophs also control PRL synthesis during adult life. Prominent among these is

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the Pit-1 protein, a POU homeodomain transcription factor that dictates the terminal differentiation of pituitary somatotrophs, lactotrophs, and thyrotrophs and regulates expression of their respective genes, GH, PRL, and TSHβ. Multiple signaling pathways ultimately converge upon Pit-1, and as such, Pit-1 serves as the main transcriptional regulator of PRL gene expression. Pit-1 integrates information from a wide range of signaling pathways (triiodothyronine, estradiol, glucocorticoids, protein kinase A (PKA), protein kinase C (PKC), and Ras) in a cell-specific manner by functionally interacting with numerous nuclear hormone receptors and coregulators (including itself, ER, TR, GR, cJun, Oct1, GATA2, P-Lim, Ptx-1, Ets), and recruiting them to Pit-1 regulated promoters. Through its interactions with specific DNA elements in target gene promoters, Pit-1 recruits coregulatory proteins that alter histone acetylation and modify the chromatin structure, providing either a permissive or repressive environment for transcription [8]. Precise homeostatic control is achieved through a network of interactions between Pit-1 and several different classes of transcription factors, including the nuclear receptors, other homeodomain proteins, Ets family proteins, and basic region-leucine zipper (B-Zip) transcription factors. Transcriptional activation of the PRL gene requires the assembly of specific coactivator complexes at Pit-1 composite DNA binding sites. There are eight Pit-1 binding sites (four within the proximal promoter, four within the enhancer) in the regulatory region of the rat prl gene, whereas there are 13 known Pit-1 binding sites (three within the proximal promoter, eight within the distal enhancer, two within the superdistal region) in the human PRL promoter [9]. CBP/p300 is required for Pit-1 activation of PRL promoter in response to PKA activation. Pit-1 contains two well-defined functional motifs that affect its activity: the transcriptional activation domain and DNA binding domain (DBD). The aminoterminal TAD contains a regulatory domain and a basal and Ras-responsive region. This latter region contains a basal activation region and an overlapping, dual-function, Ras-responsive and inhibitory segment. Distinct coactivators mediate basal and Ras-activated Pit-1 TAD activity, with CBP/p300 being a key effector of Pit-1’s basal TAD, and steroid receptor coactivator-1 (SRC-1) a mediator of Ras responsiveness [10]. DNA binding of Pit-1 is achieved through a carboxyterminal DBD composed of two motifs, referred to as the POU-specific (POUS) and POU-homeo (POUHD) domains, both of which are necessary for high-affinity DNA binding. The combination of this bipartite DBD and specific DNA binding sites generates a complex “code” which enables various subdomains within Pit-1

to adopt specific configurations; these subsequently control the recruitment of various coregulators to finetune PRL gene transcription [11]. A Pit-1 splice isoform known as Pit-1β contains a 26-amino-acid motif inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain, resulting from an alternative splicing event that uses an in-frame acceptor 78 nucleotides upstream of exon 2. In contrast to the activation of anterior pituitary hormone promoters by Pit-1, Pit-1β represses GH, PRL, and TSHβ promoters in a pituitary cell-specific manner [12]. The Pit-1β repression is potent as it is capable of inhibiting the oncogenic Ras response of the rprl promoter. Mechanistically, this Pit-1β motifdependent transcriptional repression results from the active recruitment of corepressors as well as interference with efficient recruitment of CBP [13,14]. Studies investigating Pit-1 regulation of PRL and pituitary gene expression have determined that several mechanisms exist that enable Pit-1 to respond to multiple diverse stimuli. For example, Pit-1 largely binds to DNA elements as a dimer, but under the influence of certain signaling events, will bind as a monomer. Crystal structure analysis reveals that Pit-1 homodimerizes in a head-to-tail fashion to achieve optimal DNA binding on an idealized palindromic sequence. The activity of Pit-1 homodimers on dimeric sites in the rprl promoter is determined by the balance between corepressor (NCoR/SMRT, mSin3A/B) and coactivator (histone acetylases, CBP, P300/CBP) complexes at the transcriptional start site. In fact, homodimerization of Pit-1 appears to be important to maximize CBP recruitment in response to insulin and other growth factor stimulation. In contrast, Ras or ERα activation of the rprl promoter at a monomeric Pit-1/ Ets composite binding site is independent of CBP and instead relies upon a p160 SRC coactivator complex. The recruitment of these specific transcriptional complexes is dependent upon structural features that are revealed when Pit-1 binds to DNA sites as a monomer or as a dimer. In addition, differences in the spacing between DNA contact points for the POUS and POUHD domains alter the structure of the linker region, such that the 4-bp spacing on a Pit-1 site in the prl promoter results in an activating Pit-1 conformation, whereas a 6-bp spacing in the gh promoter coverts Pit-1 to a repressor in pituitary lactotrophs [15]. Finally, phosphorylation of Pit-1 by PKA, PKC, and cyclindependent kinases modifies its conformation on DNA recognition elements and thus alters its DNA binding affinity. Phosphorylation specifically inhibits binding of Pit-1 to monomeric DNA sites (i.e., those used by Ras and estradiol) and consequently decreases its transcriptional activity in response to these signaling events, whereas phosphorylation has no effect on

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basal, dimeric Pit-1 binding [16]. Thus, a plethora of mechanisms, including monomeric versus dimeric Pit1 binding, relative spacing of the Pit-1 POUS and POUHD binding sites, and the phosphorylation status of Pit-1 altogether generate a unique structural interface for the binding of distinct coactivator/corepressor complexes to regulate the expression of the PRL gene in pituitary lactotrophs.

Estrogen Estrogen is an essential physiological activator of both PRL gene synthesis and lactotroph proliferation. At present, three identified ERs are expressed in the adenohypophysis: two classical nuclear ERs, ER alpha (α), and ER beta (β), which function as ligandactivated nuclear transcription factors, and G proteincoupled estrogen receptor 1 (GPR30, GPER1), a seventransmembrane G-protein-coupled receptor (GPCR) that binds with high affinity to estradiol and mediates rapid signaling events. Both ERα and ERβ are expressed in human and rat lactotrophs. The mouse pituitary expressed ERα, but not ERβ. GPR30 expression in rodent adenohypophysis has been confirmed by in situ hybridization and immunohistochemistry, but detailed information regarding specific cell-type localization of this receptor, or possible functional roles in mediating or contributing to estradiol effects in lactotrophs, are not known. The in vivo function of ERα in regulating prl expression has been demonstrated in ERα2/2 mice. Although the specification of the lactotroph lineage occurs normally in these animals, there is a 10 20-fold reduction in prl mRNA levels and a decrease in the number of lactotrophs [17]. Curiously, plasma PRL levels are only slightly reduced, probably as a result of compensatory mechanisms, such as heightened sensitivity to PRL secretagogues. Pituitaries from ERβ 2/2 mice exhibit normal prl expression, but the lack of ERβ expression in wild-type mouse pituitary precludes the ability to draw conclusions about the role of ERβ in human PRL expression and/or lactotroph function on the basis of this genetic mouse model [18]. The ER selectively binds to a single estrogen response element (ERE) located within the distal rPRL enhancer adjacent to the monomeric Pit-1d site at approximately 1.5 kb upstream from the transcription initiation site. This ERE has been shown to mediate a dramatic synergistic interaction between Pit-1 and ER that results in a 60-fold induction of rPRL transcription in response to estrogen. Binding of Pit-1 to this distal enhancer site as a monomer dictates the use of a specific aminoterminal transactivation domain that is necessary to synergize with ERα in a cell-specific manner. Interestingly, this Pit-1 synergy domain is not required for synergistic events on the GH promoter. Estrogen-induced PRL

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expression also requires an intact mitogen-activated protein kinase (MAPK) signaling transduction pathway as interfering with MAPK activation ablates the ability of estrogen to induce PRL expression [19]. A degenerate yet functional ERE sequence also exists in the hPRL promoter at 21189 bp relative to the transcription start site. This ERE differs significantly from the rprl ERE, although it is located within the corresponding sequence. ERα and Pit-1 exert synergistic transcriptional effects on both the rat and human PRL promoters, suggesting that Pit-1 and ER are involved in the formation of a multiprotein complex at this site. The hPRL ERE has a relatively low binding affinity for ERα, and when stimulated by estradiol alone, exhibits modest transcriptional activity. However, a marked synergistic transcriptional effect is observed in the presence of estradiol and TNFα and it appears that this particular ERE sequence is essential for the TNFαinduced, NFκB-mediated activation of the hPRL promoter [20]. Thus, the ERE sequence in the hPRL promoter appears to be a target for at least two signaling pathways and, as such, may represent an important converging point for integrating multiple physiological endocrine signals in vivo.

Ets Members of the Ets transcription factors family are key regulators controlling PRL gene expression. Analyses of the rprl promoter in somatolactotroph cell lines have identified two critical Ets binding sites (EBS): a composite Ets-1/Pit-1 binding site located at 2212 and a more proximal EBS located at 296. The composite Ets-1/Pit-1 binding site confers synergy between the two proteins and mediates stimulation by the Ras/MAPK signaling transduction pathway, including those initiated by fibroblast growth factor (FGF) and thyrotrophin-releasing hormone (TRH). Although Pit-1 and Ets interact directly, the synergistic activation of PRL gene expression does not necessitate this physical interaction, but does require the assembly of distinct Pit-1 transcriptional activation domains, as well as the specific sequence of the composite site. The synergy between Pit-1 and Ets-1 can be prevented by an Ets-2 repressor factor apparently by preventing Pit1 from binding to the composite site. The proximal EBS centered at 296, the target of several growth factor signaling pathways, is recognized and activated by Ets factors GA-binding protein α and β.

Other Transcription Factors Several additional transcription-regulating proteins identified in the pituitary have been implicated as key elements in the regulation of the PRL promoter. These

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include Pitx factors, thyroid hormone receptor (via activating protein-1 [AP-1] transcription factor), CCAAT/enhancer-binding protein (C/EBPα), SMAD4, and Ikaros. The Pitx family is a class of bicoid homeodomain proteins required for the development of several organs. Among the three members of this family, Pitx1 and Pitx2 are expressed in the anterior pituitary and in several pituitary cell lines. Both Pitx1 and Pitx2 can interact and synergize with Pit-1 in activating pituitary-specific promoters. The synergy between Pitx2 and Pit-1 is achieved by Pit-1 binding to the carboxy-terminal tail of Pitx2, which relieves the autorepression imposed by this region, thereby increasing DNA binding of Pitx2 to a canonical bicoid site. Two bicoid sites, B1 and B2, located at 227 and 2110, respectively, have been identified in the human PRL proximal promoter; the B2 site and two Pit-1 binding sites are necessary for the synergistic interaction of Pitx2 and Pit-1 [21]. Although an intact B2 Pitx binding site is necessary for full responsiveness to several signaling pathways regulating the hPRL promoter, Pitx factors play a secondary role to factors such as Pit-1 and Ets in the regulation of hPRL gene expression, since mutation of the B1 and B2 sites has only modest inhibitory impact on promoter activity [21]. Triiodothyronine (T3) downregulates transcription of hPRL gene. The hPRL promoter contains two T3 responsive regions. The first, located in the proximal promoter, mediates a strong negative effect, while the second, located in the distal promoter, mediates a weak positive effect. The overall effect of the two combined regions is negative. T3 exerts its inhibitory effect by binding to thyroid hormone receptor (TR) and interfering with the AP-1 transactivation mediated by an AP-1-binding site located in the proximal hPRL promoter. The T3/TR complex has different effects on prl expression in the rat, where it mediates an overall activation of the rprl promoter [22]. C/EBPα, a member of the bZip family of transcription factors, also synergizes with Pit-1 to stimulate the rprl promoter and the rgh promoter [23]. The importance of C/EBPα interactions with Pit-1 is underscored by the observation that mutations in Pit-1 that disrupt physical binding between Pit-1 and C/EBPα lead to combined pituitary hormone deficiency in humans. The DNA binding site utilized by the Pit-1-C/EBPα complex in the prl promoter overlaps with the proximal EBS which is recognized by GABPα/GABPβ [23]. Bone morphogenetic protein-4 (BMP-4), one of the members of the TGF-β superfamily, plays a role in pituitary development from the initial induction of Rathke’s pouch to cell specification in the anterior lobe and differentiation of the lactotroph lineage [24]. BMP4, which is also overexpressed in different rodent prolactinoma models, exerts proliferative effects on postnatal lactotrophs through a complex mechanism

involving crosstalk among intracellular signaling pathways of BMP-4, Smad-4, and estradiol. In addition to effects on lactotroph proliferation, BMP-4 and estradiol synergistically activate transcription of the rprl promoter independently of EREs, but in a manner dependent upon a Smad binding element located between 22000 and 21500 bp relative to the pituitary transcriptional start site. In mammosomatotroph cells that express both GH and PRL, the zinc finger transcription factor Ikaros suppresses gh but activates prl gene expression. The mechanism for this differential effect on hormone expression appears to rely on the state of chromatin accessibility for Pit-1, as mediated in part by Ikaros. Access of Pit-1 is modulated in a gene-specific manner such that Ikaros selectively deacetylates histone 3 residues on the GH promoter, and therefore restricts the access of Pit-1 [25]. In contrast, Ikaros acetylates histone 3 on the proximal prl promoter and thereby facilitates Pit-1 binding to this region in the same cells. Thus, Ikaros-mediated histone acetylation and chromatin remodeling provides one potential mechanism for the selective regulation of pituitary GH and PRL gene expression in cells that are capable of elaborating both.

Signaling Pathways That Converge Upon the PRL Promoter PRL expression is dynamically regulated by neurotransmitters, hormones, and growth factors through activation or inhibition of GPCRs and receptor tyrosine kinases that feed into several signal transduction pathways (Fig. 5.2). These positive and negative signals ultimately converge upon the regulatory promoter region. The intracellular signal transduction pathway linking dopamine (DA) to the PRL gene involves inhibition of adenylate cyclase and the cAMP/PKA pathway. PKA-dependent cAMP signals typically activate the transcription factor cAMP response element (CRE)binding protein (CREB), leading to its homodimerization on DNA complexes. However, the human and rat PRL promoters lack functional cAMP DNA response elements (CREs), and although a one-half CRE is located in the rPRL promoter, CREB does not bind to this sequence with high affinity. Therefore alternative undefined molecular mechanisms are involved in cAMP-dependent PRL gene regulation [26]. Growth factors and hormones, such as insulin, epidermal growth factor (EGF), and TRH, utilize PKC-dependent pathways to directly phosphorylate the coactivator CBP, which stimulates its subsequent recruitment to Pit-1, to regulate transcription of the PRL gene. Other signaling pathways involving phosphoinositide 3-kinase (PI3K)-Akt phosphorylation of CREB have also been proposed to mediate induction of the PRL

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FIGURE 5.2 Schematic diagram of the membrane receptors and corresponding signal transduction pathways in pituitary lactotrophs that are involved in PRL gene regulation. The dopamine D2 receptor (D2R) exerts the main inhibitory influence upon PRL synthesis. TRHR, thyrotrophin-releasing hormone receptor; VIPR, vasoactive inhibitory peptide receptor; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; PLC, phospholipase C; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C; AKT, protein kinase B; Creb, cAMP response element binding protein; ER, estrogen receptor.

promoter by insulin and prolactin-releasing peptide. In this latter pathway, CREB probably regulates PRL promoter activity through interaction with an Ets family member. Extracellular signal-regulated kinase-1/2 (Erk-1/2) activation serves as a point of convergence for PRL gene regulation by numerous stimuli, including vasoactive intestinal peptide (VIP), insulin-like growth factor 1 (IGF-1), pituitary adenylyl cyclase activating polypeptide (PACAP), and fibroblast growth factor 2 (FGF2). These stimuli initially signal through pathways characterized by differential utilization of monomeric G proteins. Thus, VIP and IGF-1 stimulate PRL gene expression through a Ras/Raf/Erk/Ets cascade, whereas PACAP signals through Rap1/Braf/Erk, and FGF2 signals through Rac-1/phospholipase C (PLC)/ PKC/Erk to control PRL gene expression [27,28]. Some of the intra- and extracellular factors that participate in lactotroph proliferation and PRL gene expression appear to participate in complex crosstalk signaling pathways when studied in vitro. For example, BMP4 and estradiol exert a synergistic effect on PRL gene expression in a manner dependent upon Smad1, but independent of EREs [29]. TGFβ, however, inhibits PRL transcription in a manner that overrides both BMP4 and estradiol stimulatory actions. Similarly, EGFR and ERα participate in overlapping

signaling pathways that modulate PRL expression and release [30]. The cytokine TNF-α activates the hPRL promoter through NF-κB signaling in a manner that is also dependent upon ERα [20,31]. Since most of these observations are based upon data derived from transformed rodent cell lines that lack normal dopaminergic control, the relevance of these crosstalk patterns for human PRL expression and lactotroph function remains to be confirmed.

HORMONE BIOSYNTHESIS Prolactin Protein Structural Characteristics and Posttranslational Modifications The mature human PRL protein is composed of 199 amino acids [32,33]. A comparison of the sequence homology at the amino acid level from different species shows that the degree of conservation is highly variable among mammalian and nonmammalian species, reflecting their phylogenetic relationships. Primate PRL has 97% homology to hPRL, whereas rodent PRL has only 61 64% homology. Moreover, rat PRL is capable of activating the human PRL receptor (PRLR), whereas

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FIGURE 5.3 Schematic illustration of PRL proteins as they exist in the serum as three forms: monomeric 23-kDa PRL ( . 95%), “big PRL,” consisting of PRL aggregates, and “big, big PRL,” or macroprolactin, consisting of PRL bound to IgG. The native PRL protein is composed of 199 amino acids. Red hatching indicates the relative locations of the three disulfide bonds. A single N-glycosylation site has been identified on human PRL at codon 31. Two putative phosphorylation sites (not depicted) have been proposed at serines 163 and 194.

mouse PRL cannot. The PRL polypeptide is arranged in a single chain of amino acids with three highly conserved intramolecular disulfide bonds between six cysteine residues (Fig. 5.3). According to nuclear magnetic resonance spectroscopy, PRL folds into four antiparallel α-helices, similar to the tertiary structure of GH and other close relatives [34]. Posttranslational modifications of the PRL polypeptide, such as glycosylation, phosphorylation, proteolytic cleavage, and polymerization influence its stability, receptor binding, measurement, and biological activity. Phosphorylated forms of PRL have been identified in most species, although it is not known whether these forms appear in the plasma in vivo. Mass spectrometry of standard human pituitary extracts indicates that B19% of human PRL exists in a monophosphorylated form, another 19% in a diphosphorylated form, and B62% is unphosphorylated. In the human pituitary, PRL is phosphorylated at serine (ser)194 and ser163; in sera, ser163 is primarily dephosphorylated. The physiologic role of phosphorylated PRL may be inferred from investigations using a recombinant artificial mutant of PRL that has been developed as a PRLR antagonist. S179D is a mimic of monophosphorylated hPRL in which the putative serine phosphorylation site is replaced by an aspartate residue [35]. Phosphorylated PRL has reduced potency in standard bioassays, and it antagonizes the proproliferative action of the predominant unphosphorylated form. As compared to unmodified PRL, S179D PRL

inhibits cell proliferation, promotes differentiation, is proapoptotic, and antiangiogenic. These biological properties may result from differential use of postreceptor signaling pathways [35]. Although both phosphomimetic S179D and unmodified PRL interact with the same PRLR, unmodified PRL preferentially activates the Jak-Stat signaling cascade, whereas S179D predominantly activates Erk 1/2. In the rat pituitary, the relative ratio of phosphorylated to nonphosphorylated PRL isoforms is altered during different phases of male reproductive development [36], and various stages of the estrous cycle, suggesting that this posttranslational modification may have functional significance. Whether phosphorylation modifications have biological relevance and, the extent to which endogenous phosphorylated forms of hPRL participate in biologically significant PRLR signaling, has not been conclusively determined. Glycosylated PRL has been identified in the pituitary glands of several mammalian and nonmammalian species at highly variable degrees (1 60%) [36]. hPRL is N-glycosylated on N31. Like other PRL variants, glycosylation lowers its biological activity as well as its receptor binding and metabolic clearance rate, glycosylated PRL may account for rare cases of mild asymptomatic unexplained hyperprolactinemia. Proteolysis Several PRL variants are generated from proteolytic cleavage of the 23-kDa protein [37]. The major 16-kDa variant is a product of cleavage occurring outside the cells in the interstitial medium and, in the vicinity of blood capillaries, which implies that tissue-specific mechanisms of regulation exist. This 16-kDa PRL variant contains only the N-terminal part of the mature protein and does not bind the PRLR. New evidence suggests that this variant is produced in several tissues. The 16-kDa variant seems to bind to endothelial cells and has inherent antiangiogenic properties, which has led to use of the term “vasoinhibin” for this protein [38,39]. Macroprolactin In addition to monomeric 23-kDa PRL, two other major forms of the protein are present in the circulation. Referred to as “big PRL,” which has a molecular weight of 48 56 kDa and “big big PRL” (also known as macroprolactin), which has a molecular weight of .100 kDa, these complexes of 23-kDa PRL and IgG autoantibodies can be detected to varying degrees by PRL immunoassays [40]; however these forms of PRL have minimal biological activity in vivo and no known pathological functions [40]. Macroprolactinemia is also termed analytical hyperprolactinemia, and its presence in the sera of patients can lead to clinical dilemmas

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due to the potential misinterpretations of biochemical testing (see Clinical testing, below). Placental, Decidual, and Lymphoblastoid Forms A variety of PRL-like proteins produced by the placenta have been identified in rodents, in addition to PL [41]. These PRL-like proteins are secreted at different times during gestation by the placenta and thus may have different functions, including alterations in blood vessel formation, hematopoiesis, and lymphocyte function. Although the expanded PRL family ligands are not conserved in the human, the cells they target are conserved and undergo fundamental pregnancy-dependent adaptations. PRL levels in maternal blood rise throughout gestation and are of pituitary origin. However, PRL concentrations in amniotic fluid are 10 100-fold higher than either maternal or fetal blood levels. Utilizing the “extrapituitary” or “decidual” PRL promoter (as detailed above), human chorion-decidual tissues synthesize and release a PRL species that is identical to pituitary PRL. Most recent reports have focused on the expression and regulation of decidual PRL in humans [6]. The regulation of decidual PRL secretion differs from that of pituitary PRL (detailed below). Dopamine, bromocriptine, and TRH have no effects on the decidual production of PRL in vitro. Decidual PRL production is increased by progesterone and progesterone plus estrogen, but not estrogen alone. Insulin, through the insulin receptor, IGF-1, through the IGF-1 receptor and relaxin, a third peptide related to insulin and IGF-1 have all been reported to stimulate synthesis and release of PRL. Although there is some evidence in animal studies that it may contribute to the osmoregulation of the amniotic fluid, fetal lung maturation, and uterine contractility, the function of decidual PRL in the human remains obscure [6]. Expression of decidual PRL is controlled by many cytokines, transcription factors, and signaling peptides that act either via well-defined regulatory pathways or by binding directly to putative control elements within the superdistal promoter regions [6,42].

HORMONE SECRETION: BIOCHEMISTRY Studies in the early 1970s demonstrated the existence of two pools of PRL within the rat lactotroph cell, one turning over rapidly and the other turning over slowly. Newly synthesized PRL is preferentially released compared to older, stored PRL in response to some stimuli and constitutes the rapidly turning over pool. However, other stimuli, such as TRH, result in a preferential release of older, stored PRL. These two types of secretion, rapid and slow, occur not so much due to differences in the type of stimulation for a given

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cell but due to functional heterogeneity of the cells so that some cells synthesize and secrete PRL rapidly while others secrete more slowly. Much of the storage pool of PRL in the pituitary appears to exist in a high-molecular-weight, disulfidebonded, poorly immunoreactive polymeric form that is converted to a releasable, immunoreactive monomeric form within the secretory granule when processed for release.

Measurement of Prolactin Assays and Bioassays PRL levels in sera are measured by two-site immunoradiometric assays and chemiluminometric assays utilizing the sandwich principle, whereby the PRL molecule reacts with an immobilized capture antibody and a labeled detector antibody at two distinct sites. Following removal of unused reagents with a wash step, the signal generated is proportional to the concentration of PRL in the sample. Most immunoassays are calibrated against the WHO third international standard for PRL, IS 84/500, consisting of human 23-kDa monomeric PRL. The standard reference bioassay for PRL and other lactogens is the Nb2 cell proliferation assay. In this assay, cultured rat lymphoma cells that are completely dependent on lactogenic hormones for growth are incubated with a biologic sample, and the rate of cell division is quantified to provide a measurement for the amount of lactogens present. This assay is highly sensitive (10 pg/mL) but has the potential theoretical disadvantage of inaccuracy due to species differences in PRLR responsiveness. Newer bioassays for human lactogens have been developed to address this issue, as well as to analyze structure function studies of human lactogen analogues, and to assess for the presence of macroprolactin (see below). These bioassays utilize cell lines that stably express the human PRLR alone, as part of a proliferation assay, or in the presence of a luciferase reporter to measure transcriptional activity [43,44]. Experience with these assays is limited and testing to determine accuracy, validity, and reproducibility in larger series of patients is awaited. In addition, a recent ultrasensitive-ELISA assay was developed to detect mouse PRL in very small volumes of whole blood [45]. Clinical Testing PRL is secreted episodically and some PRL levels obtained during the day may rise above the upper limit of normal established for a given laboratory. Thus the finding of minimally elevated levels in blood requires confirmation in several samples. Several

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nonhypothalamic pituitary conditions can cause moderate PRL elevations, generally to levels ,250 ng/mL. A careful history and physical examination, screening blood chemistries, thyroid function tests, and a pregnancy test will identify virtually all causes except for hypothalamic pituitary disease. When there is no obvious cause of the hyperprolactinemia from routine screening, radiologic evaluation of the hypothalamic pituitary area is mandatory to exclude a mass lesion. This includes patients with even mild PRL elevations. Magnetic resonance imaging with gadolinium enhancement is the preferred study for pituitary imaging (see chapter: Prolactinoma). It must be emphasized that it is essential to distinguish between a large nonfunctioning tumor causing modest PRL elevations (usually ,250 ng/mL) from a PRL-secreting macroadenoma (PRL levels usually .. 250 ng/mL), as the management approaches to these two entities are different. Most PRL-secreting macroadenomas generally respond readily to DA agonist therapy with size reduction, whereas only about 10% of nonsecreting pituitary tumors respond in this manner. It is also important to be aware of potential artifacts in PRL measurement that may lead to misdiagnoses. Stimulation and suppression tests using TRH, hypoglycemia, chlorpromazine, domperidone, and other medications yield nonspecific results and reveal no more information than simple measurement of basal PRL levels. Thus, consensus has developed that such stimulation and suppression tests are not recommended in the differential diagnosis of hyperprolactinemia [46]. Artifacts HOOK EFFECT

Although current PRL immunoassays are highly sensitive and specific, artifacts due to saturation of the antibodies at excessively high PRL concentrations, which prevent antibody PRL antibody sandwich formation may nevertheless occur (Fig. 5.4). This phenomenon, referred to as the “hook effect,” grossly underestimates the true PRL concentration leading the laboratory to report a falsely low value. St.-Jean et al. noted this high-end “hook effect” in 5.6% of 69 patients who were thought to have clinically nonfunctioning adenomas [47]. Interference from the “hook effect” generally becomes problematic when PRL levels exceed 100,000 mU/L [48], but the threshold for this effect varies among immunoassay platforms. Therefore, in patients with large macroadenomas if there is suspicion regarding the susceptibility of an assay to this phenomenon, PRL assessments should be performed in both undiluted and 1:100 diluted serum to exclude the “hook effect” [49].

FIGURE 5.4 Schematic diagram illustrating the measurement artifact referred to as the “hook effect.” Under usual circumstances (left side), PRL is detected in immunoassays by a solid-phase capture antibody and a labeled detection antibody. When PRL levels are grossly elevated (right side), the PRL protein saturates both antibodies, preventing sandwich formation and quantitative detection.

MACROPROLACTIN

A major diagnostic conundrum facing laboratories and clinicians is the differentiation of patients with true hyperprolactinemia from those with macroprolactinemia [50]. Based on several clinical series, the estimated incidence of macroprolactin accounting for a significant proportion of hyperprolactinemic sera is approximately 10 20% [48,51]. Current best practice recommends that serum is subfractionated using polyethylene glycol precipitation to provide increased quality of the measurement of bioactive monomeric PRL [52]. With this pretreatment step, the larger molecular weight forms of PRL are removed by precipitation, leaving the residual monomeric forms in the supernatant [48]. Clinicians should be aware that due to methodological issues, PEG precipitation is not technically feasible on all commercial immunoassays. Normative ranges have been published for sera treated with PEG for several of the most widely used assay systems [53]. Nevertheless, disagreement persists regarding the threshold concentration of residual monomeric PRL at which a hyperprolactinemic sample should be designated as being attributable to macroprolactin. Some investigators assert that in a patient with hyperprolactinemia, if the recovery of monomeric PRL left following PEG precipitation is less than 40% or 50% of the initial total value, then the hyperprolactinemia is due to macroprolactin [51]. Others suggest that since some macroprolactin is present in normal serum and/or some monomer is precipitated with PEG, that the “normal” reference range should be recalculated from normal samples after PEG treatment; therefore, hyperprolactinemia is attributable to macroprolactin only when the level of nonprecipitated PRL is within the normal range [54]. It is clear that in many

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cases, especially when sera contain high concentrations of PRL, even when the amount of nonprecipitated PRL is less than 40% of the total, the residual PRL level still exceeds the normal range. An additional issue that remains unresolved is a reliable estimation of the bioactivity of macroprolactin, as it is presumed that its presence in serum does not confer biologic significance since it is unable to pass through the capillary endothelial barrier. The bioactivity of macroprolactin as measured by the classic rat Nb2 cell proliferation assay is comparable to the bioactivity of monomeric PRL. However, when bioactivity is measured using human PRLR constructs as tested in Ba/F-3 or human embryonic kidney-derived 293 (HEK-293) cell lines, the bioactivity of macroprolactin is reduced [43,44]. Technical pitfalls could potentially account for the findings in either of these cell-based systems. For example, under the bioassay conditions employed in the Nb2 cell assay, dissociation of macroprolactin into its constituents may free monomeric PRL to react, yielding a result suggestive of hormonal activity [55]. On the other hand, transfection conditions used to express exogenous human PRLR plasmids could alter the structure or function of the PRL added from the sample. In most clinical series investigating suspected or possible macroprolactinemia, symptoms are fewer and less severe in patients whose hyperprolactinemia is attributable to macroprolactin than in those patients who are determined to have true hyperprolactinemia [48,56]. In studies in which patients with suspected macroprolactinemia (often in retrospect) are treated with DA agonists, galactorrhea, when present, generally disappears, but oligo/amenorrhea is variably responsive [56]. Long-term follow-up studies of patients diagnosed with macroprolactinemia indicate that PRL levels show considerable instability (up to fivefold) [56]. In clinical practice, if a patient has typical symptoms, such as galactorrhea, amenorrhea, or impotence and is found to have mild hyperprolactinemia, the usual conditions should be excluded (medications, hypothyroidism, elevated creatinine, pregnancy) to be followed by pituitary MRI, primarily to exclude a large lesion such as a craniopharyngioma or clinically nonfunctioning adenoma. In patients with mild hyperprolactinemia who have equivocal symptoms (such as headaches or decreased libido) but normal menses and no galactorrhea, assessment for macroprolactin using PEG precipitation is reasonable. A decision that hyperprolactinemia is due to macroprolactin then would depend on demonstrating an abnormal amount of PRL precipitated by PEG, and a residual PRL monomer level that falls within the normal range. Under these circumstances, pituitary imaging may not be indicated, but would warrant continued clinical and biochemical assessment of such patients on a periodic basis.

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Physiology Metabolic Clearance and Production Rates of Prolactin Using a labeled PRL method, the metabolic clearance rate has been found to be 46 6 4 and 40 6 6 mL/ min/m2 and the calculated production rates using the labeled PRL method were 200 6 63 and 536 6 218 μg/ day/m2 in two studies [57]. Studies in patients with chronic renal failure have shown the MCR to be reduced by 33%; increased uptake by the liver has been found in nephrectomized rabbits. Hormone Secretion Patterns PRL is secreted episodically (Fig. 5.5). There is an innate pulsatility to pituitary PRL secretion with an interpulse interval of about 8 minutes, as determined by studies of media obtained from primate pituitaries cultured in vitro. When plasma is sampled from normal individuals in whom hypothalamic function is superimposed upon this innate pulsatility, it becomes apparent that there are 4 14 secretory episodes per day. Using cluster analysis, 13 14 peaks per day in young subjects were found with a peak duration of 67 76 minutes, a mean peak amplitude of 3 4 ng/mL, and an interpulse interval of 93 95 minutes [58]. Disinhibition caused by hypothalamic tumors causes an increase in basal PRL levels due to an increase in pulse amplitude and not pulse frequency. There is an increase in the amplitude of the PRL secretory pulses that begins about 60 90 minutes after sleep onset; secretory pulses increase with non-REM sleep and fall prior to the next period of REM sleep. The lowest PRL concentrations are found during REM sleep and the highest concentrations are found during non-REM sleep. When subjects are kept awake to reverse the sleep waking cycle, PRL levels do not rise until sleep onset. Thus, the diurnal variation of PRL secretion is not an inherent rhythm but depends on the occurrence of sleep. Interestingly, the diurnal variation

FIGURE 5.5 PRL levels throughout the day in a single individual superimposed upon the range from five normal individuals. Note the episodic nature of secretion and the nocturnal rise.

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of PRL with sleep-induced rises persists despite other powerful physiologic influences such as breastfeeding. There is an increase in circulating PRL levels of 50 100% within 30 minutes of meals that is due to the amino acids generated from the protein component of the meals, phenylalanine, tyrosine, and glutamic acid being the most potent in this regard. Evidence was provided that this stimulatory action of these amino acids is centrally mediated by showing that large neutral amino acids such as valine inhibit the transport of phenylalanine across the blood brain barrier and blunt the stimulatory action of this amino acid. Changes in Prolactin With Age PRL levels are elevated almost 10-fold in infants following delivery but then gradually decrease so that levels achieve normal ranges by 3 months of age. These high levels of PRL at birth are probably related to the stimulatory effect of high maternal estrogen levels. PRL levels are lowest between the ages of 3 months and 9 years and then rise modestly during puberty to adult levels. In some studies, there is a gradual fall of basal PRL levels with age but in others no changes with age have been found. In hyperprolactinemic women, estrogen replacement therapy does not alter PRL levels. PRL levels are lower by 55% in older men compared to younger men due to both a decreased basal secretion as well as the amount secreted with each secretory burst. Changes in Prolactin Levels during the Menstrual Cycle Some, but not all, women have higher levels at midcycle and lower levels in the follicular compared to the luteal phase. In most of these studies, no correlations were found between PRL and estradiol, progesterone, LH, and FSH levels. However, some studies have shown that PRL and LH secretion are often synchronous in the luteal phase and that very small doses of gonadotrophin-releasing hormone (GnRH) can cause the secretion of both PRL and LH at this time. Changes in Prolactin Levels During Pregnancy The profile of PRL release during human pregnancy is entirely different from that in rodent lactation [30]. It involves three independently regulated compartments: maternal, fetal, and decidual. Maternal serum PRL levels start rising at 6 8 weeks gestation. Basal PRL levels gradually increase throughout the course of pregnancy to reach 200 300 ng/mL at term. Simultaneously, the pituitary gland enlarges due to increases in lactotroph size and number. Indirect evidence suggests that increased PRL release and lactotroph hyperplasia are driven by estrogens, which presumably suppress

hypothalamic dopamine and stimulate lactotroph proliferation [30]. PRL begins to rise in the fetal circulation at 20 24 weeks, increasing from week 30 to term, when it reaches levels similar to maternal serum PRL. The PRL rise is fetal autonomous. Unique to humans, the decidua produces very large amounts of PRL, which accumulates in the amniotic fluid, attaining peak levels as high as 4000 5000 ng/mL between 16 22 weeks gestation and reducing to 400 500 ng/mL at term. Despite such profound changes in PRL in the fetal compartment, there is little knowledge of its importance in human fetal physiology. These elevated PRL levels found at term prepare the breast for lactation. Changes in Prolactin Levels With Postpartum Lactation Within the first 4 6 weeks postpartum basal PRL levels remain elevated in lactating women and each suckling episode triggers a rapid release of pituitary PRL resulting in a 3 5-fold increase in serum PRL levels, peaking about 10 minutes after the end of suckling. Following termination of suckling PRL levels gradually fall to reach prenursing levels by about 3 hours after the beginning of the suckling episode. Over the next 4 12 weeks, basal PRL levels gradually fall to normal and the PRL increase which occurs with each suckling episode decreases. Eventually there is little or no rise in PRL with suckling, despite continued milk production. The decreases in basal and stimulated PRL levels between 3 and 6 months postpartum are largely the result of decreased breastfeeding as formula is introduced into the baby’s diet. If intense nursing behavior is maintained, basal PRL levels remain elevated and postpartum amenorrhea persists. Eighty minutes of nursing per day with a minimum of six nursing episodes will usually result in persistent hyperprolactinemia and amenorrhea. However, delayed onset of menses is more associated with high suckling duration and frequency than with a specific PRL level. Highintensity lactation-induced failure to ovulate and menstruate has been used as a method of contraception in some developing countries for many years. To support a period of hyperprolactinemia during lactation, and thereby promote milk production, there is an apparent loss of sensitivity of the short-loop feedback system during late pregnancy and lactation [59]. This is a remarkable example of adaptive plasticity within a neuroendocrine control network, allowing a sustained period of high PRL secretion to be maintained unused by a regulatory feedback pathway [60]. Breast stimulation may cause an increase in PRL levels in some nonbreastfeeding normal women, but

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chronic nipple stimulation with nipple rings has been reported to cause sustained galactorrhea [61]. PRL is also produced directly by human breast glandular and adipose tissue [62,63]. Although progesterone inhibits the glandular PRL production, it has no effect on the adipocyte PRL production. It is unclear whether the PRL present in breast milk is of local or systemic origin. Changes in Prolactin Secretion With Stress PRL is one of the pituitary hormones released by stress, along with adrenocorticotrophic hormone and GH. Stress-induced PRL generally occurs with a doubling or tripling of PRL levels and lasts less than 1 hour. In humans, prolonged critical illness does not cause a sustained elevation of PRL; rather there is a reduction in the pulsatile secretion with an overall lowering of levels. The teleological significance for these stress-induced changes in PRL is not clear. The neuroendocrine mediation of the acute stress response is probably multifactorial but does not include a decrease in DA. It was attempted to dissect out the neurotransmitter regulation of the PRL stress response in humans by administering various blocking agents immediately prior to surgery. Blockade of histamine H1 receptors using chlorpheniramine, serotonin receptors using cyproheptadine, and DA receptors using pimozide had little effect on the peak PRL level reached during surgery. Blockade of opiate receptors with high-dose naloxone resulted in a significant blunting, but not complete inhibition, of the PRL response. These studies imply that the endogenous opiate-like peptidergic pathways may play a role in the PRL stress response. On the other hand, in

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humans, naloxone has generally not been found to be able to block the PRL response to hypoglycemia. Hypoglycemia has been regarded as a form of stress, and PRL does indeed rise with hypoglycemia. Acute exercise also results in an acute, transient increase in PRL levels. Although chronic, high-level exercise often results in menstrual disturbance, it is not associated with sustained hyperprolactinemia.

NEUROENDOCRINE REGULATION The hypothalamus exerts a predominantly inhibitory influence on PRL secretion through one or more PRL inhibitory factors (PIF) that reach the pituitary via the hypothalamic pituitary portal vessels (Fig. 5.6). There are PRL-releasing factors (PRF) as well. Disruption of the pituitary stalk leads to a moderate increase in PRL secretion as well as to decreased secretion of the other pituitary hormones.

Prolactin-Inhibiting Factors Dopamine In the 1950s, it was demonstrated that the luteotrophic properties of the pituitary, due to PRL, were increased when pituitary glands were transplanted to beneath the renal capsule, a site away from the regulation by the hypothalamus, thus demonstrating the predominance of the inhibitory component of hypothalamic regulation of PRL secretion. Later, it was demonstrated that tuberoinfundibular DA (TIDA) released into the hypothalamic pituitary portal vessels in the

FIGURE 5.6 Neuroendocrine regulation of PRL secretion. TRH, thyrotrophin-releasing hormone; VIP, vasoactive intestinal peptide.

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median eminence was the physiologic PIF with direct action on the pituitary [64]. A number of experiments firmly established that DA is the predominant, physiologic PIF, including the findings that the concentration of DA found in the pituitary stalk plasma (about 6 ng/mL) was sufficient to decrease PRL levels in rats and that stimuli which result in an acute release of PRL usually also result in an acute decrease in portal vessel DA levels [64]. However, in many experiments it was found that the PRL increase obtained by simply reducing DA was considerably less than the elevation of PRL achieved by simultaneous stimulation by a PRF; similarly, the PRL level achieved with the simultaneous stimulation by a PRF with the reduction in DA is usually greater than that achieved by a PRF alone. It is likely that in most physiologic circumstances that cause a PRL rise, such as lactation, there is a simultaneous fall in DA along with a rise in a PRF, such as VIP, although there may well be circumstances in which various PRFs may stimulate PRL release with no concomitant lowering of DA levels or DA may be lowered with no concomitant increase in a PRF. Newer work with mice in which the D2 receptor (D2R) or DA transporter (DAT) have been “knocked out” has confirmed these earlier studies that employed pharmacologic methods or lesioning. Thus, D2r2/2 mice develop lactotroph hyperplasia and sustained hyperprolactinemia, followed by lactotroph adenomas in aged mice, demonstrating that a chronic loss of neurohormonal DA inhibition promotes a hyperplasia neoplasia sequence in adenohypophysial lactotrophs [65]. DA action within the synapse terminates by DA reuptake by the DA-secreting neurons via the DA transporter. In contrast to the findings with the D2r2/2, Dat2/2 mice have increased dopaminergic tone and lactotroph hypoplasia [66]. Although such mice have normal circulating levels of PRL, they cannot increase these levels with various stimuli and are unable to lactate [66]. Although much of the direct work demonstrating DA in hypothalamic pituitary portal vessels and the effects of DA on PRL release in vitro have been done in animals, it is clear that DA is the primary PIF in humans as well. Infusion of DA causes a rapid suppression of basal PRL levels that can be reversed by metoclopramide, a DA receptor blocker. Dopamine also blocks the PRL increments induced by various stimuli. Studies with low-dose DA infusions in humans have shown that DA blood concentrations similar to those found in rat and monkey hypothalamic pituitary portal blood are able to suppress PRL secretion. Blockade of endogenous DA receptors by a variety of drugs, including phenothiazines, butyrophenones, metoclopramide, and domperidone causes a rise in PRL [67].

The axons responsible for the release of DA into the median eminence originate in perikarya in the dorsomedial portion of the arcuate nucleus and inferior portion of the ventromedial nucleus of the hypothalamus [30]. This pathway is known as the TIDA pathway. The DA that traverses the TIDA pathway binds to D2 receptors on the lactotroph cell membrane [64]. As discussed above, activation of this receptor results in (1) an inhibition of adenyl cyclase with lowered intracellular cAMP levels, (2) inhibition of phosphoinositide metabolism, and (3) decreased intracellular calcium mobilization and inhibition of calcium transport through calcium channels. It has been proposed that these different actions of DA may actually be mediated by multiple similar D2 receptors that are produced by alternative RNA splicing [64]. The inhibitory action of DA on PRL secretion is partially blocked by estrogen administration. This may be largely due to the direct action of estrogen on the ERE of the PRL gene (see above). However, there may also be other mechanisms but with considerable interspecies differences. Estradiol is able to block the inhibitory action of DA on PRL release from rat lactotroph cells in vitro. In studies in humans, the same dose of infused DA results in a greater suppression of PRL during the early follicular phase, when estrogen levels are low, compared to the late follicular or periovulatory phases, when estrogen levels are higher. Estrogens result in a decrease in DA receptor abundance in rats but the DA receptor population showed no sex-related differences in a limited number of human pituitaries. Gonadotrophin-Associated Peptide Whether DA alone can account for all of the PIF activity of the hypothalamus has long been a question. In 1985 Nikolics et al. reported the PRL-inhibiting ability of a 56-amino-acid polypeptide that is in the carboxyterminal region of the precursor to GnRH and which they termed GAP [68]. However, the GAP sequences in human and rat have 17 amino acid differences and subsequent studies showed that GAP has no PRL-suppressing activity when tested against human prolactinomas cultured in vitro. To date, there is no evidence that GAP has physiologic significance in humans as a PIF. γ-Aminobutyric Acid γ-Aminobutyric acid (GABA) has an inhibitory effect on PRL secretion in vivo and in vitro in rats and high-affinity GABA receptors are present on lactotrophs. A tuberoinfundibular GABAergic system has been described with perikarya located in the arcuate nucleus and nerve endings demonstrated in the

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median eminence and GABA has been demonstrated to be present in portal blood. Studies of the GABA system in humans have yielded conflicting results in studies of widely differing experimental designs. GABA itself causes a modest decrease in PRL levels when given to humans for several days and activation of the endogenous GABAergic system with sodium valproate causes a suppression in the PRL rise induced by mechanical breast stimulation in puerperal women. The physiologic role of GABA remains to be fully elucidated in the human.

PRL levels are not low basally but the PRL response to TRH is markedly blunted and returns to normal with correction of the hyperthyroidism. The above conflicting data from passive immunization studies, trh2/2 mice, observation of TSH levels during lactation, and examination of PRL levels in various thyroid states support a role for TRH as a physiologic PRF, albeit not the primary one or even one of major importance. Moreover, a TRH stimulation test is often used to diagnose hyperprolactinemia in patients, but its physiological importance as a regulator of PRL release in humans is unclear.

Prolactin-Releasing Factors

VIP and Peptide Histidine Methionine (PHM)/PHI

Thyrotrophin-Releasing Hormone TRH binds to type 1 TRH receptors expressed in both thyrotrophs and lactotrophs. Shortly after its initial isolation and characterization, TRH causes a rapid release of PRL from rat pituitary cell cultures and in humans after intravenous injection. Release of PRL is biphasic, the initial peak being mediated by activation of intracellular phosphoinositide pathways with IP3 generation and mobilization of intracellular calcium causing release of stored hormone; the second, more sustained phase is mediated influx of extracellular calcium through calcium channels, which causes sustained secretion and synthesis of new hormone. A number of different experimental approaches have failed to clarify the physiologic role of TRH as a PRF. The smallest dose of TRH that releases TSH also releases PRL in humans. Immunoneutralization of endogenous TRH with TRH antisera causes a 50% suppression of basal PRL levels in rats in some studies but not in others. The trh2/2 mice became hypothyroid with elevated levels of TSH with reduced biological activity but had normal PRL levels, further casting doubt on the essential role of TRH in PRL regulation. If TRH mediates the PRL response to suckling, even in part, it ought to be accompanied by an increase in TSH, unless there were a concomitant increase in somatostatin. Studies in humans failed to show any elevations of TSH with suckling. Very small doses of TRH given systemically were effective in releasing PRL and TSH in lactating rats and women in those studies, however, so it is unlikely that failure to show a rise in TSH was due to an increase in somatostatin. In hypothyroidism, TRH synthesis is increased, portal vessel TRH levels are increased, and there is an increased number of TRH receptors. In human hypothyroidism, basal TSH and PRL levels are increased as are their responses to injected TR. Correction of the hypothyroidism with thyroid hormone corrects both the elevated TSH and PRL levels and their responses to TRH. Conversely, in hyperthyroidism in humans,

VIP stimulates PRL release and is found in neuronal perikarya in the parvocellular region of the paraventricular nucleus with axons terminating in the external zone of the median eminence. Its effects are selective for PRL and additive to TRH in causing PRL release at concentrations found in hypothalamic pituitary portal blood. The effects of VIP appear to be mediated by stimulation of adenyl cyclase, although recent evidence suggests that transport of calcium through membrane calcium channels may also be important. In addition to stimulating PRL release, VIP also stimulates pituitary PRL mRNA content and PRL synthesis. In conditions of increased PRL synthesis, such as lactation, hypothalamic VIP mRNA levels are also increased. Intravenously administered VIP has also been shown to increase PRL levels in humans at serum levels similar to those demonstrated in rat portal blood. A number of experiments have been performed using passive immunoneutralization techniques to determine the physiologic role of VIP as a PRF. AntiVIP antisera administered to rats have been shown to partially inhibit the PRL responses to suckling and ether-induced stress. Part of the 20-kDa 170-amino-acid VIP precursor is another similarly sized peptide known as PHM. PHM and VIP colocalize in the hypothalamus and median eminence. PHM given to humans has caused a PRL increment in some experiments but not others. Further complicating the role of VIP as a PRF is the finding that VIP is actually synthesized by anterior pituitary tissue. Antisera to VIP inhibit basal PRL secretion from dispersed pituitary cells in vitro, suggesting a local “autocrine” role for VIP in PRL regulation within the pituitary. The physiologic role of VIP as a PRF appears to be warranted by the experimental data. The precise roles of VIP versus. PHM and hypothalamic VIP versus. pituitary VIP still are not clear. How VIP/PHM interact with other PRFs such as TRH are additional areas requiring clarification. The general current consensus is that VIP is not a potent PRL secretagogue in humans [30].

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Serotonin A considerable number of experiments have demonstrated a role for serotonin as a neurotransmitter involved in the release of PRL. Most serotoninergic neuronal perikarya are in the dorsal and median raphe nuclei and their axons project forward to the hypothalamus and other limbic and cortical areas. Lesions of the dorsal but not the median raphe nuclei decrease forebrain serotonin levels and basal and stimulated serum PRL levels. Studies in humans also suggest a role for serotonin in PRL secretion. Infusion of the serotonin precursor, 5-hydroxytryptophan, elicits a prompt increase in PRL levels. Nocturnal PRL secretion is inhibited by cyproheptadine. On the other hand, pizotifen, a specific, nonergot serotonin antagonist, had no effect on suckling-induced PRL in postpartum women. Whether serotonin effects are mediated solely through brain pathways or whether it has direct effects on the pituitary is controversial. One possibility is that serotonin causes a decrease in hypothalamic DA generation. Synaptic junctions between serotoninergic nerve terminals and dopaminergic perikarya in the arcuate nucleus have been demonstrated. Furthermore, intraventricular injections of serotonin decrease portal vessel DA concentrations. It has also been proposed that serotonin acts by increasing VIP and oxytocin via effects at the paraventricular nucleus. Serotonin has been found within the anterior pituitary serotoninergic nerve terminals and has been demonstrated within the median eminence. High-affinity S2 serotonin receptors have been found in the anterior pituitary as well as uptake of labeled serotonin into cells of the pituitary. In direct tests of the effects of serotonin on pituitary PRL release, serotonin has been found to increase basal and stimulated PRL secretion from pituitaries in vitro and this appears to be mediated by the serotonin subtype 4 receptor [69]. Thus, although it is possible that serotonin is a direct secretagogue for PRL, via transport from the hypothalamus by the portal vessels or through an autocrine action within the pituitary, its role in this regard is still uncertain. It may mediate the nocturnal surge of PRL and may well participate in the sucklinginduced rise in PRL via the ascending serotoninergic pathways from the dorsal raphe nucleus and mediated by activation of VIP release. Serotonin reuptake inhibitors are widely prescribed antidepressants but only rarely have cases been reported of hyperprolactinemia due to their use. Opioid Peptides A detailed description of the various opioid peptides, their receptors, and their neuronal pathways is

beyond the scope of this discussion. Approaches to determining the roles of the opioid peptides and pathways in the regulation of PRL secretion have focused on using opioid agonists and antagonists in experimental animals and humans. In rats, morphine, Met- and Leu-enkephalin, β-endorphin, dynorphin, and leumorphin injected systemically or intracerebroventricularly have all been shown to elicit PRL release. Subsequent studies employing specific agonists and antagonists operative on the μ, δ, and κ opioid receptors and antibodies directed against several opioid peptides have shown that it is the μ receptor that is the predominant one involved in PRL release, the κ receptor is involved to a lesser extent, and the δ receptor is not at all involved. Opioids stimulate PRL release by inhibiting DA turnover and release by the TIDA pathway. Orphanin FQ (also called nociceptin), which binds to an opioid-like orphan receptor, also causes an increase in PRL levels in rats when administered intracerebroventricularly. In humans, morphine and morphine analogues increase PRL release acutely and chronically. However, blockade of the μ receptor with naloxone has minimal to no effect on PRL levels either basally or with stimulation by hypoglycemia, exercise, sleep, TRH, or physical stress. Overall, it appears that the endogenous opioid pathways play at most only a minor role in the regulation of PRL secretion, especially in humans. Growth Hormone-Releasing Hormone A number of studies have found GHRH to have PRL-releasing properties. The initial clue to this effect of GHRH was the finding that many of the patients with acromegaly due to GHRH-secreting tumors were hyperprolactinemic and PRL levels fell in parallel with GH following excision of the GHRH-secreting tumor. Large doses of GHRH have been reported to release PRL in vivo in normal humans. Chronic therapy with GHRH in children with GH neurosecretory dysfunction results in a sustained elevation of PRL levels. In rat pituitary cell cultures, GHRH causes PRL and GH release but an increase only in gh mRNA and not prl mRNA, indicating no stimulation of PRL synthesis. The similarity of GH and PRL responses to a variety of stimuli, such as exercise, stress, hypoglycemia, arginine infusion, and sleep and the pathological conditions of renal failure and hepatic cirrhosis suggest but do not prove that GHRH may serve as a physiologic PRF under some circumstances. Posterior Pituitary, Oxytocin, and Vasopressin Studies in animals have shown that oxytocin, in doses found in the hypothalamic pituitary portal vessels can stimulate PRL release when added to the medium of pituitary cell cultures or incubations or

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when given intravenously, but it lowers PRL levels when directly injected into the third ventricle. Studies in which endogenous oxytocin was eliminated by passive immunization with oxytocin antisera or by oxytocin antagonists show a reduction and a delay in the suckling and cervical-stimulation-induced PRL surges in some but not all studies [70]. Experimental data also support the possibility that the oxytocin-induced PRL increase in these experimental paradigms is mediated by a decrease in DA [70]. Very limited studies in humans suggest that oxytocin administered intravenously has no effect on basal PRL levels and causes only a minimal increase in TRH-stimulated PRL levels. It is likely that oxytocin plays at most a minimal role even in suckling-induced PRL secretion in humans [30]. Vasopressin also has PRL-releasing properties when injected intravenously into normal rats and sheep and rats with pituitaries transplanted to the renal capsule but not sheep with hypothalamic pituitary disconnection. The neurophysin portions of the precursors to oxytocin and vasopressin also stimulate PRL secretion in rats. There are no studies to date of the effects of vasopressin on PRL secretion in humans. Whether other PRFs in the posterior pituitary exist in addition to oxytocin, vasopressin, and their respective neurophysins has been a matter of controversy, but isolation of such substances has not been successful. Overall, it is felt that the posterior pituitary may play only a minor role in the regulation of PRL secretion [30].

cells and in PRL-secreting adenomas. Angiotensin II incubated with rat pituitary cells stimulates release of PRL, an effect blocked by AT1 but not AT2 antagonists. However, in humans, blockade of ACE with enalapril results in no change in basal PRL levels, no change in the TRH and metoclopramide 5 induced PRL rises and only a minimal decrease in the PRL response to hypoglycemia. It is unlikely, therefore, that the endogenous renin angiotensin system of the hypothalamus and pituitary has significant physiologic effects on PRL regulation.

Gonadotrophin-Releasing Hormone

This neurotransmitter plays an uncertain role in PRL regulation. Histamine neuronal perikarya are present in the posterior hypothalamic region and axons project to almost all of the nuclei of the hypothalamus. Although some experiments show an effect of histamine via hypothalamic mechanisms, it has no effect on PRL release from pituitaries in vitro or in stalk-sectioned rats. In humans, intravenous H2 but not H1 blockers cause a rise in PRL levels but prolonged oral administration of H2 blockers does not result in sustained PRL elevation. The fact that the administration of high doses of H2 blockers increases PRL levels in humans, that histamine cannot cross the blood brain barrier and that histamine has no effect on pituitaries in vitro suggests that histamine may play a physiologic facilitatory role in PRL secretion within the median eminence. The roles of other bioamines in the regulation of PRL secretion are even less well established. Central adrenergic α2-agonists such as clonidine usually have no effect on PRL secretion although both increases and decreases have been reported, the differences being due to the doses used. α-Methyl dopa, another central adrenergic agonist, causes a sustained elevation of PRL

GnRH was initially found to release PRL from rat pituitary cells in vitro. Subsequently, GnRH has been found to cause a release of PRL in anovulatory women. Postmenopausal women also have a PRL response to GnRH that is augmented with estrogen supplementation. There is no PRL release in response to GnRH in normal, eugonadal males but such a release does occur with high doses of estrogen pretreatment (given to transsexual men). Analysis of PRL and LH secretory pulses suggests a high degree of concordance in women, thereby arguing for a physiologic role for GnRH in PRL secretion. A subset of human prolactinomas that also contain the glycoprotein α subunit has been shown to bind GnRH specifically and with high affinity and to release PRL in response to GnRH in vitro. Renin Angiotensin System Angiotensin-converting enzyme (ACE) and angiotensin II receptors and activity have been identified in the rat pituitary and median eminence of the hypothalamus. In the human pituitary, renin, ACE, and angiotensinogen have been detected in normal lactotroph

Other Neuroactive Peptides and Neurotransmitters Somatostatin receptors are expressed on human PRL as well as GH-secreting adenomas. Somatostatin inhibits adenyl cyclase activity of rat anterior pituitary homogenates and spontaneous and stimulated PRL release [71]. Furthermore, administration of somatostatin antiserum to rats causes a rise in PRL levels, implying a physiologic inhibitory action of somatostatin basally. In humans, however, somatostatin administered exogenously has no effect on TRH-induced PRL release. A number of other peptides (neurotensin, substance P, cholecystokin, bombesin, calcitonin, endothelin, galanin, gastrin, transforming growth factor β) and bioamines such as glutamine have been found to have varying effects on PRL levels in rats in different experimental paradigms, but very limited studies in humans have shown no effect. HISTAMINE

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but this may be due to inhibition of the synthesis of norepinephrine or DA centrally by inhibiting the enzyme l-aromatic acid decarboxylase, which is responsible for conversion of dopa to DA and by acting as a false neurotransmitter to decrease DA secretion or synthesis by a local feedback inhibitory action. However, monoamine oxidase inhibitors increase PRL levels. Such an elevation is unexpected, since these drugs should increase levels of synaptic norepinephrine and DA.

found of suppression of TSH, LH, or FSH levels with short-term administration of human PRL which resulted in a 2 3-fold elevation of PRL levels. However, these acute studies do not rule out the possibility that such feedback may occur with more prolonged states of hyperprolactinemia. Alternatively, such feedback might occur via other mechanisms, such as a decrease in a PRF such as VIP.

PROLACTIN ACTION

ACETYLCHOLINE

This neurotransmitter inhibits adenyl cyclase and cAMP accumulation, lowers intracellular free calcium levels, and decreases PRL release from pituitary cell cultures, acting through muscarinic and not nicotinic receptors. Although atropine, a muscarinic receptor antagonist blocks acetylcholine inhibition of PRL in vitro, it had no effect on basal or TRH-induced PRL release in humans and pirenzepine, another muscarinic receptor blocker actually caused a modest decrease of PRL levels in humans in vivo. The widespread presence of acetylcholine as a neurotransmitter in the CNS and the possibility that pituitary tissue itself may synthesize acetylcholine, making interpretation of studies testing this system difficult and the true role of acetylcholine in the regulation of PRL secretion is uncertain. Prolactin Short-Loop Feedback Considerable evidence in rats suggests that PRL feedsback negatively on its own secretion (short-loop feedback or autofeedback) [59]. Most evidence suggests that such feedback occurs via augmentation of hypothalamic TIDA turnover, including direct measurements of DA in portal vessels. Electrophysiological data have demonstrated rapid actions of PRL on the electrical activity of TIDA in mice [72,73] or in rats [60,74]. Studies using Prl2/2 mice show that they have markedly decreased DA in TIDA neurons, along with hyperplasia of lactotrophs that do not synthesize PRL [75,76]. Direct evidence for such PRL short-loop feedback in the human has not been demonstrated. In a number of reports, however, it has been suggested that altered regulation of gonadotrophin and TSH secretion in hyperprolactinemic patients may constitute indirect evidence of PRL-induced augmented TIDA activity. In hyperprolactinemic patients, decreases in gonadotrophin pulse amplitude and frequency are usually found, being attributed to altered gonadotrophinreleasing hormone secretion (see below). Such an alteration of GnRH secretion has been postulated to be due, in part, to PRL-induced DA increase. In a direct test of this hypothesis, however, no evidence was

PRL has a great diversity of actions in many species of animals from fish and birds to mammals, including osmoregulation, growth and developmental effects, metabolic effects, actions on ectodermal and integumentary structures, and actions related to reproduction [39,77]. However, in humans it has as its primary physiologic action the preparation of the breast for lactation in the postpartum period. A number of effects of increased levels of PRL may be seen on many tissues. Although the roles of physiologic levels of PRL in such tissues are quite speculative, considerable clarification of these roles has been elucidated from studies in Prl22 [76] and Prlr2/2 [78] mice.

Prolactin Receptor The prolactin receptor (PRLR) belongs to the class I cytokine receptor superfamily, a family of single-pass transmembrane proteins that transduce signals following phosphorylation by cytoplasmic kinases [79]. The human PRLR (hPRLR) gene is located on chromosome 5p14-p13.2 and consists of eight or nine coding exons and two noncoding exons, which constitute the 5’ UTR. Six alternative forms of the first exon (hE1N1hE1N5) are expressed in a tissue-specific manner, and are spliced into the noncoding exon 2. Exons 3 10 encode the full-length activating long form of the receptor. Multiple PRLR transcripts, resulting from alternative splicing and transcriptional start sites, give rise to intermediate and short forms of the receptor (described below). Transcripts from exon 11 are only present in the short forms of the receptor [80]. The entire hPRLR locus spans a region that exceeds 200 kb. The hPRLR cDNA is 1869 nucleotides long and encodes a protein of 622 amino acids, 24 of which represent the signal peptide. The PRLR is ubiquitously expressed. Regulation of PRLR protein levels occurs at both transcriptional and post-transcriptional levels. Transcription of hPRLR is controlled by multiple promoters, with each of the alternative noncoding exons 1 utilizing a separate promoter. The preferentially utilized, generic promoter

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1/exon-1 (PIII/hE13) contains functional Sp1 and C/EBP sites that bind transcription factors Sp1/Sp3 and C/EBPβ, respectively. Estradiol, operating through a nonclassical ERα signaling mechanism, activates the hPRLR hPIII promoter in breast cancer cells through ERα-mediated recruitment of Sp1 and C/EBPβ, and assembly of a coactivator complex consisting of p300, SRC-1, and pCAF [81]. PRLR levels are negatively regulated at a posttranslational level by proteolytic degradation via receptor ubiquitination, facilitated by the SCF β-Trcp E3 ubiquitin ligase, and targeting to the lysosomal complex [82,83]. Alterations in PRLR degradation may contribute to transformation of breast cells [84]. The PRLR contains an extracellular domain (ECD), required for ligand binding, a transmembrane domain, and an intracellular domain (ICD), required for signal transduction (Fig. 5.7). The hPRLR exists as at least nine recognized isoforms, which have different signaling properties. Mice and rats also express multiple PRLR isoforms [85]. Within a species, the ECDs of most PRLR isoforms are identical, whereas the ICDs are of variable length and composition. The long PRLR serves as the canonical sequence, and is the only isoform that signals properly. The intermediate form results from a frameshift, and leads to absence of a portion of the ICD. The ΔS1 isoform lacks exons 4 and 5, and has reduced affinity for hormone, but displays effective signal transduction. Short forms of the hPRLR known as S1a, S1b, and Δ4-S1b are derived from alternative splicing. These forms have similar binding affinity for PRL as the long form, but cannot transduce signal and exhibit dominant negative activity when coexpressed with the long form. In addition to membrane-anchored receptor, a soluble, freely circulating form of the receptor, PRL receptor binding protein (PRLRBP), is generated by proteolytic cleavage of the long hPRLR [86].

The functional significance of the various isoforms has not been determined conclusively, but some insights into their function have been gained through the phenotypic analysis of genetic mouse models in which a short PRLR isoform is overexpressed on a long Prlr null background (Prlr2/2;rstg) [87]. These mice display premature ovarian follicular development followed by massive follicular cell death. On a molecular level, PRL signaling through this particular short PRLR isoform in ovarian tissue represses transcription of several genes, including Foxo3a and Galt, which are important for normal follicular development. Moreover, this short PRLR isoform does not activate Jak/Stat, but instead utilizes a unique intracellular signaling pathway involving calmodulin-dependent protein kinase (CamK) [88]. The phenotypic alterations demonstrated in Prlr2/2rstg ovaries are reversed by the re-coexpression of the long PRLR, suggesting that the two isoforms inhibit the activity of each other, and the proper ratio of their expression is important for the normal physiological development of ovarian follicles. Whether improper ovarian PRLR isoform expression occurs in humans, and contributes to ovarian failure is not known, but remains an important question. The short PRLR isoforms also appear to inhibit long PRLR function through heterodimerization with long PRLR isoforms resulting in dominant negative activity, and by enhancing PRLR degradation [89,90]. Alterations in the expression of PRLR isoforms and ratio of short to long PRLR isoforms have been observed in breast tumor tissue and cancer cell lines compared with the normal breast and control mammary cells. Accordingly, abnormal signaling as a consequence of these changes could theoretically contribute to breast tumor development and/or progression. The PRLR has three defining domains, two within the ECD and one in the ICD. The ECD contains two signature motifs: an aminoterminal region (S1) and a FIGURE 5.7 Schematic illustration of eight of the most common human PRLR isoforms. The PRLR gene (not depicted) undergoes alternative splicing to yield several transcripts, and subsequently proteins, of variable length. In general, the extracellular and transmembrane domains are nearly identical, whereas the intracellular domains vary in length and composition. Motifs, such as the disulfide bonds, WSXWS, box 1, and box 2 are highly conserved.

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FIGURE 5.8 Schematic illustration demonstrating two models for PRLR activation. (A) In the ligand-dependent dimerization model, one molecule of PRL binds to a PRLR monomer at binding site 1, which leads to the recruitment of the second PRLR monomer to bind PRL at site 2. Dimerization of the two PRLRs leads to phosphorylation of Janus kinase and signal transducer and activator of transcription 5a (Stat5). (B) In the ligand-independent dimerization model, the PRLRs exist as preformed dimers at the cell membrane in the absence of ligand. PRL binding to the dimeric PRLR receptor induces conformational changes that subsequently activate the receptor.

membrane-proximal region (S2). Two pairs of disulfide bonds in S1 are highly conserved and critical for tertiary receptor folding of the ligand binding domain. A highly conserved WSXWS motif constitutes the S2 motif and may be involved in receptor trafficking. The PRLR ECD, as resolved by X-ray crystallography, contains two subdomains, which are related to the type III repeats of fibronectin. Each domain is composed of seven β-strands folded into two antiparallel β-sheets [79]. The third defining motif, which lies in the ICD of the PRLR, is referred to as “box 1” and consists of an eight-amino-acid proline-rich hydrophobic sequence that directly interacts with intracellular tyrosine kinases. Mutations in residues of box 1 completely disrupt Jak/Stat PRLR signaling. Increased PRL concentrations promote binding to monomeric or dimeric forms of the PRLR and induce structural changes in the ECD [79]. The active PRL/ PRLR complex has a stoichiometry of one hormone bound to two receptors (Fig. 5.8). As such, two ECDs of the PRLR interact with two asymmetric ligand binding sites located opposite to each other within the receptor core. The formation of 1:2 complexes is an essential first step for subsequent signal transduction. Two different mechanisms of PRL binding to PRLR have been proposed. The conventional view holds that binding of PRL to a monomeric first receptor induces the sequential recruitment and dimerization of a second receptor [91]. This leads to activating changes in the ICD and initiates signal transduction. Recent data garnered from coimmunoprecipitation and

bioluminescence resonance energy transfer (BRET1) analyses support an alternative model in which the PRLR dimerizes independently of ligand binding [92]. It is now recognized that, like GH receptor (GHR) dimers [93], human PRLR dimers exist constitutively on the cell surface [89,92]. These dimers are linked by transmembrane domains. PRLR homodimers cannot drive signal transmission in the absence of the PRL ligand. Heterodimerization of long and short PRLR isoforms produces inactive complexes. The mechanism of GHR activation was elucidated by Brooks et al. [94]. It appears that the same mechanism could apply to all class I cytokine receptors, which include PRLR. PRLR, like GHR and other homologous cytokine receptors, does not possess intrinsic tyrosine kinase activity but transmits its signal through associated cytoplasmic proteins such as Janus protein kinase2 (Jak2) [95] The initial event is the binding of a single ligand molecule to predimerized receptor monomers on the membrane surface, via two extracellular interaction sites. This triggers a change in the conformation of the receptor dimer that enables signal transduction to occur. For GHR, Brooks et al. propose a fascinating multistep scissor-like mechanical model in which movements of the receptor dimer ultimately lead to receptor activation [94]. These recent findings show that, in the absence of hormone, helices of the transmembrane domains of the predimerized monomers lie parallel to each another. Binding of hormone converts the helices into a crossover state that induces their separation at the lower transmembrane boundary, hence inducing

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cytoplasmic Box 1 separation. This helix state transition requires rotation of the receptors, but the key outcome is separation of the Box 1 sequences. Indeed, in the basal state (in the absence of ligand), the Box 1 domains are attached to the N-terminal domain of Jak2, which is a pseudo-Jak2 kinase sequence whose function is to inhibit the inherent kinase activity of Jak2. Distancing of the two Box 1 domains allows separation of the pseudokinase inhibitory domain of one Jak2 molecule, which blocks the kinase domain of the other Jak2, and vice versa, allowing Jak2 activation. This may be a general mechanism underlying class I cytokine receptor action [96].

PRLR Signal Transduction Ligand binding to the PRLR results in rapid activation of Jak2, which is constitutively associated with box 1 of the PRLR. Jak2 activation is the most proximal event in the intracellular events that occur after ligand binding (Fig. 5.9). Jak2 phosphorylates tyrosine residues on the PRLR ICD and autophosphorylates residues within itself. Receptor-associated Jak2 also phosphorylates cytoplasmic signal transducer and activator of transcription proteins. Four Stat family members (Stat 1, Stat 3, Stat 5a, Stat 5b) serve as the central transducer molecules of the signal transduction pathways initiated by PRLR activation, but Stat 5a and, to a lesser extent, Stat 5b, are especially important for mammary gland development and lactogenesis [97]. A phosphorylated tyrosine residue of the activated PRLR

interacts with the SH2 domain of a Stat protein. Following phosphorylation by activated Jak2, Stat proteins hetero- or homodimerize, translocate to the nucleus, and transactivate γ-interferon activation sequence (GAS) consensus elements on target genes. The tyrosine phosphatase short heterodimer partner (Shp)-2 promotes PRL stimulated assembly of the JakPRLR complex, and is a required component for Stat 5a activation during pregnancy and lactation. Jak2-Stat signaling is attenuated through an intracellular negative-feedback system involving the action of several negative regulators, including (1) suppressor of cytokine signaling (SOCS) proteins, which inhibit Jak2 kinases; (2) cytokine-inducible SH2-containing (CIS) proteins, which compete with Stat proteins for docking sites on the PRLR; and (3) protein tyrosine phosphatases, PTP1B1 and TC-PTP, which dephosphorylate PRL-activated Stats [77]. Although the Jak/Stat pathway mediates most physiological actions of PRL in mammary development and lactation, binding of PRL to its receptor also activates several additional intracellular cascades to promote specific cellular responses. Effectors of these cascades engage in signaling crosstalk, and likely operate as a complex network rather than hierarchically depending upon cell type and context. Phosphotyrosine residues of the PRLR can serve as docking sites for adapter proteins (Shc/Grb2/SOS) connecting the receptor to the Ras/Raf/MAPK cascade [79]. This pathway appears to mediate at least some of PRL mitogenic effects. Activation of PRLR also facilitates docking of Src family kinases, which

FIGURE 5.9 Schematic illustration depicting intracellular signal transduction pathways downstream of the PRLR. Following activation of the PRLR, Janus kinase 2 tyrosine kinase becomes activated through auto- or transphosphorylation. This triggers association with signal transducer and activator of transcription protein 5a (Stat5) followed by Stat5 phosphorylation, dimerization, and nuclear translocation. Association with adaptor proteins such as Shc leads to signaling through the mitogen-activated protein kinase (MAPK) pathway to stimulate mitogenesis. Association with Src family kinases triggers phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) signaling to affect cell proliferation and survival. Members of the suppressors of cytokine signaling (SOCS) and cytokine-inducible inhibitor of signaling (CIS) proteins decrease the actions of Jak.

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couple to multiple signaling effectors, including phosphatidylinositol (PI) 3’-kinase/AKT and Erk 1/2, linking PRLR activation to cell survival and proliferation [98,99]. The requirement of Src as an essential mediator of PRLR signaling in normal mammary tissue is underscored by findings in female Src2/2 mice, which demonstrate lactation failure and precocious mammary gland involution [100]. As they become available, detailed characterization of mouse genetic models lacking components of PRLR signaling cascades should provide information on their physiologic roles and relevance.

Female Reproductive Tissues PRL Effects on Breast PRL plays a dominant role in several aspects of the breast, including growth and development of the mammary gland (mammogenesis), synthesis of milk (lactogenesis), and maintenance of milk secretion (galactopoiesis). Development of the mammary gland is a tightly coordinated process involving several hormones, in addition to PRL, that occurs in defined stages that include embryonic, prepubertal, pubertal, and pregnancy [101]. During puberty, the epithelial cell compartment, which consists of a branched ductal system, expands while, during pregnancy, the lobuloalveolar compartment differentiates and develops. Mammary gland development during the embryonic and prepubertal stages occurs independently of the actions of PRL, and PRL plays only a minor role in the pubertal stage. However PRL, together with ovarian steroids, local growth factors, and cytokines, is essential for the morphologic changes that occur in the mammary gland during pregnancy and lactation [102]. The combination of experimental mouse genetics and transcriptomic profiling has been useful for confirming the roles of genes and identifying PRLdependent signaling pathways that control mammary development. At the onset of puberty, the ovarian steroids estradiol and progesterone and pituitary GH initiate and drive ductal morphogenesis [103]. Mammary gland development in Prl2/2 and Prlr2/2 mice is arrested at the stage of ductal elongation and these mice completely lack lobuloalveolar units, indicating that it is at this stage and beyond that PRL exerts developmental influences [76,78]. PRL indirectly influences the process of ductal side branching during puberty by promoting ovarian progesterone synthesis [104,105]. In contrast to these relatively minor effects, PRL plays a major role in the morphologic and functional changes that occur in the breast during pregnancy. During pregnancy, high concentrations of estradiol and progesterone, produced by the placenta,

coupled with high levels of PRL and hPL promote proliferation of the lobuloalveolar epithelium. During and after parturition, progesterone, estradiol, and hPL levels decline, whereas PRL levels rise. These hormonal changes, together with the effects of local growth factors such as RANK-ligand and insulin-like growth factor 2 (IGF-2) induce the lobuloalveolar epithelium to convert into secretory acini [106,107]. Epithelial PRLRs are required for this process, as Prlr2/2 mammary transplants fail to develop lobuloalveoli, and cannot produce milk proteins during pregnancy [105]. PRL also acts in concert with insulin and hydrocortisone to induce differentiation of pluripotent mammary epithelial cells that produce progeny that subsequently grow into alveolar structures [108]. Members of the PRLR signaling pathway whose functions are essential for mediating PRL effects on alveolar morphogenesis include the transcription factors Stat5a, Id2, Socs-2, Gata-3, and Elf5 [101,109 112]. Both lactogenesis and galactopoiesis require pituitary PRL, since hypophysectomy during pregnancy prevents or stops, respectively, lactation. Prl22 and Prlr2/2 mice cannot produce milk as a result of defective mammogenesis. PRL is also essential to maintain sustained lactation. During lactation, PRL regulates the synthesis of milk proteins, including β-casein, lactoglobulin, lactalbumin, and whey acidic protein [113]. PRL also regulates the synthesis of enzymes involved in lipid metabolism, including lactose synthetase, lipoprotein lipase, and fatty acid synthase [113]. Galactorrhea Clinically, nonpuerperal galactorrhea has been regarded as being a sign of possible hyperprolactinemia. The presence of even minute amounts of milk expressible from one or both breasts indicates a diagnosis of galactorrhea. Its persistence for more than 1 year after normal delivery and cessation of breastfeeding or its occurrence in the absence of pregnancy generally is regarded as a definition of inappropriate lactation. If the material expressible from the nipple looks like milk, it probably is milk; if there is any uncertainty, examination of the breast secretion by staining of fat globules with Sudan IV is diagnostic. The incidence of galactorrhea has been variously reported in normal women as ranging from 1 to 45% of subjects tested. This variability is probably due to differences in the techniques used to express milk from the breast and the way in which nonmilky secretions are classified. The volume of milk expressed does not correlate with PRL levels. However, in individuals with hyperprolactinemia, lowering the blood PRL level to normal almost always will lead to a marked decrease in or abolition of lactation. Inappropriate lactation may be an important clue to the presence of

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pituitary hypothalamic disease, especially if accompanied by amenorrhea. Clinical experience suggests that galactorrhea may be present in about 5 10% of normally menstruating women and basal PRL levels are normal in more than 90% of these women. PRL and Breast Cancer Apart from hyperprolactinemia, which is the most widely characterized disorder in humans that is related to PRL signaling, the role of PRL and its receptor in the initiation and/or progression of human breast cancer remains an active area of debate. A wide range of studies has attempted to address the possible contribution of PRL to breast cancer through epidemiological analyses, cellular and molecular studies, and transgenic mouse models. Overall, epidemiological studies examining the relationship between serum PRL levels and the risk of breast cancer in women have shown conflicting results. The Nurses’ Health Study, the largest prospective cohort study reported, found a significantly (34%) increased risk of breast cancer when comparing top to bottom quartiles of serum PRL in postmenopausal women and a nonsignificant 30% increase in risk comparing top to bottom PRL quartiles in premenopausal women [114] (reviewed in [39]). These findings are similar to results of previously reported smaller studies that found nonsignificant increases in breast cancer risk [115]. The clinical relevance of these epidemiological studies is limited by observations that serum levels of PRL in the study participants remained within the normal range and were not associated with clinical symptoms. In women with obvious hyperprolactinemia, two studies failed to show an association of this state with the risk of breast cancer [116,117]. The number of studies investigating associations between genetic variability in the PRL or PRLR genes and the risk of breast cancer are limited, but favor lack of association. Analysis of high-density single-nucleotide polymorphism (SNP) data from the Multiethnic Cohort Study which included 1600 cases of breast cancer and 1900 controls, did not find a significant association between PRL and PRLR haplotypes or individual SNPs in relation to breast cancer risk [118]. Recent attention has turned to the question as to whether local production of PRL within breast tissue plays an autocrine or paracrine role in the etiology or progression of breast cancer [30,119]. Accumulating data from in vitro and animal studies have suggested that PRL and/or actions of the PRLR may be involved in mammary tumorigenesis by promoting cell proliferation and survival, increasing cell motility, supporting tumor vascularization, and/or contributing to cell transformation [84]. Transgenic mice that universally overexpress PRL systemically or locally within the

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mammary epithelia develop mammary carcinomas at a long latency, whereas transient overexpression of PRL in differentiated, lactating mammary tissue leads to the development of benign mammary adenomas. By contrast, genetic ablation of the PRLR delays, but does not prevent or reduce the incidence of, the development of SV40 large T-antigen-induced breast carcinomas [120]. Taken together, findings from these murine models support a modulatory role for PRLR function in mammary neoplasia. In humans, a substantial percentage of primary human breast carcinomas express PRL and PRLR, but immunostaining for the receptor does not correlate well with clinicopathological stage or disease-free survival. A heterozygous nonsynonymous gain-of-function PRLR variant in the PRLR (I146L substitution in the ECD) has been identified in a small percentage (B5.6%) of human benign breast tumors [121]. However, this variant has also been reported as a common polymorphism that occurs in B2.4% of European and US populations, which argues against a role of this variant in this disorder. In light of the above-listed observations that have suggested a role for PRL or PRLR in promoting mammary tumor growth, efforts have been initiated to investigate the potential usefulness of pharmacologic inhibition of the PRLR for the treatment of breast neoplasms or other conditions associated with hyperprolactinemia [122]. Preclinical studies using competitive PRLR antagonists have demonstrated proof-ofprincipal PRLR antagonism and inhibition of cell proliferation, but have been associated with unfavorable pharmacokinetics and the potential adverse effects of PRLR inhibition, as observed in Prlr2/2 mice, has not been ascertained. PRL Effects on Gonadotrophin Secretion The effects of normal circulating PRL levels on gonadotrophin secretion are not known. Prl2/2 and Prlr2/2 female mice are sterile, and both types have disordered estrous cycles, but whether the sterility in these cases is due to cell-autonomous effects in the gonads, altered gonadotrophin secretion, or to a combination of these is not clear from these global knockout mice (see PRL effects on the ovary and PRL effects on the testes, below) [76,78]. Prl2/2 male mice have reduced plasma LH levels; gonadotrophin levels in Prlr2/2 male mice are not altered. In normal women treated with short-term bromocriptine to lower PRL levels to about 5 ng/mL, there is no change in the pulsatile secretion of LH and FSH but estradiol levels are higher during the last 3 days of the follicular cycle, and progesterone levels lower during the luteal phase. Hyperprolactinemia, on the other hand, has a number of effects on various steps in the reproductive axis (Fig. 5.10). Hyperprolactinemia has been found in most

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FIGURE 5.10

Diagram demonstrating how hyperprolactinemia induces anovulatory infertility. As PRLRs are not expressed on GnRH neurons, hyperprolactinemia induces infertility via its actions on other intermediate cells. Increased serum PRL levels result in decreased kisspeptin expression in hypothalamic Kiss1 neurons (arcuate [ARC] and anteroventral periventricular [AVPV] nuclei), mediated by PRLR expressed on these cells. Suppression of kisspeptin reduces hypothalamic GnRH secretion. This leads to reduced LH and FSH secretion and loss of ovarian stimulation, which result in infertility. PRL may also have direct effects on other GnRH afferent neurons. Moreover, the intermediate participation of other non-neural factors acting on Kiss1 and GnRH neurons secretions cannot be excluded.

studies to suppress LH pulsatile secretion by decreasing pulse amplitude and frequency. At the menopausal transition in humans, hyperprolactinemia can prevent the expected rise in gonadotrophins; normalization of PRL levels with bromocriptine results in a rise in gonadotrophin levels and hot flashes. Hyperprolactinemia is a well established cause of hypogonadotrophic hypogonadism and anovulatory infertility [123], but the mechanism by which PRL inhibits hypothalamic secretion of GnRH-I was unclear. Recently, it was demonstrated that this inhibition involves metastasis suppressor kisspeptin-1 neurons that express PRLR. Mice rendered hyperprolactinemic do not ovulate, have low circulating levels of LH and FSH, and exhibit reduced hypothalamic expression of the Kiss1 gene, which encodes kisspeptin-1. Intraperitoneal injections of kisspeptin-1 restored both hypothalamic GnRH-I and gonadotrophin secretion, as well as ovarian cyclicity suggesting that kisspeptin-1 neurons have a major role in hyperprolactinemic anovulation [124]. These experiments suggest PRLmediated inhibition of GnRH-I occurs, in part, through decreased secretion of kisspeptin-1. PRL might also have direct effects on other GnRH-I afferent neurons, and the possibility that other non-neural factors could act on kisspeptin-1 and GnRH-I neuronal secretion cannot be excluded. Likewise, during lactation, a state of physiological hyperprolactinemia, a selective loss of kisspeptin-1 input to GnRH-I neurons has been observed in mice [125] and PRL contributes to

inhibition of kisspeptin-1 in the arcuate nucleus and anteroventral periventricular nucleus. PRL Effects on the Ovary and Fertility The effects of PRL on ovarian function and fertility are complex and to some extent species-specific. PRL is an essential luteotrophic hormone in rodents, but not in humans. Instead, in humans, pituitary LH supports luteal development and steroidogenesis during the menstrual cycle, and the embryonic trophoblast sustains the corpus luteum during pregnancy. The action of PRL on the ovaries has been fairly wellcharacterized in rats, but the role of PRL in normal ovarian physiology is not well-defined in humans. A comparison of PRL profiles across the estrous and menstrual cycles in rodents and humans, respectively, reveals marked differences, supporting the notion that the function of PRL during these cycles differs between the two species. In humans, serum PRL levels remain relatively stable throughout the menstrual cycle, except for a slight increase during the luteal phase, the functional significance of which is not known. By contrast, in rats, PRL levels rise in a triphasic manner showing a sharp rise just prior to ovulation, followed by a plateau and an extended termination phase [30]. The PRLR is expressed in human granulosa cells and the human ovary produces its own PRL, but the specific roles of circulating versus autocrine-derived PRL on ovarian function are unknown. PRL is found in human follicular fluid

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where it has been shown to stimulate ovarian endothelial cell proliferation [126]. Although the role(s) of normal physiologic levels of PRL on ovarian function and female fertility are not fully known in humans, it is well established that the actions of PRL and the activity of its receptor are essential for fertility in female mice. Both Prl2/2 and Prlr2/2 female mice are completely infertile. Female Prl2/2 mice have irregular estrous cycles. Whether this is due to an ovarian or hypothalamic defect is not known for certain, but obvious histological defects in the ovaries are not observed. Prlr2/2 female mice, on the other hand, display multiple reproductive abnormalities, including reduced rates of mating, fertilization, and ovulation, as compared to wild-type mice. Prlr-deficient ovaries contain fewer primary follicles and those eggs that do become fertilized develop poorly as oocyte development is arrested almost immediately after fertilization. In these mice, the corpus luteum regresses, and is unable to support implantation and placental development [127]. Administration of progesterone to Prl2/2 and Prlr2/2 females rescues implantation and early embryonic development. Thus, regulation of sustained progesterone production, permitting the proper expression of progesterone-dependent genes, is the essential function of PRL that is required for normal implantation in Prl2/2 and Prlr2/2 mice in early pregnancy. The cause of late embryonic lethality is not known for certain, but may be a result of the absence of local decidual PRL production, leading to derepression of two genes, IL-6 and 20α-hydroxysteroid dehydrogenase (20αHsd), whose expression is detrimental for the normal progress of pregnancy. The phenotypic differences between the Prl2/2 and Prlr2/2 mouse models could reflect compensatory action by numerous murine PRLlike proteins that are capable of activating the PRLR, whereas Prlr2/2 mice cannot respond to any of the PRL family members. The major mechanism by which PRL performs its luteotrophic action in rodents is through stimulation of progesterone production by luteal cells [128]. Specifically, PRL downregulates the expression of 20α-Hsd, which prevents the catabolism of progesterone to an inactive metabolite. This serves to increase progesterone secretion from the corpus luteum. Paradoxically, in some experimental settings, PRL can also induce luteolysis. In human granulosa cells, PRL stimulates expression of type II 3β-hydroxysteroid dehydrogenase, the enzyme responsible for catalyzing the final step in progesterone biosynthesis, and increases IGF-2 secretion. Perfusion studies of human ovaries in vitro show that PRL directly suppresses progesterone and estrogen secretion. PRL inhibits estrogen formation by (1)

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antagonizing the stimulatory effects of FSH on aromatase activity and (2) directly inhibiting aromatase synthesis itself. In fact, PRL is required at low doses (,20 ng/mL) for progesterone production by granulosa cell cultures, but at higher concentrations (i.e., those that correlate with hyperprolacinemia in women), PRL inhibits progesterone production. These in vitro findings are in line with in vivo studies of luteal function in women where treatment with bromocriptine to lower normal PRL levels to hypoprolactinemic levels resulted in lowered progesterone levels and shorter luteal phases. As noted above, during pregnancy, human maternal PRL secretion rises gradually beginning at 6 8 weeks gestation until term. In the human fetal circulation, PRL rises slightly starting at around 10 weeks, plateaus, and then rises again very sharply at 30 weeks until term. The decidua produces and secretes high amounts of PRL into the amniotic fluid beginning at 12 weeks, peaking at around 20 weeks, and then gradually declining until term. The exact function of decidual or amniotic fluid PRL is not known, although several functions of decidual PRL have been postulated. Putative functions of decidual PRL, such as facilitation of trophoblast growth, inhibition of myometrial contractility, and regulation of angiogenesis, have been suggested but limited data are available to support these theories [129]. Human PL shows a similar pattern of rise to that of PRL, only on a larger scale, such that by 30 weeks, PL levels exceed PRL levels by 10-fold. Clinical Effects of Hyperprolactinemia on Menstrual Function Elevated serum PRL levels (hyperprolactinemia) in women cause oligomenorrhea or amenorrhea. The amenorrhea caused by hyperprolactinemia is typically secondary, but primary amenorrhea can occur if the disorder begins before the usual age of puberty. In patients with primary amenorrhea due to hyperprolactinemia, estrogen deficiency and failure to develop normal secondary sexual characteristics may be the presenting problem. Galactorrhea is variable in this setting because the breast may not have been exposed to appropriate priming with estrogen and progesterone. Patients with primary amenorrhea tend to have macroadenomas more commonly than those with secondary amenorrhea, for uncertain reasons. When amenorrhea or oligomenorrhea is associated with galactorrhea, it usually is a manifestation of hyperprolactinemia. Hyperprolactinemia is found in many women with a short luteal phase. It is likely that a short luteal phase is the first evidence of interference in the normal cycle by hyperprolactinemia. Infertility also may be a

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presenting symptom of patients with hyperprolactinemia and is invariable when gonadotrophin levels are suppressed with anovulation. In three series of women (combined number of 367 cases) studied for infertility, one-third were found to have hyperprolactinemia. Most of these women presented with amenorrhea and galactorrhea as well, but hyperprolactinemia without other symptoms was found in five of the 22 hyperprolactinemic women in one series of 113 cases of infertility. That PRL excess may be important in this type of patient is suggested by the finding that treatment of similar patients with dopamine agonists restored fertility. In some infertile women, transient hyperprolactinemia lasting for 1 2 days during the cycle can be documented; this subset usually responds to bromocriptine with increased progesterone during the luteal phase and improved fertility. Reduced libido and orgasmic dysfunction are found in most hyperprolactinemic amenorrheic women when such complaints are specifically elicited. Reduction of PRL levels to normal restores normal libido and sexual function in most of these women. PRL levels have been reported as elevated in 13 50% of women with polycystic ovary syndrome (PCOS) [130]. The relationship between hyperprolactinemia and PCOS is debated. Recent studies in PCOS patients with increased PRL levels have implicated other causes of hyperprolactinemia, because hyperprolactinemia is not a clinical manifestation of PCOS [131,132]. The presence of stress-related hyperprolactinemia and macroprolactinemia may interfere with the diagnosis of PCOS and leads to unnecessary and expensive diagnostic and therapeutic approaches [133].

Male Reproductive Tissues While PRL clearly plays an essential reproductive role in females, the role of PRL in male reproductive function is less defined. A conclusive determination regarding the involvement of PRL in male fertility has not emerged from Prl2/2 and Prlr2/2 mouse models. The deletion of the PRLR does not alter fertility parameters, sperm reserves, plasma gonadotrophin levels, testosterone levels, or weight or histology of the testes or epididymides [134]. Deficiency of PRL itself in Prl2/2 mice is associated with reduced plasma LH levels, but not with effects on male fertility parameters or basal plasma testosterone levels [135]. In other rodents and humans, indirect evidence supports the view that PRL plays a subtle role in testes and/or germ cell function. PRL promotes Leydig cell proliferation and differentiation in prepubertal hypophysectomized rats and is involved in the maintenance of Leydig cell morphology, upregulation of LH

receptor expression, and potentiation of LH-induced steroidogenesis. In humans, PRLRs are expressed in germ cells undergoing spermatogenesis in seminiferous tubules, and in Leydig cells, vas deferens, epididymis, prostate, and seminal vesicles. Human semen contains significant quantities of PRL. The functional significance of PRL or PRLR expression at many of these sites is still unclear. However, the presence of PRLR in differentiating germ cells in the testes is supportive of data that PRL acts as a prosurvival factor for human spermatozoa by preserving motility, suppressing sperm capacitation, and enhancing vitality by inhibiting entry into the cell death pathway [136]. Moreover, reduction of normal PRL levels by administration of dopamine agonists in men results in suppression of basal and hCG-stimulated testosterone levels, implying a physiologic role for PRL in testosterone production in humans. A substantial body of data from in vitro and in vivo studies supports mitogenic or prosurvival roles for PRL in the prostate. PRL and PRL isoforms are expressed and functional in normal human prostate epithelia and malignant human prostate tissue [137]. Some studies in human prostate cancer cell lines have shown that autocrine-derived PRL promotes prostate cancer cell growth [138], whereas others show that exogenous PRL inhibits apoptosis. In rodents, elevated levels of PRL are associated with increased prostate growth. Ubiquitous transgenic overexpression of PRL in mice leads to prostatic hyperplasia with elevated serum testosterone levels [139]. Prostate-specific transgenic overexpression of PRL leads to stromal hyperplasia, ductal dilatation, focal epithelial dysplasia, but without changes in serum androgen levels, indicating that the abnormal prostate findings are not consequential to hyperandrogenemia, and that autocrine-derived PRL is at least a contributing factor to these effects. By contrast, the prostate glands of Prl2/2- mice are B30% smaller than those of wild-type mice, and, as found in murine models in the breast, PRLR deficiency reduces the incidence of SV40 T-antigen-induced prostate carcinoma [140]. Thus, these studies, which support proproliferative and/or antiapoptotic properties for PRL in the prostate, suggest potential (patho)physiological roles for PRL in human prostate development and/or disease, possibly elaborated at an autocrine level. However, data on the frequency of prostate hyperplasia or carcinoma in humans with sustained systemic hyperprolactinemia are lacking. Clinical Effects of PRL in Males Chronic hyperprolactinemia in males results in impotence and decreased libido in over 90% of cases [124]. Other findings of hypogonadism, such as decreased beard growth and strength are less commonly encountered.

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Galactorrhea in men is reported in 10 20% of cases of hyperprolactinemia, and is virtually pathognomonic of a prolactinoma. The frequency of hyperprolactinemia among men with complaints of impotence or infertility as assessed by surveys ranges between 2 and 25% among various series. However, only 1 5% of men with infertility have been found to be hyperprolactinemic. Hyperprolactinemia in men is associated with decreased pulsatile secretion of LH and FSH (as noted above), and low or low-normal testosterone levels. The testosterone response to stimulation with hCG has been reported to be both decreased and normal; in those with decreased responses there is improvement in the response when PRL levels are lowered with bromocriptine. If there is sufficient normal pituitary tissue, reduction of elevated PRL levels to normal usually results in a return of normal testosterone levels [141]. Although some studies have suggested that drug-induced hyperprolactinemia partially inhibits the enzyme 5-αreductase, resulting in reduced dihydrotestosterone (DHT) levels, hyperprolactinemia in men with prolactinomas is not associated with this effect. Hyperprolactinemia has an effect on impotence that is independent of testosterone levels: Testosterone therapy of hyperprolactinemic men does not always correct the impotence until PRL levels are brought down to normal. Whether this is due to a decrease in DHT levels has not been verified directly. Elevated PRL levels have adverse effects on male germ cell and testes function. Sperm counts and motility are decreased with an increase in abnormal form [142]. Histology of the testes reveals abnormal seminiferous tubule walls and altered Sertoli cell ultrastructure. Hyperprolactinemia may have a sustained effect on male reproductive function, as the semen analysis does not always return to normal despite therapy that successfully normalizes testosterone and PRL levels [142].

Carbohydrate Metabolism and Adiposity PRL functions as a metabolic regulator in two chief areas: (1) pancreatic β-cell development and function; (2) appetite regulation and adiposity. The phenotypic assessment of Prlr2/2 mice indicates that signaling through the PRLR, which serves as a common receptor for both PL and PRL, is important to fully attain normal β-cell mass, insulin content, and insulin secretory capacity The expansion of β-cell mass during both embryogenesis and the postnatal period is impaired in this mouse model [143]. Prlr2/2 newborns display a 30% reduction of β-cell mass, consistent with reduced proliferation index at the 18.5th day of embryonic development. Reduced pancreatic Igf-II expression in both rat and mouse models suggests that this factor

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may constitute a molecular link between PRL signaling and cell ontogenesis [143]. Upregulation of the functional β 2 cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on PLs and PRLR that play a particularly central role in the adaptation of islets to pregnancy, as illustrated by studies in heterozygous Prlr1/2 female mice. Pregnant Prlr1/2 female mice exhibit impaired glucose clearance, reduced glucosestimulated insulin release, and lower insulin levels as a result of impaired islet expansion [144]. In support of the critical role of PRLR for islet expansion during gestation, it has been shown that Stat 5, PI3 kinase, MAPK, and pathways involving the endocrine tumor suppressor menin all collaborate to mediate the proliferative effects of PRL and PL on pancreatic islets during pregnancy [145]. Furthermore, a strong lactogendependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet β-cells during pregnancy. Since newly formed serotonin is rapidly released, this lactogen-induced β-cell function may serve local or endocrine tasks, the nature of which remains to be identified [146]. Moreover, male mouse islets, when exposed to the environmental conditions of pregnancy, undergo similar changes in gene expression as do female transplanted islets. These results corroborate the finding that the key environmental factor driving the phenotypic plasticity of β-cells is a rise in circulating PL that activates PRLR on β-cells. This conclusion is supported by experiments of cultured and transplanted islets and results obtained from Prlr2/2 mice [147]. Although studies in humans are limited, in vitro experiments confirm that PRL increases β-cell number and stimulates insulin secretion in cultured human islets. A protective effect of lactogens has been demonstrated on human β-cell apoptosis. The improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are relevant for improvement of the islet isolation procedure and for clinical islet transplantation [148]. With regard to appetite regulation and adiposity, in vivo studies in rodents and humans support a modest orexigenic effect of PRL. In rats, higher PRL levels are associated with greater food intake and body weight, while suppression of PRL levels leads to the opposite effects [149]. Prlr2/2 mice exhibit a subtle reduction in parametrial and subcutaneous adipose tissue mass as compared to wild-type littermates, although no differences are observed in overall body weight [150]. Prlr2/2 mice are highly resistant to high fat diet-induced obesity, owing to the emergence of a brown adipose-like phenotype in peculiar white fat depots. This finding is associated with a concomitant

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increase of Prdm16, Pgc1α, AdRβ3, and Foxc2 that constitutes a molecular switching mechanism. This converges toward an activation of thermogenic brown capacity with the final increase of UCP1 responsible for heat dissipation and resistance to high-calorie weight gain. Then, PRL signaling represents an additional determinant of energy homeostasis during physiological and pathophysiological conditions [151]. Prl2/2 mice exhibit normal body weight and adiposity [152], suggesting that PLs or other ligands act in conjunction with PRL to influence adiposity. In humans, patients with hyperprolactinemia have been described to exhibit altered energy metabolism and are candidates to obesity. Treatment of these patients to normalize their PRL levels is accompanied by a reduction of body weight and an improvement of glucose tolerance and insulin sensitivity [153]. In contrast to these results, suppression of PRL levels with bromocriptine has no effect on glycemic control in normoprolactinemic subjects with insulindependent diabetes. A new preparation of bromocriptine, which has been approved in the US for the treatment of diabetes [154], has been shown to cause a modest reduction in plasma glucose and hemoglobin A1c levels. However, the safety of lowering normal PRL levels to subnormal levels, with respect to fertility and sexual function, has not been proven. Other in vitro studies suggest a possible role for PRL in adipogenesis. PRL itself is produced in small amounts by human adipocytes, including sources from the breast, visceral, and subcutaneous adipose tissue. The PRLR is expressed in both brown and white adipose tissue [149]. PRL upregulates the mRNA expression of its receptor, and two transcription factors principally involved in adipocyte differentiation, CEBP/β and PPARγ, and it also stimulates the conversion of NIH-3T3 fibroblasts into adipocytes. Complex interactions between PRL and several adipokines, such as leptin and adiponectin, have been described. However, the data regarding effects of exogenous PRL, transgenic overexpression of PRL, and PRL deficiency on leptin levels in vivo are inconsistent and preclude reliable conclusions [155].

activity relies on a crosstalk between Jak2 and the PKA signaling pathway through control of the stability of the activated CREB protein [157]. Plasma dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) levels are mildly elevated in about 50% of women with hyperprolactinemia in some series. In most of these studies, however, the investigators did not correlate androgen levels with the presence of hirsutism or other indices of virilization. The abnormal androgen levels return to normal with correction of the hyperprolactinemia by dopamine agonists.

Calcium and Bone Metabolism PRL may have a physiologic role in calcium and bone metabolism, and increases intestinal calcium absorption even in vitamin D-deficient rats and can stimulate 1-α-hydroxylation of renal 25-hydroxyvitamin D, resulting in increased plasma 1,25 (OH)2D levels. In humans, however, plasma 1,25 (OH)2D levels and intestinal calcium absorption are normal in hyperprolactinemic subjects. Prlr2/2 mice have decreased bone formation rates and bone mineral density in association with increased parathyroid hormone levels but decreased estradiol and progesterone levels so that it is difficult to delineate the contribution of a lack of PRL [158]. The initial observation by Klibanski et al. that hyperprolactinemic women have decreased bone mineral density [159] was confirmed by others but whether this effect is mediated by estrogen deficiency or is a direct effect of the hyperprolactinemia has been controversial. Correction of hyperprolactinemia results in increased bone mass. Studies of hyperprolactinemic women who were not amenorrheic and hypoestrogenemic have shown that bone mineral density is normal, confirming the initial hypothesis that estrogen deficiency mediates bone mineral loss. A similar, androgen-dependent loss of bone mineral is reported in hyperprolactinemic men, which is reversible with reversal of the hypoandrogenic state.

Immune System Adrenal Cortex It has been suggested that PRL acts on the adrenal cortex to regulate adrenocortical function even though the molecular link between PRL/Stat5 and steroidogenic genes has not yet been established [156]. Using Prlr2/2 mice and both cell lines and adrenocortical primary culture, it was shown that PRL has no significant effect on adrenal steroidogenesis in vivo and in vitro. However, Jak2 controls adrenal steroidogenesis. This

Hypophysectomized rats evince thymic involution and decreased cell-mediated immune function, both of which are reversed by the administration of ovine PRL [160]. Similarly, hypoprolactinemia induced in animals by bromocriptine or anti-PRL antibodies has been shown to lead to impaired lymphocyte proliferation and macrophage-activating factor production, again reversed by ovine PRL. PRL has long been proposed as an immune-stimulating and detrimental factor in autoimmune disorders. In the murine model of

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systemic lupus erythematosus, bromocriptine was shown to suppress immunoglobulin levels, autoantibodies, and immune-complex glomerulonephritis, and to improve survival rates. Interestingly, however, Prl2/2 and Prlr2/2 animals have shown that PRL is not essential for normal immunity, as they have normal development, distribution, and function of T-lymphocytes, B-lymphocytes, and NK cells [160]. It has been suggested that other cytokines may be compensating for the lack of PRL in these knockout models or that PRL has significant effects on the immune system only under conditions of stress. A critical overview of data supporting a role for PRL in the regulation of immune responses has been recently published. In addition, studies are focused on the involvement of PRL in autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis, in light of the recently outlined regenerative properties of this hormone [161]. The work performed in experimental models of autoimmune disorders has revealed that PRL can promote, be dispensable, or even protect against autoimmunity. The involvement of PRL in autoimmunity appears more complex than being solely restricted to immune stimulation, and might be the result of a fine interplay between immune-modulating and regenerative properties of this hormone. Studies of patients with hyperprolactinemia of various etiologies have suggested an increased rate of autoantibodies (including antithyroid, anti-dsDNA, anti-ro, anticardiolipin, and antinuclear antibodies [ANA]) without clinical evidence of autoimmune disease [160]. Conversely, elevated levels of PRL have been found in patients with lupus, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Reiter syndrome, primary Sjo¨gren syndrome, psoriasis, and uveitis, leading to hypothesized causal relationships and a possible therapeutic target. Multiple trials of varying designs with DA agonists, primarily bromocriptine, have shown some clinical improvement in some of these disorders. However, it has not been firmly established in humans that there is a definitive role for PRL in immunomodulation and whether DA agonists have therapeutic utility in some of these disorders [160].

Acknowledgments The author extends her thanks to Dr. Mark E. Molitch who authored the previous version of this chapter.

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[120] Oakes SR, Robertson FG, et al. Loss of mammary epithelial prolactin receptor delays tumor formation by reducing cell proliferation in low-grade preinvasive lesions. Oncogene 2007;26(4):543 53. [121] Bogorad RL, Courtillot C, et al. Identification of a gain-offunction mutation of the prolactin receptor in women with benign breast tumors. Proc Natl Acad Sci USA 2008;105 (38):14533 8. [122] Clevenger CV, Zheng J, Jablonski EM, Galbaugh TL, Fang F. From bench to bedside: future potential for the translation of prolactin inhibitors as breast cancer therapeutics. J Mammary Gland Biol Neoplasia 2008;13(1):147 56. [123] Melmed S, Casanueva FF, et al. Diagnosis and treatment of hyperprolactinemia: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011;96(2):273 88. [124] Sonigo C, Bouilly J, et al. Hyperprolactinemia-induced ovarian acyclicity is reversed by kisspeptin administration. J Clin Invest 2012;122(10):3791 5. [125] Liu X, Brown RS, Herbison AE, Grattan DR. Lactational anovulation in mice results from a selective loss of kisspeptin input to GnRH neurons. Endocrinology 2014;155 (1):193 203. [126] Castilla A, Garcia C, Cruz-Soto M, Martinez dlE, Thebault S, Clapp C. Prolactin in ovarian follicular fluid stimulates endothelial cell proliferation. J Vasc Res 2010;47(1):45 53. [127] Grosdemouge I, Bachelot A, Lucas A, Baran N, Kelly PA, Binart N. Effects of deletion of the prolactin receptor on ovarian gene expression. Reprod Biol Endocrinol 2003;1:1 12. [128] Stocco C, Telleria C, Gibori G. The molecular control of corpus luteum formation, function and regression. Endocr Rev 2007;28(1):117 49. [129] Jabbour HN, Critchley HO. Potential roles of decidual prolactin in early pregnancy. Reproduction 2001;121(2):197 205. [130] Falaschi P, Pozo del E, et al. Prolactin release in polycystic ovary. Obstet Gynecol 1980;55(5):579 82. [131] Escobar-Morreale HF. Macroprolactinemia in women presenting with hyperandrogenic symptoms: implications for the management of polycystic ovary syndrome. Fertil Steril 2004;82(6):1697 9. [132] Filho RB, Domingues L, Naves L, Ferraz E, Alves A, Casulari LA. Polycystic ovary syndrome and hyperprolactinemia are distinct entities. Gynecol Endocrinol 2007;23(5):267 72. [133] Hayashida SA, Marcondes JA, et al. Evaluation of macroprolactinemia in 259 women under investigation for polycystic ovary syndrome. Clin Endocrinol (Oxf) 2014;80(4):616 18. [134] Binart N, Melaine N, et al. Male reproductive function is not affected in prolactin receptor-deficient mice. Endocrinology 2003;144(9):3779 82. [135] Steger RW, Chandrashekar V, Zhao W, Bartke A, Horseman ND. Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 1998;139(9):3691 5. [136] Pujianto DA, Curry BJ, Aitken RJ. Prolactin exerts a prosurvival effect on human spermatozoa via mechanisms that involve the stimulation of Akt phosphorylation and suppression of caspase activation and capacitation. Endocrinology 2010;151 (3):1269 79. [137] Nevalainen MT, Valve EM, Ingleton PM, Nurmi M, Martikainen PM, Harkonen PL. Prolactin and prolactin receptors are expressed and functioning in human prostate.. J Clin Invest 1997;99(4):618 27. [138] Dagvadorj A, Collins S, et al. Autocrine Prolactin Promotes Prostate Cancer Cell Growth via Janus Kinase-2-Signal Transducer and Activator of Transcription-5a/b Signaling Pathway. Endocrinology 2007;148(7):3089 101.

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[139] Kindblom J, Dillner K, et al. Prostate hyperplasia in a transgenic mouse with prostate-specific expression of prolactin. Endocrinology 2003;144(6):2269 78. [140] Robertson FG, Harris J, et al. Prostate development and carcinogenesis in prolactin receptor knockout mice. Endocrinology 2003;144(7):3196 205. [141] Molitch ME, Elton RL, et al. Bromocriptine as primary therapy for prolactin-secreting macroadenomas: results of a prospective multicenter study. J Clin Endocrinol Metab 1985;60 (4):698 705. [142] De Rosa M, Ciccarelli A, et al. The treatment with cabergoline for 24 month normalizes the quality of seminal fluid in hyperprolactinaemic males. Clin Endocrinol (Oxf) 2006;64 (3):307 13. [143] Auffret J, Freemark M, et al. Defective prolactin signaling impairs pancreatic beta-cell development during the perinatal period. Am J Physiol Endocrinol Metab 2013;305(10): E1309 18. [144] Huang C, Snider F, Cross JC. Prolactin receptor is required for normal glucose homeostasis and modulation of beta-cell mass during pregnancy. Endocrinology 2009;150(4):1618 26. [145] Karnik SK, Chen H, et al. Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational diabetes mellitus. Science 2007;318(5851):806 9. [146] Schraenen A, Lemaire K, et al. Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy. Diabetologia 2010;53(12):2589 99. [147] Goyvaerts L, Lemaire K, et al. Prolactin receptors and placental lactogen drive male mouse pancreatic islets to pregnancyrelated mRNA changes. PLoS One 2015;10(3):e0121868. [148] Terra LF, Garay-Malpartida MH, Wailemann RA, Sogayar MC, Labriola L. Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways. Diabetologia 2011;54(6):1388 97. [149] Carre N, Binart N. Prolactin and adipose tissue. Biochimie 2014;97:16 21. [150] Flint DJ, Binart N, Boumard S, Kopchick JJ, Kelly P. Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects

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upon proliferation, differentiation and hormone sensitivity. J Endocrinol 2006;191(1):101 11. Auffret J, Viengchareun S, et al. Beige differentiation of adipose depots in mice lacking prolactin receptor protects against high-fat-diet-induced obesity. FASEB J 2012;26(9):3728 37. Lapensee CR, Horseman ND, Tso P, Brandebourg TD, Hugo ER, Ben Jonathan N. The Prolactin-deficient mouse has an unaltered metabolic phenotype. Endocrinology 2006;. Naliato EC, Violante AH, et al. Body fat in nonobese women with prolactinoma treated with dopamine agonists. Clin Endocrinol (Oxf) 2007;67(6):845 52. Scranton R, Cincotta A. Bromocriptine--unique formulation of a dopamine agonist for the treatment of type 2 diabetes. Expert Opin Pharmacother 2010;11(2):269 79. Brandebourg TD, Bown JL, Ben Jonathan N. Prolactin upregulates its receptors and inhibits lipolysis and leptin release in male rat adipose tissue. Biochem Biophys Res Commun 2007;357(2):408 13. Jaroenporn S, Nagaoka K, Ohta R, Watanabe G, Taya K. Prolactin induces phosphorylation of the STAT5 in adrenal glands of Hatano rats during stress. Life Sci 2009;85 (3 4):172 7. Lefrancois-Martinez AM, Blondet-Trichard A, et al. Transcriptional Control of Adrenal Steroidogenesis: novel connection between Janus Kinase (JAK) 2 protein and Protein Kinase A (PKA) through stabilization of cAMP response element-binding protein (CREB) transcription factor. J Biol Chem 2011;286(38):32976 85. Clement-Lacroix P, Ormandy C, et al. Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice. Endocrinology 1999;140:96 105. Klibanski A, Neer RM, Beitins IZ, Ridgway EC, Zervas NT, McArthur JW. Decreased bone density in hyperprolactinemic women. N Engl J Med 1980;303(26):1511 14. Chuang E, Molitch ME. Prolactin and autoimmune diseases in humans. Acta Biomed 2007;78(Suppl. 1):255 61. Costanza M, Binart N, Steinman L, Pedotti R. Prolactin: a versatile regulator of inflammation and autoimmune pathology. Autoimmun Rev 2015;14(3):223 30.

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C H A P T E R

6 Thyroid-Stimulating Hormone Virginia D. Sarapura and Mary H. Samuel

INTRODUCTION

ONTOGENY OF THYROTROPH CELLS

Thyroid-stimulating hormone (TSH) is a glycoprotein produced by the thyrotroph cells of the anterior pituitary gland. TSH, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), as well as the placental hormone chorionic gonadotrophin (CG), consist of a heterodimer of two noncovalently linked subunits, α and β. The β-subunit is unique to each and confers specificity of action while the α-subunit is common to all four glycoprotein hormones. Each TSH subunit is encoded by a separate gene located on a different chromosome and is transcribed in a coordinated manner responsive mainly to the stimulatory effect of thyrotrophin-releasing hormone (TRH) and the inhibitory effect of thyroid hormone. Production of bioactive TSH involves a process of cotranslational glycosylation and folding that enables combination between the nascent α- and β-subunits. TSH is stored in secretory granules and released into the circulation in a regulated manner responsive mainly to the stimulatory effect of TRH. Circulating TSH binds to specific cell surface receptors on the thyroid gland where it stimulates the production of thyroid hormones, L-thyroxine (T4), and L-triiodothyronine (T3), which act on multiple organs and tissues to modulate many metabolic processes as well as result in a negative inhibition of TSH output. The introduction of sensitive TSH assays has allowed accurate measurement of the level of circulating TSH and has led to the recognition of abnormal production of TSH related with abnormal function of the thyroid gland reflecting a wide range of metabolic derangements.

Thyrotrophs comprise only 5% of the cells in the anterior pituitary gland, yet these cells are solely responsible for synthesizing the α- and β-subunits of TSH, the key pituitary hormone that circulates in serum and controls the growth and function of the thyroid gland. The distinct cell types of the anterior and intermediate lobes of the pituitary are defined by the hormone they produce and secrete, these include thyrotrophs (TSH), gonadotrophs (LH, FSH), corticotrophs adrenocorticotrophic hormone (ACTH), somatotrophs growth hormone (GH), lactotrophs prolactin, and melanotrophs melanocyte stimulating hormone(MSH). The anterior pituitary develops from Rathke’s pouch, an invagination of oral ectoderm located at the anterior neural ridge, directly contacting the emerging infundibulum [1]. The close association between these tissues suggests that inductive interactions are apt to be very important [2]. Pituitary organogenesis involves the proliferation of common progenitor cells and their subsequent differentiation by a series of precisely controlled extrinsic and intrinsic signals that regulate cell proliferation, lineage commitment, and terminal cell differentiation [3]. Many of the key genes initiating and regulating these developmental pathways continue to be uncovered and include transcription factors, signaling molecules, and cell surface receptors. Many of these factors act transiently during pituitary development while expression of others persists in the mature differentiated cell. Signals derived from Rathke’s pouch and the adjacent infundibulum at embryonic day 9.5 (e9.5) in the mouse initiate the

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temporal and spatial organization of the different pituitary cell types. The key factors involved in this initial phase include sonic hedgehog (Shh) and members of fibroblast growth factor (FGF), bone morphogenetic protein (BMP), Notch and Wnt families of morphogens/growth factors [4]. These factors themselves are not specific to the pituitary but play additional roles in the patterning of other organ systems. Expression of dorsal factors such as FGF8/10/18 act by opposing the developmental actions of the ventral BMP2/Shh signals [3]. Several of the genes critical for regulating pituitary development have been identified through the characterization of hereditary mouse and human pituitary endocrine deficiencies [5]. Distinct cell lineages emerge as a result of signaling gradients of transcription factors formed in a spatially and temporally specific fashion (Fig. 6.1) [6]. The end result is overlapping programs of transcription factor expression [7]. The stereotypic pattern of activation of these early transcription factors as well as an assemblage of other tissue-restricted factors are critical for determination of the cells that produce the glycoprotein hormone α-subunit (αGSU), which is common to the dimeric pituitary hormones TSH, LH, and FSH. The αGSU, glycoprotein hormone alpha is the first pituitary hormone gene expressed during early development [8] at mouse e10.5. Wnt5a and BPM4, which are expressed in the adjacent neuroepithelium, provide the initiating signals followed by expression of Hesx1, Ptx1/2, and Lhx3/4 [2]. TSHβ expression begins in the rostral tip of the pituitary at mouse e12.5 and correlates with expression of thyrotroph embryonic factor which is restricted to the pituitary at this embryonic stage [9]. By birth, TSHβ expression in the rostral tip has

FIGURE 6.1 Thyrotroph cell origin during anterior pituitary development. Schematic representation of the regions of the pituitary where Pou1F1 and GATA2 transcripts are detected. Somatotrophs and lactotrophs evolve from Pou1F1(1)/GATA2(2) cells, gonadotrophs from POU1F1(2)/GATA2(1) cells, and thyrotrophs from Pou1F1(1)/GATA2(1) cells. The thyrotrophs in the rostral tip (Tr) appear at an earlier stage in the region where TEF is expressed, but do not persist in the adult. Source: Modified from Dasen JS, O’Connell SM, Flynn SE, Treier M, Gleiberman AS, Szeto DP, et al. Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell 1999;97:587 98.

disappeared and another population of thyrotrophs arises by e15.5 in the caudomedial region, following expression of Pou1F1 (Pit-1), a POU-homeodomain transcription factor restricted to thyrotrophs, somatotrophs, and lactotrophs [10]. Both POU1F1 and TSH β-subunit expression are present in the wild-type but not in the Snell dwarf mouse, which has a POU1F1 gene mutation that renders it inactive [11]. These data suggest that the second population of thyrotrophs, associated with Pou1F1, is likely the source of mature thyrotrophs. In addition, Pou1F1 mutations have also been reported in humans [12,13] and are associated with a lack of thyrotrophs, somatotrophs, and lactotrophs, analogous to the Snell dwarf mouse phenotype. Pou1F1 synergizes with Lhx3 to activate the TSH α-subunit promoter [14]. Pou1F1 expression, in turn, depends on the expression of another transcription factor, Prophet of Pou1F1 (Prop1). Mutations in this factor have been associated with many cases of combined pituitary hormone deficiency (CPHD) in humans, affecting not only thyrotroph, somatotroph, and lactotroph but also gonadotroph expression [15]. Mutations in the Prop1 gene were also found in the Ames dwarf mouse that exhibits a similar phenotype [16]. When complexed with β-catenin it acts as a transcriptional activator of Pou1F1 and can also work to repress Hesx1. An additional enhancing factor, Atbf1, also activates early Pou1F1 expression along with Prop1 [17]. Of note, both Atbf1 and Prop1 only persist in the pituitary for a limited time period between e10.5 and e14.5. Thus precise temporal regulation is critical for proper pituitary development [3]. However, other cell-type-restricted factors must be involved in the initiation of thyrotroph-specific gene expression, since the presence of both Pou1F1 and Lhx3 in somatotrophs and lactotrophs does not result in TSH production by these cells. Mechanisms exist that establish combinatorial codes which specify distinct cell phenotypes. In many cases, such a code involves reciprocal synergistic or inhibitory protein protein interactions between two or more cell-type-restricted transcription factors. Studies have suggested that a zinc finger transcription factor, Gata2, plays a critical role in thyrotroph differentiation [6]. Gata2 is transcribed in the developing anterior pituitary as early as e10.5 and persists in an expression pattern coincident with the glycoprotein hormone α-subunit. Gata2 binds and transactivates the αGSU promoter [18] and acts synergistically with Pou1F1 to activate the TSHβ gene [19]. A ventral dorsal gradient of Gata2 occurs early in development in response to BMP2: the intermediate cells that express both Gata2 and Pou1F1 activate the thyrotroph-specific genes, whereas the more ventral cells that express Gata2 and not Pou1F1 become gonadotrophs and the more dorsal cells that express Pou1F1 and not Gata2 become somatotrophs and lactotrophs [6]. The in vivo function of

I. HYPOTHALAMIC PITUITARY FUNCTION

TSH SUBUNIT GENES

Gata2 in pituitary development has been examined by targeted inactivation of Gata2 in a transgenic mouse model using Cre recombinase directed by the αGSU promoter/enhancer [20], which is active early in pituitary development. The Gata2 knockout mice in the pituitary have a decreased thyrotroph cell population at birth and lower levels of circulating TSH and FSH in the serum of adults. This demonstrated the role of Gata2 in the production of both TSHβ and αGSU subunits. Thyroid ablation and castration studies demonstrated a decreased capacity of mutant thyrotrophs and gonadotrophs to mount the appropriate response to the loss of negative feedback by thyroid hormones and steroid hormones, respectively. These studies showed that Gata2 is important for optimal thyrotroph and gonadotroph function but not for thyrotroph and gonadotroph cell fate specification [20]. Studies in zebrafish have identified a role for Sox4b in specification of thyrotroph and gonadotroph cells and induction of Gata2 [21] A population of multipotent stem cells in the adult pituitary [22,23] are distinct from the embryonic precursor cells. These nestin- and Sox2-containing stem cells reside in a localized niche within the perilumenal region of the gland, have the capacity to expand into all of the terminally differentiated pituitary cell types after birth, and may contribute to pituitary tumors [22]. These newly discovered cells may, in fact, contribute to the dynamic changes in cell growth that occur in the pituitary gland under certain physiologic or pathologic states, such as the marked thyrotroph hyperplasia/hypertrophy seen following severe hypothyroidism [24].

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promoter, recruit specific chromatin-modifying enzymes, and initiate transcription when the DNA is in an accessible state or silence the gene if inaccessible to the transcriptional machinery.

TSH β-Subunit Gene Structure The human TSH β-subunit gene has been isolated and its structure characterized [25]. The gene is 4527 base pairs (bp) in size, and is located on the short arm of chromosome 1 at position 13.2 [26]. The gene structure consists of three exons and two introns (Fig. 6.2, top panel). The first exon of 37 bp contains the 5’ untranslated region of the gene. It is separated from

TSH SUBUNIT GENES The TSH α- and β-subunits are encoded by two separate genes located on different chromosomes. Since the isolation and characterization of these genes, much information has been gained regarding the molecular events that result in the regulated production of TSH α- and β-subunit messenger RNAs (mRNAs) and protein. Most of this information has been obtained from studies performed in mouse thyrotrophic tumors and rodent pituitary glands. Thyrotroph cells are believed to contain specific transcription factors that bind to the regulatory regions of the genes and interact with ubiquitous factors to initiate transcription. Regulation of TSH subunit gene transcription, mainly activation by TRH or inhibition by thyroid hormone, is achieved by modulating the activity of the specific and ubiquitous factors. Extensive biochemical studies show that activation and/or repression of these genes within thyrotrophs is fundamentally determined by modifications of the chromatin state at each TSH subunit gene. Following an activating or inhibitory stimulus to the cell, factors bind to the

FIGURE 6.2 Structural organization of the human TSH subunit

genes and mRNAs. The two panels show the TSH β- (top) and α-subunit (bottom) genes. Shown are the relative locations and sizes of the exons and introns. The TATA box, important for positioning the RNA transcriptional start, is located in the promoter close to exon 1. Following transcription, introns are spliced out, exons precisely joined, and a polyA tail added to the 3’ end of the mature mRNA. Source: Adapted from De Groot LJ, Jameson JL, editors. Endocrinology. 6th ed. Chapter 73, Thyroid-stimulating hormone: physiology and secretion. Elsevier.

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the second exon by a large first intron of 3.9 kb. The coding region is contained in the second (163 bp) and third (326 bp) exons, which are separated by a 0.45 kb intron, while the 3’ untranslated region is contained in the third exon. DNA sequences close to the transcriptional start site in the promoter of the TSHβ gene reporter contain elements responsible for initiating transcription and regulating expression. A consensus TATA box, a sequence that is important for positioning RNA polymerase II activity, is located 28 bp upstream of the transcriptional start site and is important for the accurate initiation of RNA transcripts. In contrast to the human gene, both the mouse and the rat genes have two transcriptional start sites. The human exon 1 is 10 bp longer than that of the mouse and rat, presumably due to an insertion that displaces the TATA box 9 bp further upstream relative to the TATA box in the mouse and rat genes. Progressive 5’ deletions of the mouse TSHβ promoter linked to a luciferase reporter following expression into thyrotroph cells defined the cis-acting sequences required for expression to the first 270 bp of the promoter [27,28]. While these sequences defined the minimal promoter, other studies have shown that enhancer sequences located more than 6 kb upstream are also required for the promoter to express in pituitary thyrotrophs in transgenic mice [29]. However, the mouse TSHβ promoter region from 2271 to 280 is sufficient to confer thyrotroph-specific activity [27] and thyrotroph transcription factors can bind to these DNA sequences [30]. Within this broad area, four regions of protein interaction have been identified by DNase footprint analysis using nuclear extracts from thyrotroph cells [28]. Two transcription factors, Pou1F11 and

Gata2, bind to adjacent sequences located within a composite cis-acting region on the proximal TSHβ promoter from the region 2135 to 288 relative to the major transcriptional start site (Fig. 6.3, top panel). This composite DNA element has a 5’ Pou1F1 site and a 3’ Gata2 site. Between these two sites are 16 bp that include overlapping putative Pou1F1 and Gata2 sites. This 16 bp intervening sequence is critical for high promoter activity, independent of the actual spacing between the flanking Pou1F1 and Gata2 sites [19]. Mechanistically, binding of Pou1F1 may provide stabilizing effects through direct contacts with Gata2, it may induce stabilizing contacts between Gata2 and DNA, or it may alter DNA conformation. The DNA behaves as a docking platform which recruits multiple components of a fundamental regulatory assembly initiated by binding of Pou1F1 and Gata2 to facilitate thyrotroph-specific transcription. An additional transcription factor, Med1 (TRAP220, PBP), was shown to be recruited to the TSHβ proximal promoter and play a role in transcriptional activation [31]. Med1 was originally defined as part of a transcriptional mediator complex that interacts with hormone-occupied thyroid/steroid hormone receptors in a ligand-dependent manner [32]. The physiological relevance of these studies originated with the observation that mice with one half the genetic complement encoding this factor were hypothyroid with a pituitary phenotype characterized by reduced levels of TSHβ gene expression [33]. Med1 is recruited to the TSHβ gene by virtue of its physical interaction with both Pou1F1 and Gata2 since the protein itself does not possess a DNA-binding domain. Cotransfections in nonpituitary CV-1 cells showed that Pou1F1, Gata2, or Med1 alone do not markedly FIGURE 6.3 Schematic representation of the human TSH β-subunit (top) and the glycoprotein hormone α-subunit (bottom) promoters. The transcriptional start site is indicated by the arrow and the TATA box is shown. The numbers above the line denote the position of the nucleotides relative to the transcriptional start site set at 11. The boxes under the line indicate the regions important for the responses to the various factors that regulate transcription, as shown (top panel). The Gata2 binding sites have only been described in the mouse TSH β-subunit gene (bottom panel). The placental-specific, gonadotroph-specific, and thyrotroph-specific activities of the glycoprotein hormone α-subunit are shown. The thyrotroph-specific regions other than the P-LIM-binding region have only been described in the mouse α-subunit gene.

I. HYPOTHALAMIC PITUITARY FUNCTION

TSH SUBUNIT GENES

stimulate the TSHβ promoter. However, Pou1F1 plus Gata2 resulted in a 10-fold activation, demonstrating synergistic cooperativity, and addition of Med1 resulted in a further dose-dependent stimulation up to 25-fold that was promoter-specific [34]. Interaction studies showed that Med1 or Gata2 each bound the homeodomain of Pou1F1, whereas Med1 interacted independently with each zinc finger of Gata2, and Med1 interacted with Gata2 and Pou1F1 over a broad region of its N-terminus. These regions of interaction were also important for maximal function. Chromatin immunoprecipitation assays have shown in vivo occupancy on the proximal TSHβ promoter [31]. Thus, the TSHβ gene is activated by a unique combination of transcription factors present in pituitary thyrotrophs, including those that act via binding to the proximal promoter as well as others recruited to the promoter via protein protein interactions.

α-Subunit Gene Structure The human glycoprotein hormone α-subunit gene is located on chromosome 6 at position 6q12-q21 [35]. The gene is 9635 bp in size and consists of four exons and three introns. It contains a consensus TATA box located 26 bp upstream of the transcriptional start site [36]. The first exon (94 bp) contains virtually all of the 5’ untranslated sequence and is separated from the second exon by a 6.4 kb intron. The second exon contains 7 bp of 5’ untranslated sequence and 88 bp of the coding region. The coding sequence continues in the third (185 bp) and fourth (75 bp) exons and the 3’ untranslated region (220 bp) is contained completely in the fourth exon (Fig. 6.2, bottom panel). The second and third introns are 1.7 kb and 0.4 kb, respectively. The genomic organization of the mouse (located on chromosome 4), rat, and cow α-subunit genes are similar, except that in the rat and cow the second intron is located 12 bp downstream, resulting in a peptide sequence that is four amino acids longer. There are also differences in the length of the 5’ untranslated sequence, which is 10 bp longer in the mouse, apparently due to a 10-bp insertion between the TATA box and the transcriptional start site [19]. The elements responsible for initiating transcription and regulating the expression are located in the 5’ flanking region of the α-subunit gene (Fig. 6.3, bottom panel). The human α-subunit gene contains a consensus TATA box located 26 bp upstream of the transcriptional start site. A single transcriptional start site has been found in the glycoprotein hormone α-subunit genes of all the species that have been studied. Analysis of the mouse α-subunit promoter in transgenic mice showed that 381 bp of the 5’ flanking region is sufficient for

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expression of a β-galactosidase reporter gene in both thyrotrophs and gonadotrophs, although hormonally and temporally regulated high levels of expression are achieved when longer promoter fragments of 4.6 kb were included [37,38]. This indicates that an enhancer region of the promoter located several thousand base pairs upstream of the transcriptional start site is required along with key cis-acting proximal promoter elements for maximal in vivo expression of the glycoprotein hormone α-subunit gene in pituitary thyrotrophs and gonadotrophs. There have been a number of proximal cis-acting elements identified by gene transfer and DNA-binding studies that have been shown to be important for α-subunit expression in pituitary and placental cells. These elements interact with cell-specific and/or ubiquitous trans-acting factors to allow regulated expression in the appropriate cell type. The glycoprotein hormone α-subunit gene is unique in that it is expressed in thyrotrophs, gonadotrophs, and placental cells, as part of the hormones (thyrotrophin (TSH), lutrophin/follicotrophin (LH/FSH) and CG, respectively). In each of those cell types it is differentially regulated. Studies from several laboratories using the human and mouse genes have demonstrated that the cell-specific expression in each cell type is dependent on vastly different regions of the promoter (Fig. 6.3, bottom panel). Whereas the region downstream of 2200 is sufficient for placental expression [39], gonadotrophs require sequences between 2225 and 2200 [40], and regions further upstream appear to be critical for thyrotroph expression [41]. The elements involved in human placental α-subunit expression extend from 2177 to 284 and include the upstream regulatory element, also called trophoblast-specific element (TSE), that binds the placental-specific protein TSEB [42], an 18-bp repeat cAMP response element (CRE) that extends from 2146 to 2111 and binds the ubiquitous protein CREB [43,44], the junctional regulatory element (JRE) that binds a 50-kDa protein [45], the CCAAT-box that binds a 53-kDa α-subunit binding factor (α CBF) [46], and a Gata motif that interacts with Gata-binding proteins [18]. Some of these regions binding to similar factors also play a role in pituitary α-subunit expression. In addition, a region from 2225 to 2200 that binds the orphan nuclear receptor SF-1 appears to be critical for gonadotroph expression of the α-subunit gene [47], but this region has no effect on thyrotroph expression [48]. Basic helix-loop-helix E-box binding proteins [49] and GATA-binding proteins [18] also appear to play a role in α-subunit expression in gonadotrophs. Transgenic mouse studies have shown that 313 bp of the bovine α-subunit 5’ flanking DNA, which contain the SF-1-binding region, targeted expression to gonadotrophs but not thyrotrophs [50], suggesting that this region is sufficient for expression in

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gonadotrophs. It was then shown that 480 bp of the mouse α-subunit 5’ flanking DNA was able to target transgenic expression to both gonadotrophs and thyrotrophs [38], in agreement with in vitro transfection studies which showed that the same promoter region mediates a high level of expression in thyrotroph and gonadotroph cells. Several sequences within the region from 2480 to 2300 appear to be important for mouse α-subunit expression in thyrotrophs but not gonadotrophs [51]. Among these is the sequence from 2434 to 2421 that interacts with the developmental homeodomain transcription factor Msx1 [52]. This factor was found to be expressed in mature thyrotrophs, and its role in α-subunit expression appears to be mediated by binding to the TATA box binding protein [53], although this needs to be further elucidated. Another important sequence is the pituitary glycoprotein hormone basal element, or PGBE, extending from 2342 to 2329, that is critical for both thyrotroph and gonadotroph expression [54]. The PGBE interacts with P-LIM (mLIM3, Lhx3), a pituitary-specific LIM-homeodomain transcription factor [55], that is important not only for thyrotroph and gonadotroph cell specification but is also important for somatotrophs and lactotrophs [56]. In gonadotrophs, gonadotrophin-releasing hormone regulates expression of the α-subunit via two elements in the proximal promoter, PGBE and a second element recognized by an ETS factor, which is activated by mitogen-activated protein kinase [57]. Other sequences within the 480-bp promoter have been found to interact with the pituitaryspecific homeodomain factor Ptx-1 [51], and a synergism between Ptx-1 and P-LIM, mediated by the coactivator C-LIM, has been described [58]. Studies with the mouse promoter also showed that an upstream DNA element located between 24.6 and 23.7 kb further enhanced transgenic expression in both thyrotrophs and gonadotrophs, by interacting with proximal sequences [37]. The active region was localized to 125 nucleotides upstream of 23700, and this same region was shown to mediate inhibition of expression in GH3 somatotrophic cells where α-subunit is not endogenously expressed [59]. The upstream 125-bp enhancer element harbors consensus-binding sites for GATA, SF1, Sp1, ETS, bHLH factors, and suggests cooperativity between factors binding both to proximal cis-acting elements and to the distal enhancer. In spite of significant advances in this area, thyrotroph-specific factors that determine α-subunit gene expression have not yet been completely identified.

TSH BIOSYNTHESIS The intact TSH molecule is a heterodimeric glycoprotein with a molecular weight of 28 kDa that is

composed of the noncovalently linked α- and β-subunits. The common α-subunit contains 92 amino acids while the specific TSH β-subunit has 118 amino acids. TSH biosynthesis and secretion by thyrotroph cells of the anterior pituitary are precisely regulated events. This section examines our understanding of the biosynthesis of TSH, including the processes of transcription, translation, glycosylation, folding, combination, and storage.

TSH transcription The TSH β- and α-subunit genes are transcribed into a precursor RNA by a series of enzymatic steps as directed by each of their promoters, with the participation of both ubiquitous and specific transcription factors. Transcribed RNAs undergo a precise series of splicing events at the exon intron junctions that lead to production of mature mRNAs, which exit the nucleus and attach, through polyA tails, to ribosomes, where they are translated into proteins.

TSH translation The next steps in TSH biosynthesis are summarized in Fig. 6.4 [60]. The mRNAs for TSH β- and α-subunits are independently translated by ribosomes in the cytoplasm. The first peptide sequences consist of “signal” peptides of 20 amino acids for TSH β and 24 amino acids for α [61]. These signal peptides are hydrophobic, allowing insertion through the lipid bilayer of the membrane of the rough endoplasmic reticulum. Translation into TSH β- and α-presubunits continues into the lumen of the rough endoplasmic reticulum, and cleavage of the signal peptide occurs before translation is completed. This results in the formation of a 118-amino-acid TSH β-subunit [62] and a 92 amino acid α-subunit. Cleavage of TSH β to a protein of 112 amino acids appears to be an artifact of purification. Synthesis of recombinant TSH β-subunit results in two products of 112 and 118 amino acids, both of which are similarly active in vitro [63].

TSH glycosylation Glycosylation of TSH has a significant impact on its biological activity [64]. The TSH β-subunit has a single glycosylation site, the asparagine residue at position 23, whereas the α-subunit is glycosylated in two sites, the asparagine residues at positions 52 and 78 [65] (Fig. 6.4). Excess free α-subunit is glycosylated at an additional site, the threonine residue at position 39 [66]. This residue is located in a region believed to be important for combination with the TSH β-subunit. It

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FIGURE 6.4 (Top panel) Oligosaccharide chains of thyroid-stimulating hormone (TSH). Shown are typical oligosaccharide chains present on the TSH heterodimer and the free α-subunit. Glycosylated asparagine (Asp) and threonine (Thr) residues are indicated. Symbols represent the oligosaccharide chain residues as indicated in the key (bottom panel). Biosynthesis of thyroid-stimulating hormone (TSH). (Schematic) Shown are the processes of translation and glycosylation within the rough endoplasmic reticulum (RER) and Golgi apparatus, divided into proximal and distal. Cleavage of the aminoterminal (H2N) signal peptide and early addition of highmannose chains (black boxes) as well as combination of α- and β-subunits occur in the RER. In the proximal Golgi, oligosaccharide chains are modified and the final steps of sulfation and sialation occur in the distal Golgi apparatus. Source: Adapted from Weintraub BD, Gesundheit N. Thyroid-stimulating hormone synthesis and glycosylation: clinical implications. Thyroid Today 1987;10:1 1.

is not known whether glycosylation at this residue is a regulated step that inhibits combination with the TSH β-subunit or whether it occurs in excess free α-subunits because this site is exposed. Extensive studies on the processes of TSH subunit glycosylation have been carried out. Glycosylation of the TSH β- and α-subunits begins before translation is completed (cotranslational glycosylation), while addition of the second oligosaccharide in the α-subunit occurs after translation is completed (post-translational glycosylation). The first step in this process involves the assembly of a 14-residue oligosaccharide, (glucose)3 (mannose)9 (Nacetylglucosamine)2 on a dolichol-phosphate carrier. This oligosaccharide is then transferred to asparagine residues by the enzyme oligosaccharyl transferase that recognizes the tripeptide sequence (asparagine) (X) (serine or threonine). This mannose-rich oligosaccharide is progressively cleaved in the rough endoplasmic reticulum and Golgi apparatus. An intermediate with only six

residues is produced, and then other residues are added resulting in complex oligosaccharides [67]. The residues added include N-acetylglucosamine, fucose, galactose, and N-acetylgalactosamine. Oligosaccharides prior to the six-residue intermediate are termed high-mannose and are sensitive to endoglycosidase H that releases the oligosaccharide from the protein, whereas the intermediate and the complex oligosaccharides are endoglycosidase H-resistant. Complex oligosaccharides usually consist of two branches (biantennary) but sometimes three or four branches are seen, as well as hybrid oligosaccharides consisting of one complex and another high-mannose branch. Sulfation and sialation occur late in the pathway, within the distal Golgi apparatus. Sulfate is bound to N-acetylgalactosamine residues, and sialic acid, or its precursor N-acetylneuraminic acid, is bound to galactoside residues [68]. Thus, the activation of the enzymes sulfotransferase and N-acetylgalactosamine transferase may be important regulatory steps

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that impact the ratio of sulfate to sialic acid. As demonstrated with LH, it appears that sulfation increases and sialylation decreases the bioactivity of TSH [68], since the exclusively sialylated recombinant glycoprotein produced in Chinese hamster ovary cells has been found to have attenuated activity in vitro [69]. Processing of complex oligosaccharides appears to occur at a slower rate for secreted glycoproteins, such as TSH, when compared to nonsecreted glycoproteins. For example, after an 11-minute pulse labeling with [35S]methionine and a 30-minute chase only a few α-subunits were endoglycosidase H-resistant and only 76% reached this stage after an 18-hour chase [70]. Secretion was observed after a 60-minute chase, and the secreted products—TSH, free α-subunit, but not free β-subunit—had mostly complex oligosaccharides associated with them [65]. It may be important to note that many of the studies described were carried out in thyrotrophic tumor tissue obtained from hypothyroid mice, and glycosylation may differ in the euthyroid as compared to the hypothyroid state. In addition, differences between species have been noted, such as the human TSH containing more sialic acid than the bovine TSH [62]. Tissue-specific TSH glycosylation has been proposed to define a distinct role for TSH produced in the pars tuberalis [71].

TSH folding, combination, and storage The elucidation of the crystal structure of human CG (hCG) [72] allowed the construction of a model of human TSH (Fig. 6.5), supported by other evidence [74,75]. This model has greatly facilitated the interpretation of structure function studies of the protein backbone. However, crystallization was only achieved with partly deglycosylated hCG, so it is likely that the conformation of the glycosylated protein may differ to some extent. Nuclear magnetic resonance studies suggested that the α-subunit carbohydrate moieties project outward and may be freely mobile [76]. Nevertheless, this model predicts that the tertiary structure of each TSH subunit consists of two hairpin loops on one side of a central knot formed by three disulfide bonds and a long loop on the opposite side. In this tertiary structure, the glycoprotein hormones share features in common with transforming growth factor β, nerve growth factor, platelet-derived growth factor, vascular endothelial growth factor, inhibin, and activin, all of which are now grouped in the family of “cystine knot” growth factors [77]. Folding of nascent peptides begins before translation is completed. It has been shown that proper folding is dependent on glycosylation, since the drug tunicamycin that prevents the initial oligosaccharide

FIGURE 6.5 Human thyroid-stimulating hormone (TSH) ribbon homology model showing domains important for activity. The schematic drawing is based on a molecular homology model built on the template of the human chorionic gonadotrophin (hCG) model derived from crystallographic coordinates obtained from the Brookhave Data Bank. The α-subunit is shown as a red line, and the TSH β-subunit as a blue line. The two hairpin loops (L1, L3) in each subunit are marked. The long loops (L2) in each subunit extend from the opposite side of the central cystine knot. The functionally important α-subunit domains are boxed: α11 20, α33 38, α40 46 (“α-helix”), α52, α64 81, and α88 92. The functionally important β-subunit domains are indicated within the line drawing: β58 69, β88-95 (the “determinant loop” or N-terminal segment of the seat belt), and β96 105 (the C-terminal segment of the seat belt). The β-subunit beyond 106 is not drawn because the corresponding region of hCG was not traceable. The oligosaccharide chains are not shown because hCG was deglycosylated before crystallization. Source: Adapted from Grossmann M, Weintraub BD, Szkudlinski MW. Novel insights into the molecular mechanisms of human thyrotropin action: structural, physiological, and therapeutic implications for the glycoprotein hormone family. Endocr Rev 1997;18:476 501 [73].

transfer to the asparagine residue results in peptides that do not fold properly and are degraded intracellularly [78]. Site-directed mutagenesis of a single glycosylation site also disrupted processing and decreased TSH secretion in transfected Chinese hamster ovary cells [79]. Folding is a critical step that allows correct internal disulfide bonding that stabilizes the tertiary structure of the protein allowing subunit combination. Combination of TSH β- and α-subunits begins soon after translation is completed in the rough endoplasmic reticulum, and continues in the Golgi apparatus [65]. Subunit combination then accelerates and modifies oligosaccharide processing of the α-subunit [80]. In fact, studies have suggested that the conformation of the α-subunit differs after combination with each type

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of β-subunit [73,81], and this may affect subsequent processing. The rate of combination of TSH β- and α-subunits has been examined in mouse thyrotrophic tumors. After a 20-minute pulse labeling with [35S] methionine, 19% of TSH β-subunits were combined with α-subunits, and this percentage increased to 61% after an additional 60-minute chase incubation [65]. Studies have also shown that the combination of the TSH β- and α-subunits, as is the case with other glycoprotein hormones, occurs after the “latching” of the disulfide “seat belt” of the β-subunit, with subsequent “threading” of loop 2 and the attached oligosaccharide of the α-subunit beneath that “seat belt” [82]. The sequence of the TSH β-subunit from amino acid 27 to 31 (CAGYC) is highly conserved among species and is thought to be important for combination with the α-subunit. In a case of congenital hypothyroidism, a point mutation in the CAGYC region (see Disorders of TSH production) results in the synthesis of altered TSH β-subunits that are unable to associate with α-subunits, with consequent lack of intact TSH production [83]. A lack of free-circulating TSH β-subunit was also observed, suggesting that combination with α-subunit is necessary for TSH β-subunit secretion. This phenomenon was also demonstrated in studies where synthesis of wild-type recombinant TSH β-subunit was carried out in the presence or absence of recombinant α-subunit [84]. Using site-directed mutagenesis, another study showed that a mutation at residue 25 in the glycosylation recognition site that substitutes a serine for a threonine does not alter glycosylation but decreases TSH production by 70%, possibly because of disruption of the nearby CAGYC region [85]. After TSH and free α-subunit are processed in the distal Golgi apparatus they are transported into secretory granules or vesicles [86]. The secretory granules constitute a regulated secretory pathway, mainly influenced by TRH and other hypothalamic factors. These granules contain mostly TSH, whereas free α-subunit is contained in the secretory vesicles that constitute a nonregulated secretory pathway.

REGULATION OF TSH BIOSYNTHESIS TSH biosynthesis is regulated by coordinated signals from the central nervous system and feedback from the peripheral circulation. The most important positive input for TSH biosynthesis is hypothalamic TRH and the most powerful negative regulator is circulating thyroid hormone levels. However, additional hypothalamic factors and circulating hormones have important modifying effects. Most of these factors

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have independent effects on the biosynthesis of the two subunits of TSH.

Hypothalamic Regulation of TSH Biosynthesis TRH is a tripeptide secreted from the hypothalamus, transported to the pituitary via the hypothalamic hypophyseal portal system, and is a major activator of TSH production with a significant 3 5-fold increase in the transcription of both TSH β- and α-subunit mRNAs [87]. TRH from maternal or fetal sources is not required for normal thyrotroph development during ontogeny and TRH-deficient mice are not hypothyroid at birth. However, TRH is required for the postnatal maintenance of TSH activation [88]. TRH binding to its cell surface receptor initiates a cascade of intracellular events. In GH3 cells, the TRH receptor complex interacts with a guanine nucleotidebinding regulatory protein (G) that then binds and activates GTP (G’). G’ binds to phospholipase C (C) and activates it (C’). C’ catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate, which results in the formation of two intracellular “second messengers,” inositol triphosphate (InsP3) and 1,2-diacylglycerol (1,2-DG). InsP3 diffuses from the cell surface membrane to the endoplasmic reticulum, where it causes the release of sequestered Ca21. This activates the movement of secretory granules to the cell surface and their exocytosis. Simultaneously with these events, there is a parallel activation of protein kinase C by 1,2DG that also leads to phosphorylation of proteins involved in exocytosis. TRH has been shown to stimulate a nuclear protein, Islet-brain-1 (IB1)/JIP-1, in the anterior pituitary gland and in cultured rat GH3 cells [89] that has been implicated in the action of TRH in stimulating the TSH β gene in thyrotrophs. Studies in somatomammotroph cells, where TRH stimulates prolactin production, have suggested that phosphatidylinositol, protein kinase C and calcium-dependent pathways may be involved [90]. Other factors implicated in TRH stimulation of the TSH β-subunit promoter include AP1 [91] and the orphan nuclear receptor NR4A1 (Nur 77) [92]. Two TRH-response regions are located from 2128 to 261 and from 228 to 18 of the human TSH β promoter [93]. The upstream region contains binding sites for the pituitary-specific transcription factor, Pou1F1, suggesting a role for this or a similar factor in the regulation of the TSH β-subunit gene by TRH. In the rat TSH β-subunit gene, responsiveness to TRH has been localized to regions upstream of 2204, where Pou1F1binding sites are also found [94]. Furthermore, it has been shown that both protein kinase C and protein kinase A pathways can phosphorylate Pou1F1 at two

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sites in response to phorbol esters and cAMP [95], and can alter the binding to Pou1F1 transactivation elements on the human TSHβ gene [96]. TRH stimulates α-subunit transcription through a mechanism involving a CREB-binding protein (CBP) that binds the region from 2151 to 2135 of the human α-subunit promoter, and a Pou1F1-like protein that binds to a more distal region from 2223 to 2190 [97]. Two other transcription factors, P-Lim and CBP, synergistically cooperate in a TRH signaling-specific manner to activate α-subunit promoter activity [98]. P-Lim binds the α-subunit promoter directly but CBP does not possess a DNA-binding domain so it is likely recruited to the promoter via interaction with another factor(s). TSH glycosylation is also modulated by TRH [99]. Primary hypothyroidism [100] and TRH administration increase oligosaccharide addition that results in enhanced TSH bioactivity, as also reported in patients with resistance to thyroid hormone. TSH glycosylation patterns were also found to differ in central hypothyroidism, TSH-producing pituitary adenomas, and euthyroid sick syndrome [101]. Changes in sulfation and sialylation of the oligosaccharide residues also modulate bioactivity [100,102,103]. Dopamine, acting via DA2 dopamine receptors, inhibits TSH α- and β-subunit gene transcription by decreasing the intracellular levels of cAMP [87]. Studies of the TSH β-subunit gene have localized two regions of the promoter necessary for cAMP stimulation, from 2128 to 261 bp and from 13 to 18 bp. The upstream region coincides with the TRH-responsive region and contains Pou1F1-binding sites. The downstream region resides within the regions responsive to T3 (13 to 137) and TRH (228 to 18). The downstream region also overlaps with an AP1-binding site (21 to 16). The sequence from 21 to 16 appears to cooperate with Pou1F1 in mediating responses to cAMP and TRH [91]. Thus, multiple interactions between transcription factors and hormonal regulators appear to converge on sequences close to the transcriptional start site. α-Subunit gene expression in thyrotrophs is inhibited by dopamine in coordination with expression of the TSH β-subunit gene, but the mechanism has not been elucidated. Circadian regulation of TSH biosynthesis may be regulated by binding of the nuclear receptor corepressor (NcoR1) and the orphan nuclear receptor NR1D1 (Rev-Erbα) to an upstream promoter region on the TSHβ subunit gene [104].

Peripheral Regulation of TSH Biosynthesis Thyroid hormone is thought to act predominantly through a classical thyroid receptor-mediated genomic model. T4 serves as a minimally active prohormone

that is converted into a metabolically active T3 by a family of tissue deiodinases termed D1, D2, and D3. These selenoprotein enzymes are membrane-bound and can activate or inactivate substrate in a time- and tissue-specific manner [105]. D2 is the major T4-activating deiodinase. It is present on the endoplasmic reticulum close to the nucleus, and produces 3,5,3’triiodothyronine (T3), by the removal of an iodine residue from the outer ring of thyroxine. D2 activity is rapidly lost in the presence of its substrate T4 by a ubiquitin proteasomal mechanism [106]. Rat pituitary thyrotrophs coexpress D2 RNA and protein and both are increased in hypothyroidism. Murine thyrotrophs in TtT-97 tumors or the TαT1 cell line have extremely high levels of D2, which account for the sustained production of T3 by thyrotrophs even in the presence of supraphysiological T4 levels [107]. Serum TSH levels in normal mice are suppressed by administration of either T4 or T3, although only T3 was effective in the mouse with targeted disruption of the D2 gene, which demonstrated the critical importance of D2 in controlling negative thyroid hormone regulation of TSH in thyrotrophs [108]. TSH β- and α-subunit gene transcription rates are markedly inhibited by treatment with triiodothyronine (T3). Studies using mouse TtT-97 thyrotrophic tumors have demonstrated that suppression of TSH β- and α-subunit mRNA transcription rates measured by nuclear run-on assays is evident by 30 minutes after treatment and is maximal by 4 hours [109]. This effect was seen in the presence of the protein synthesis inhibitor cycloheximide, indicating that it did not require the modulation of an intermediary protein. Other studies using mouse and rat pituitaries along with mouse thyrotrophic tumors have demonstrated that steadystate mRNA levels of TSH β- and α-subunit are dramatically decreased by T3 [110]. The mechanism of action of T3 involves interaction with nuclear receptors that act mainly at the transcriptional level. The transcriptional response to T3 is proportional to the nuclear receptor occupancy [111], and the time course of T3 nuclear binding and transcriptional inhibition are also in agreement (Fig. 6.6) [112]. Abundant information exists as to the mechanisms involved in positive gene regulation by T3 [107 110]. In contrast, molecular mechanisms involved in negative T3 regulation, such as for TSH subunit genes, have not been completely characterized. The T3 inhibitory effect on the TSH β gene requires binding to the T3 receptor β isoform (TR β), as shown by the lack of TSH inhibition in cases of thyroid hormone resistance [113]. Although thyrotroph cells contain all TRs: TRα1, TRβ1, and TRβ2, as well as non-T3 binding variant α2, TRβ2 is expressed predominantly in the pituitary and T3responsive TRH neurons and is most critical for the regulation of TSH [114]. Moreover, TRβ2-deficient

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FIGURE 6.6 The effect of thyroid hormone on the transcription of the thyroid-stimulating hormone (TSH) β- (blue circles) and α-subunit (red circles) genes. Murine thyrotrophic tumor explants were incubated for up to 4 h with 5 nmol T3 for transcription measurements or with 5 nmole 125I T3, with or without 1000-fold excess of unlabeled T3 for binding measurements. Transcription rates were measured in pools of isolated nuclei. There is an inverse relationship between T3 binding and TSH β- and α-subunit mRNA synthesis. Source: Adapted from Shupnik MA, Ridgway EC. Triiodothyronine rapidly decreases transcription of the thyrotropin subunit genes in thyrotropic tumor explants. Endocrinology 1985;117:1940 6.

mice exhibit a phenotype consistent with pituitary thyroid hormone resistance, with increased TSH and thyroid hormone levels, even in the presence of TRβ1 and TRα1, showing the lack of compensation between TR isoforms [115]. However, TRβ1 and TRα1 may still play a role, since they are able to form heterodimers with TRβ2. An intact DNA-binding domain of TRβ is required for negative regulation of the TSHβ gene in vitro [116] and in vivo [117]. A combination of Pou1F1 and Gata2 activate a human TSHβ (2128/ 1 37) reporter construct along with vectors containing TRβ1 constructs in the absence or presence of T3. Unliganded TRβ1 does not stimulate promoter activity, whereas a mutation lacking the N-terminus and DNA-binding domain of TRβ1 lost the ability of T3-treated cells to negatively regulate TSHβ promoter activity. This demonstrated the importance of modular domains constituting the molecular structure of TRs. Moreover, using a gene-targeting approach in transgenic mice, replacement of the wild-type TRβ gene with a mutant that abolished DNA-binding in vitro did not alter ligand and cofactor interactions [117]. Homozygous mutant mice demonstrated central thyroid hormone resistance with 20-fold higher serum TSH in the face of 2 3-fold higher T3 and T4 levels that were similar to those of TRβ homozygous null mice. TR interacts with specific cis-acting DNA sequences close to the transcriptional start. T3 response elements

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have been reported to be located between 13 and 137 of the human TSHβ gene [118]. There are two T3 receptor binding sites, from 13 to 113 and 128 to 137 that may mediate T3 inhibition. T3 responses can be mediated through receptor monomers, homodimers, or heterodimers involving retinoid X receptors (RXR) [119,120], constituting heterodimeric complexes that may exhibit different affinities for specific DNA sequences and different functional activities. A particular RXR isoform, RXRγ1, is uniquely expressed in thyrotrophs and mediates inhibition through a region extending from 2200 to 2149 of the mouse TSH β-subunit promoter, an area upstream and distinct from that mediating negative regulation by thyroid hormone, in TtT-97 thyrotrophic tumor cells [121] and in cultured TαT1 thyrotrophs [122]. This finding has been confirmed in vivo and resulted in central hypothyroidism (low T4 and low TSH) in cancer patients treated with the retinoid bexarotene [123]. The RXRselective retinoid (LG 268) decreased circulating TSH and T4 levels in mice with marked lowering of pituitary TSHβ mRNA without decreasing TRH, suggesting a direct effect on thyrotrophs [122]. Other proteins that interact with TR include the coactivators, such as the glucocorticoid-receptor-interacting protein-1 and the steroid receptor coactivator-1 (SRC-1) [124], and corepressors, such as the silencing mediator for retinoid receptors and thyroid hormone receptors (SMRTs) and the nuclear receptor corepressor (NCoR) [125,126]. These coactivators and corepressors modulate effects of several members of the steroid thyroid hormone receptor superfamily. Studies with genetic knockout mouse models where both TRH and TRβ genes were removed, have shown an unexpected dominant role for TRH in vivo in regulating the hypothalamic pituitary thyroid axis. The presence of both TRβ and TRH is necessary for a normal thyrotroph response during hypothyroidism, suggesting that unliganded TRβ stimulates TSH subunit gene expression [127]. Other studies have disputed the requirement of the negative response element located in exon 1 of the human gene since its deletion did not eliminate T3 suppression of TSHβ promoter activity in a reconstitution system [128]. These studies showed that liganded TRβ can associate with Gata2 in vitro and in vivo via direct interaction between the zinc fingers of Gata2 and the DNA-binding domain of TRβ. In addition, T3 occupied TRβ can physically interact with the LxxLL domain of Med1/Trap220, in competition with the corepressors SRC1/SRC2 [129]. Thus, interference with the transactivation function of the Pou1F1/Gata2/ Med1 complex on the proximal TSHβ promoter likely plays an important role in T3-negative regulation. Thyroid hormone inhibition of α-subunit gene transcription is observed in thyrotrophs in coordination

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with that of the TSH β-subunit (Fig. 6.6). The T3 response element of the human α-subunit gene promoter is located from 222 to 27 [130]. Similar to the TSH β-subunit gene, the T3 response elements of the human as well as the mouse [41] and rat [131] α-subunit genes are located close to the transcriptional start. T3 inhibition may be mediated by different isoforms of the T3 receptor [132] in combination with corepressors SMRT and NCoR [133]. Mutations of the T3 response element of the human α-subunit promoter that eliminate TR binding do not abrogate the inhibitory effect of T3, suggesting that protein protein interactions may be more important than protein DNAbinding [134]. Liganded TRβ has been reported to recruit histone deacetylase 3 (HDAC3) and reduce histone H4 acetylation resulting in a fully repressed chromosomal state of TSH subunit genes [135]. Using chromatin immunoprecipitation assays with the α-subunit promoter it was shown that T3 decreased transcription and increased histone acetylation of the promoter, mediated directly by TRs. Overexpression of nuclear receptor corepressor (NCoR) and HDAC3 increased ligand-independent basal transcription. T3 caused release of a corepressor complex, composed of NCoR, HDAC3, and a transducin β-like protein. Unexpectedly, histone acetylation was increased and coincided with lowered rates of α-subunit transcription. These data show that participation of similar complexes and overlapping epigenetic changes can participate in both positive and negative T3 regulation of the αGSU promoter [136]. Post-transcriptional effects of T3 include decreased size of the poly(A) tail of the TSH β-subunit mRNA, but not of the α-subunit [137 139], and it also decreased binding of both the TSHβ- and α-subunits to ribosomes, resulting in decreased translation rates [140]. TSH glycosylation is also modulated by thyroid hormone [99]. In primary hypothyroidism there is increased oligosaccharide addition that results in an increased bioactivity of TSH [100]. TSH glycosylation patterns, as well as sulfation and sialylation of the oligosaccharide residues, also differ in several pathological states, such as resistance to thyroid hormone, central hypothyroidism, TSH-producing pituitary adenomas, and euthyroid sick syndrome [100 103]. Those effects may result from change in T3 levels or TRH stimulation, as described under hypothalamic control of TSH biosynthesis. Thyroid hormone directly increases TSH bioactivity, and this correlates with decreased sialylation [141]. Steroid hormones, specifically glucocorticoids, inhibit TSH production but TSH subunit mRNA levels do not change significantly [142]. Their major effect

may be at the secretory level. Estrogens mildly reduce both α- and TSH β-subunit mRNA in hypothyroid rats compared with euthyroid controls [143]. In this study, estrogen also abolished the early rise in subunit mRNA levels seen following T3 replacement. Other studies showed that E2 inhibits the up-regulation of αGSU and TSHβ mRNA levels in the pituitary of hypothyroid rats [144] and that ovariectomy increased pituitary TSHβ mRNA levels [145]. In the thyrotroph cell line, TαT1, estrogen treatment reduced TSHβ mRNA levels as measured by reverse-transcription PCR and suggested that Gata2 may be prevented from gene activation by an interaction with liganded ERα [146]. Finally, testosterone has been shown to increase TSH β-subunit mRNA in castrated rat pituitary and mouse thyrotrophic tumor [147]. Transcriptional inhibition of α-subunit gene transcription by glucocorticoids may be mediated by binding the glucocorticoid receptor (GR) to sequences between 2122 and 293 of the human α-subunit gene. However, no direct binding was detected in other studies suggesting that the GR inhibits transcription by interfering with other transactivating proteins [148]. Androgen inhibition and androgen receptor binding have also been localized to that region, and negative regulation by estrogen was described in the gonadotrophs of transgenic mice expressing a reporter gene under the control of both human and bovine promoters, but no binding of these regions to the estrogen receptor (ER) was detected, suggesting an indirect effect [149]. However, other studies using rat somatomammotrophs have found positive regulation by estrogen localized to the proximal 98 bp of 5’ flanking DNA of the human α-subunit gene and binding of the ER to the T3 response element from 222 to 27 [150]. Similar studies in thyrotroph cells have not been reported. Leptin and neuropeptide-Y (NPY) have opposite effects on TSH biosynthesis. Leptin is the product of the ob gene, found mainly in adipose tissue that regulates body weight and energy expenditure [151]. NPY, a neuropeptide synthesized in the arcuate nucleus of the hypothalamus, plays many roles in neuroendocrine function [152]. In dispersed rat pituitary cells, leptin stimulated and NPY inhibited TSHβ mRNA levels in a dose-related manner [153]. In contrast, both agents increased α-subunit steady-state mRNA levels.

TSH SECRETION In euthyroid humans, the production rate of TSH is between 100 and 400 mU/day, the plasma halflife is approximately 50 minutes, and the plasma clearance

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rate is approximately 50 mL/min [154,155]. The distribution space of TSH is slightly greater than the plasma volume. In hypothyroid subjects, TSH secretion rates increase by 10 15 times normal rates, while the clearance rates decrease slightly. In hyperthyroid subjects, TSH secretion is suppressed and metabolic clearance is accelerated.

Ontogeny of TSH Levels At 8 10 weeks of gestation in the human, TRH is measurable in the hypothalamus, with progressive increases in TRH levels until term. By 12 weeks of gestation, immunoreactive TSH cells are present in the human pituitary gland, and TSH is detectable in the pituitary and the serum [156]. Serum and pituitary TSH levels remain low until week 18, when TSH levels increase rapidly, followed by increases in serum T4 and T3 concentrations. Fetal serum TSH and T4 concentrations continue to increase between 20 and 40 weeks of gestation. Pituitary TSH begins to respond to exogenous TRH early in the third trimester, while negative feedback control of TSH secretion develops during the last half of gestation and the first 1 2 months of life [157]. An abrupt rise in serum TSH levels occurs within 30 minutes of birth in term infants. This is followed by an increase in serum T3 concentrations within 4 hours and a lesser increase in T4 levels within the first 24 36 hours. The initial increase in serum TSH levels appears to be stimulated by cooling in the extrauterine environment. Serum TSH levels fall to the adult range by 3 5 days after birth, and serum thyroid hormone levels stabilize by 1 2 months. Serum TSH levels in healthy premature infants (less than 37 weeks gestational age) are quite variable, but tend to be lower at birth compared to term infants. TSH levels decrease slightly during the first week of life, followed by a gradual increase to normal term levels. Serum TSH levels are even lower in ill premature infants, but rise towards normal levels during recovery [157 159]. In adults, 24hour TSH secretion is independent of age, although feedback control of TSH secretion may be less robust in older subjects (155).

Patterns of TSH Secretion TSH is secreted from the pituitary gland in a dual fashion in infants and adults, with secretory bursts (pulses) superimposed upon basal (apulsatile) secretion [160 162] (Fig. 6.7, upper panel). Basal TSH secretion accounts for 30 40% of the total amount released into the circulation, and secretory bursts account for

FIGURE 6.7 Serum thyroid-stimulating hormone (TSH) levels measured every 15 minutes in a healthy subject (upper panel), in two subjects with primary hypothyroidism (middle panel) and in a subject with hypothyroidism due to a craniopharyngioma (lower panel). Significant TSH pulses were located by cluster analysis, a computerized pulse detection program, and are indicated by asterisks. Source: Adapted from Samuels MH, Veldhuis JD, Henry P, Ridgway EC. Pathophysiology of pulsatile and copulsatile release of thyroidstimulating hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit. J Clin Endocrinol Metab 1990;71:425 32 [160].

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the remaining 60 70%. TSH pulses occur approximately every 2 3 hours, although there is considerable variability among individuals [160]. TSH pulses appear to directly stimulate T3 secretion from the thyroid gland, as cross-correlation analysis has shown that a free T3 peak occurs between 0.5 and 2.5 hours after a TSH peak [163]. However, changes in free T3 levels from nadir to peak are only 11% of mean free T3 levels, probably because T3 has a long serum halflife, and most T3 does not arise from the thyroid gland [163]. In healthy euthyroid subjects, TSH is secreted in a circadian pattern, with nocturnal levels increasing up to twice daytime levels [164] (Fig. 6.7, upper panel). Peak TSH levels occur between 23:00 and 05:00 hours in subjects with normal sleep wake cycles, and nadir levels occur at about 11:00 hours. The TSH circadian rhythm emerges between 1 and 2 months of life, and is well-established in healthy children [165]. TSH pulsatile and circadian secretion is then maintained throughout adulthood [164]. The circadian variation in TSH levels is due to increased mass of TSH secreted per burst at night, as well as slight increased frequency of bursts and more rapid increase to maximal TSH secretion within a burst [164]. The nocturnal increase in TSH levels can precede the onset of sleep, and sleep deprivation enhances TSH secretion. Therefore, in contrast to other pituitary hormones with a circadian variation, the nocturnal rise in TSH levels is not sleepentrained. Instead, there is a sleep-related inhibition of TSH release that is of insufficient magnitude to counteract the nocturnal TSH surge. Subjects with primary hypothyroidism have increased TSH pulse amplitude with disordered secretion and attenuation of the circadian variation in TSH levels [166]. Pulse amplitude increases in proportion to the degree of hypothyroidism, while normal patterns of TSH secretion are re-established with thyroid hormone therapy [167,168]. In contrast, patients with hypothalamic pituitary causes of hypothyroidism secrete less TSH over a 24-hour period, with loss of the nocturnal TSH surge in pulse amplitude (Fig. 6.7, lower panel) [169]. This may be one explanation for the presence of hypothyroidism despite a “normal” daytime TSH level in these patients. A similar pattern of reduced 24-hour TSH secretion occurs in critical illness [170]. The origin of pulsatile and circadian TSH secretion is not known. Thyroid hormones alter TSH pulse amplitude, but have little effect on pulse frequency, and therefore are unlikely to participate in TSH pulse generation. The TSH pulse generator may reside in the hypothalamus, with TRH neurons acting in concert to stimulate a burst of TSH secretion from the pituitary gland. However, constant TRH infusions do not change TSH pulse frequency in humans, which casts

doubt on this theory [171]. Somatostatin and dopamine suppress TSH pulse amplitude, but neither agent has any major effect on TSH pulse frequency, and therefore somatostatin and dopamine do not appear to control pulsatile TSH secretion. There is a diurnal variation in the activity of anterior pituitary 5’-monodeiodinase in the rat, which may control circadian TSH secretion [172]. However, this has not been confirmed in the human. Physiologic serum cortisol levels affect circadian TSH secretion, although cortisol does not appear to affect TSH pulse frequency. When subjects with adrenal insufficiency were studied under conditions of glucocorticoid withdrawal, daytime TSH levels were increased, and the usual TSH circadian rhythm was abolished. When these subjects were given physiologic doses of hydrocortisone in a pattern that mimicked normal pulsatile and circadian cortisol secretion, daytime TSH levels were decreased, and the normal TSH circadian rhythm was re-established. Hydrocortisone infusions at the same dose given as pulses of constant amplitude throughout the 24-hour period also decreased 24-hour TSH levels, but there was no circadian variation [173]. Similarly, when healthy subjects were given metyrapone (an inhibitor of endogenous cortisol synthesis), TSH levels increased during the day, leading to abolition of the usual TSH circadian variation [174]. These data suggest that the normal early-morning increase in endogenous serum cortisol levels decreases serum TSH levels and leads to the observed normal circadian variation in TSH.

REGULATION OF TSH SECRETION TSH secretion is a result of complex interactions between central (hypothalamic) and peripheral hormones (Fig. 6.8).

Hypothalamic Regulation of TSH Secretion TRH directly affects TSH secretion in vivo and in vitro at concentrations that exist in the pituitary portal blood [174,175]. Immunoneutralization of TRH in animals leads to a decline in thyroid function [176], and TRH knockout mice have a reduced postnatal TSH surge, followed by impaired baseline thyroid function with a poor TSH response to hypothyroidism. Lesions of the paraventricular nuclei (PVN) decrease circulating TRH and TSH levels in normal or hypothyroid animals and cause hypothyroidism [177], while electrical stimulation of this area causes TSH release. Although baseline levels of TSH are reduced in animals with lesions of the PVN, TSH levels still show

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FIGURE 6.8 Neuroendocrine and peripheral control of thyroidstimulating hormone (TSH) secretion. T4, thyroxine; T3, triiodothyronine; TRH, thyrotrophin-releasing hormone; TRHR, TRH receptor; SSTRs, somatostatin receptors; D2R, dopamine receptor type 2; D2, deiodinase type II; TNF, tumor necrosis factor; IL-6, interleukin-6.

appropriate responses to changes in circulating thyroid hormone levels. Thus, TRH likely determines the setpoint of feedback control by thyroid hormones. Acute intravenous administration of TRH to human subjects causes a dose-related release of TSH from the pituitary. This occurs within 5 minutes and is maximal at 20 30 minutes. Serum TSH levels return to basal levels by 2 hours [178]. More prolonged (2 4-hour) infusions of TRH lead to biphasic increases in serum TSH levels in humans and animals [179]. The early phase may reflect release of stored TSH, while the later phase may reflect release of newly synthesized TSH. Interpretation of TSH responses to even more prolonged TRH infusions is complicated by the increase in serum T3 levels, which feedback to suppress further TSH release [170]. Continuous TRH administration in vitro also causes desensitization of TSH responses, which may further explain decreased TSH levels with long-term TRH exposure [180]. Somatostatin (SS) in humans and animals inhibits basal and TRH-stimulated TSH secretion in vivo and in vitro at concentrations that exist in the pituitary portal blood [181]. In the hypothalamus, the highest concentrations of SS occur in the anterior paraventricular region. From this region, axonal processes of SScontaining neurons project to the median eminence. Animals that have undergone sectioning of these fibers have depletion of SS content of the median eminence and increased serum TSH levels [181]. Similarly, immunoneutralization of SS in animals increases basal TSH levels and TSH responses to TRH [182]. In humans, SS infusions suppress TSH pulse amplitude, slightly decrease TSH pulse frequency, and abolish the

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nocturnal TSH surge [183]. Thus, TSH secretion is probably regulated through a simultaneous dualcontrol system of TRH stimulation and SS inhibition from the hypothalamus. SS binds to specific, high-affinity receptors in the anterior pituitary gland. SS receptor subtypes (SST) 1 and 5 have been localized to thyrotrophs [184]. Binding of SS to its receptor inhibits adenylate cyclase via the inhibitory subunit of the guanine nucleotide regulatory protein, which lowers protein kinase A activity and decreases TSH secretion. SS may also exert some effects by cAMP-independent actions on intracellular calcium levels. Hypothyroidism reduces the efficacy of SS in decreasing TSH secretion in vitro, which is reversed by thyroid hormone administration [185]. Further studies in mouse thyrotrophic tumors indicate that both SST1 and 5 are markedly downregulated in hypothyroidism and are induced by thyroid hormone [184]. Although short-term infusions of SS lead to pronounced suppression of TSH secretion in humans, long-term treatment with SS or its analogues does not cause hypothyroidism [186]. This probably reflects compensatory mechanisms in the thyroid hormone feedback loop. GH deficiency is associated with increased TSH responses to TRH, while GH administration or endogenous GH excess (acromegaly) decrease basal, pulsatile and TRH-stimulated TSH secretion [187,188], possibly due to GH stimulation of hypothalamic SS release. Dopamine also inhibits basal and TRH-stimulated TSH secretion in vivo and in vitro at concentrations that exist in the pituitary portal blood [189]. In humans, dopamine infusions rapidly suppress TSH pulse amplitude, do not affect TSH pulse frequency and abolish the nocturnal TSH surge [183], while administration of a dopamine antagonist has the opposite effect [190]. Dopamine also has direct effects on hypothalamic hormone secretion that may indirectly impact TSH secretion. For example, dopamine and dopamine-agonist drugs stimulate both TRH and SS release from rat hypothalami [191]. In the hypothalamus, dopamine is secreted by neurons in the arcuate nucleus. From the arcuate nucleus, neuronal processes project to the median eminence. Dopamine acts by binding to type 2 dopamine receptors (DA2) on thyrotroph cells [192]. This leads to inhibition of adenylate cyclase, which decreases the synthesis and secretion of TSH. The inhibitory effects of dopamine on TSH secretion vary according to sex steroids, body mass, and thyroid status. Dopamine antagonist drugs cause greater increases in serum TSH levels in women than in men. Recent studies show that obesity is associated with enhanced TSH secretion, which may be mediated via blunted central dopaminergic tone [193]. Dopamine inhibition of TSH release

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is greater in patients with mild hypothyroidism than in normal subjects, although subjects with severe hypothyroidism may be less responsive [194]. Although short-term infusions of dopamine lead to pronounced suppression of TSH secretion, long-term treatment with dopamine agonists does not cause hypothyroidism. This probably reflects compensatory mechanisms in the thyroid hormone feedback loop. Adrenergic effects have also been reported in vivo and in vitro. α-Adrenergic activation stimulates TSH release directly from the rat pituitary gland at physiologic concentrations of catecholamines [195]. α-Adrenergic agonists stimulate TSH release in rats, while blockade of norepinephrine synthesis or treatment with adrenergic receptor blockers decrease TSH levels [196]. It is unclear whether these effects are mediated via changes in TRH and/or SS levels. In humans, there are limited data regarding adrenergic effects on TSH secretion. α-Adrenergic blockade diminishes serum TSH responses to TRH [197]. However, administration of epinephrine does not alter TRH-stimulated TSH secretion [198]. These data suggest that endogenous adrenergic pathways do not have a major role in TSH secretion. Noradrenergic stimulation of TSH secretion is mediated by highaffinity α1-adrenoreceptors linked to adenylate cyclase [197]. Therefore, dopamine and epinephrine appear to exert opposing actions on thyrotrophs by opposite effects on cAMP generation. Opioid administration to rats suppresses basal or stimulated TSH levels, and the opioid receptor antagonist naloxone reverses these effects [199]. Acute opiate administration in humans may slightly stimulate TSH levels, while acute naloxone administration has little effect [200]. In contrast to these acute studies, when naloxone is given over 24 hours, the 24-hour TSH secretion decreases, primarily due to a decrease in nocturnal TSH pulse amplitude [201]. TSH responses to TRH are also decreased. Serum T3 levels are decreased as well, suggesting that the magnitude of TSH suppression is sufficient to affect thyroid gland function. These findings suggest that endogenous opioids may play a role in tonic stimulation of TSH secretion.

Peripheral Regulation of TSH Secretion Thyroid hormones directly block pituitary secretion of TSH. Acute administration of T3 suppresses TSH levels within hours in animals and humans [202,203], while chronic administration leads to further suppression. Slight changes in serum thyroid hormone levels within the normal range alter basal and TRHstimulated TSH levels, confirming the sensitivity of the pituitary gland to thyroid hormone feedback. Recent

genetic studies have suggested that genetic variations that influence thyroid hormone production and the conversion of T4 to T3 may affect these endogenous TSH levels in humans [204,205]. Thyroid hormones alter tonic TSH secretion and TSH pulse amplitude without affecting pulse frequency, since subjects with primary hypothyroidism have a near-normal number of TSH pulses, and T4 replacement leads to a decrease in TSH pulse amplitude without much change in pulse frequency [166]. In addition to direct effects on TSH secretion, thyroid hormones have other actions that impact on TSH secretion. In particular, recent studies in transgenic animals show that there is a central role for feedback inhibition of TRH by thyroid hormones in the normal hypothalamic pituitary thyroid axis [206]. In addition, hypothalamic SS content is decreased in hypothyroid rats, and is restored by T3 treatment [207]. These combined effects of thyroid hormones on TRH and SS decrease TRH release from the hypothalamus, and indirectly decrease TSH secretion. Glucocorticoids at pharmacologic doses or high endogenous cortisol levels (Cushing’s syndrome) suppress basal and pulsatile TSH levels, blunt TSH responses to TRH, and diminish the nocturnal TSH surge in humans and animals [208 210]. Glucocorticoidinduced changes in TSH levels are due to decreased TSH pulse amplitude without alteration in TSH pulse frequency, with more profound suppression of nocturnal TSH secretion and abolition of the TSH surge. Physiologic glucocorticoid levels also affect TSH secretion [173]. Untreated patients with adrenocortical insufficiency can have elevated serum TSH levels that resolve with steroid replacement. Complementary studies of metyrapone (an inhibitor of cortisol synthesis) administration to healthy subjects confirm that endogenous cortisol levels suppress TSH secretion, and physiologic hydrocortisone replacement in patients with adrenal insufficiency decreases daytime TSH levels back to those seen in healthy subjects. Glucocorticoid suppression of TSH levels may occur directly at the pituitary gland. Animal studies suggest that glucocorticoids exert direct effects on thyrotrophs to impair TSH secretion, although these appear to be highly dependent on dose and time course of administration [211,212]. Glucocorticoids do not appear to directly affect TSH gene transcription. In humans, TSH pulse frequency is maintained during glucocorticoid administration, while TSH pulse amplitude is reduced and TSH responses to exogenous TRH are attenuated, suggesting a direct effect on TSH secretion. In addition to direct pituitary effects, it appears that glucocorticoids may have hypothalamic actions that affect TSH levels. Dexamethasone increases hypothalamic TRH levels, while circulating TRH levels are decreased [213].

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Patients with Cushing’s syndrome or subjects receiving prolonged courses of glucocorticoids may have low serum T4 as well as TSH levels. Whether such patients have true hypothyroidism and whether they should be treated with thyroid hormone, is unclear; however, patients with acute or chronic illnesses and similar abnormalities in thyroid hormone levels do not appear to benefit from thyroid hormone therapy. Leptin is primarily a product of adipocytes, although it is also located in thyrotrophs. It regulates food intake and energy expenditure, decreasing acutely with fasting in animals and humans [151]. Exogenous leptin administration to fed rats raises serum TSH levels, probably by increasing TRH gene expression and TRH release directly and indirectly via the proopiomelanocortin/CART-expressing neurons of the arcuate nucleus [214,215]. Similarly, leptin administration to fasted rats or humans reverses fasting-induced decrements in TSH levels, also by increasing TRH gene expression and release [216]. This suggests that fasting-related reductions in leptin levels play a role in suppressing TSH secretion. However, immunoneutralization of leptin increases TSH levels, and therefore endogenous leptin may inhibit TSH release, at least in rats. In healthy adults and children, 24-hour TSH and leptin secretory patterns are tightly coupled, while in a leptin-deficient patient, the TSH rhythm was disorganized, suggesting that endogenous leptin plays a role in regulating patterns of TSH secretion [217,218]. Sex steroids may account for higher serum and pituitary TSH concentrations in male compared to female rats. TSH content is reduced by castration and is restored by androgen administration [151], which also increases basal and TRH-stimulated serum TSH levels [219]. In contrast, androgen administration to intact female rats does not alter serum or pituitary levels of TSH [220]. Estrogen administration to euthyroid rats does not alter serum TSH levels. In euthyroid humans, most studies suggest that changes in endogenous or exogenous sex steroid levels do not affect basal or TRH-stimulated TSH levels [221]. There is no significant gender difference in the basal mean and pulsatile secretion of TSH [163]. Therefore, sex steroids do not appear to play a major regulatory role in TSH secretion in humans. Cytokines are circulating mediators of the inflammatory response that are produced by many cells and have systemic effects on the hypothalamic pituitary thyroid axis [222 224]. Administration of tumor necrosis factor (TNF) or interleukin-6 (IL-6) decreases serum TSH levels in healthy human subjects, and TNF and interleukin-1 (IL-1) decrease TSH levels in animals. Administration of these cytokines recapitulates the alterations in thyroid hormone and TSH

levels seen in acute nonthyroidal illness. In rats, TNF reduces hypothalamic TRH content and pituitary TSH gene transcription. IL-1 stimulates type II 5’-deiodinase activity in rat brain, which may decrease TSH secretion by increasing intrapituitary T3 levels. Autocrine and paracrine peptides may alter regulatory pathways within the pituitary gland for TSH secretion, acting in concert with the central and peripheral factors described above [225]. Peptides that have been implicated in this role include neurotensin, opioidrelated peptides, galanin, substance P, epidermal growth factor (EGF), FGF, IL-1, and IL-6. Of particular interest is neuromedin B, a mammalian peptide structurally and functionally related to the amphibian peptide bombesin [226]. Neuromedin B is present in high concentrations in thyrotroph cells, with levels that change according to thyroid status. Administration of neuromedin B to rodents decreases TSH levels, while intrathecal administration of neuromedin B antiserum increases TSH levels. Knockout mice lacking the neuromedin B receptor have slightly elevated serum TSH levels, exaggerated TSH responses to TRH administration, and blunted TSH increases with hypothyroidism [227,228]. Therefore, neuromedin B appears to act as an autocrine factor that exerts a tonic inhibitory effect on TSH secretion. Further data suggest that neuromedin B may modulate the action of other TSH secretagogues and release inhibitors, including TRH and thyroid hormones.

ACTION OF TSH TSH acts on the thyroid gland by binding to the TSH receptor. An excellent review of this subject has been published [229]. This receptor is located on the plasma membranes of thyroid cells and consists of a long extracellular domain, a transmembrane domain, and a short intracellular domain. Knowledge of the molecular structure of the receptor has allowed a better understanding of the mechanism of action of TSH that results in the production of thyroid hormone.

TSH Receptor Gene The human TSH receptor gene is located on chromosome 14 locus q31 and spans a region greater than 60 kb in size containing 10 exons [230]. Exons 1 9 have 327, 72, 75, 75, 75, 78, 69, 78, and 189 bp, respectively, and encode part of the extracellular domain, whereas exon 10 is greater than 1412 bp and encodes the rest of the extracellular domain as well as all of the transmembrane and the intracellular domains. The promoter region of the human TSH receptor gene has also been partially characterized [230]. The major transcriptional

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start site, designated as 11, is located 157 bp upstream of the translation initiation codon ATG. There are no consensus CAAT or TATA boxes but there are degenerate CAAGGAAAGT and TAGGGAA boxes located at positions 286 and 243, respectively. The regions of the promoter important for tissue-specific expression and those responsive to TSH and cAMP, the two main regulators that have been shown to inhibit the rat TSH receptor gene expression [231], are yet to be defined. Northern blot analysis has revealed two major transcripts of the human TSH receptor of 3.9 and 4.6 kb in size that differ only in the length of the 3’ untranslated region [232,233].

TSH Receptor Structure The TSH receptor is synthesized as a single polypeptide chain of 764 amino acids that includes a 20amino-acid signal peptide [234]. However, the TSH receptor has been found to exist on the cell surface as a single chain and also as a two-subunit form, produced by internal cleavage apparently at two sites, releasing a potentially immunogenic 5 7-kDa peptide [235,236]. Cleavage of the TSH receptor has been found to depend on cell cell contacts [237]. The aminoterminal half of the protein contains 16 hydrophilic leucinerich repeats (LRRs) that form the extracellular domain and include six potential glycosylation sites. The asparagine-linked oligosaccharides appear to be important for correct folding, membrane targeting, and receptor function [238,239]. The LRRs are the common feature of the superfamily of LRR proteins. One of these, the ribonuclease inhibitor, has been cocrystallized with its ligand [240], and this has allowed the construction of a model of the extracellular domain of the TSH receptor bound to TSH [241], as shown in Fig. 6.9. This model has been confirmed by analysis of the crystal structure of the extracellular domain of the TSH receptor bound to a TSH receptor antibody [242], which shows remarkable similarity\ies to the receptor binding features of FSH and LH. The carboxylterminal half of the protein was modeled after the structure of the G-protein-coupled receptors, based on rhodopsin [243]. This region contains seven hydrophobic transmembrane segments, three extracellular loops, three cytoplasmatic loops, and a short cytoplasmatic tail of 82 amino acids. Molecular modeling studies have identified structural features of the TSH receptor transmembrane helices 2 and 5 [244].

Determinants of TSH Receptor Binding The entire extracellular domain and parts of the transmembrane domain of the TSH receptor contribute

FIGURE 6.9 Schematic model of the human thyroid-stimulating hormone (TSH) TSH receptor complex. The receptor (black) is depicted in accordance with models based on the leucine-rich repeats (LRR)-containing ribonuclease inhibitor (203k) and G-protein-coupled rhodopsin (203 n). In the center, the α-subunit (red ribbon) and TSH β-subunit (blue ribbon) are shown folded and combined, with the α-hairpin loops oriented toward the extracellular loops of the transmembrane domain of the receptor, and the β-hairpin loops toward the concave surface of the LRR. Source: Adapted from Grossmann M, Weintraub BD, Szkudlinski MW. Novel insights into the molecular mechanisms of human thyrotropin action: structural, physiological, and therapeutic implications for the glycoprotein hormone family. Endocr Rev 1997;18:476 501 [73].

to TSH binding. However, two regions, from residues 201 to 211 and 222 to 230, are particularly important in TSH-specific binding [245]. Inactivating TSH receptor mutations, a rare cause of congenital hypothyroidism in humans [246], frequently map to the aminoterminal extracellular domain [247]. In contrast, a different region of the extracellular domain, called the hinge region, from residue 287 to 404, appears to be more important for binding of TSH receptor antibodies [248]. Other authors have reported considerable overlap of TSH binding regions and antibody epitopes [249], although binding to the hinge region was studied using bovine TSH and was found to be dependent on positive-charged residues [250], which are less common in human TSH [251]. Interestingly, studies using rat FRTL-5 cells have shown that thyroid-stimulating autoantibodies (TSAbs or TSI) stimulate whereas TSH binding inhibitory antibodies inhibit TSH-mediated gene expression, suggesting that these antibodies must act on different epitopes of the receptor that differ in their signal transduction mechanism [231]. Recently the crystal structure of the extracellular domain of the TSH receptor bound to a stimulating TSH receptor monoclonal antibody was determined to be similar to the LH-FSH receptor crystal structure, but the hinge region of the TSH receptor was not included [242]. The transmembrane domain of the TSH receptor also

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appears to be important in ligand binding. A point mutation in the fourth transmembrane domain of the TSH receptor gene has been described in the hyt/hyt congenitally hypothyroid mouse that abolishes TSH binding [252]. Specificity of TSH binding is conferred by the TSH β-subunit. It appears that amino acid residues from 58 to 69, within the βL3 loop, and from 88 to 105, the “seatbelt” region of the TSH β-subunit [253], play an important role in binding to and activation of the TSH receptor. The carboxyl-terminal end of TSHβ contains multiple lysine residues (positions 101, 107, and 110) and a cysteine at position 105 that are critical for the ability to bind to the receptor [254]. Congenital hypothyroidism due to biologically inactive TSH was found to result from a frameshift mutation with loss of β-cysteine105 [255] (see Disorders of TSH production). Several regions of the α-subunit are also important for TSH activity, particularly the residues α11 20 and α88 92 [74,79]. In addition, the oligosaccharide chain at position α-asparagine52 plays an important role in both binding affinity and receptor activation. A mutant TSH lacking the α-asparagine52 oligosaccharide showed increased in vitro activity, although this same mutation had the opposite effect on CG binding to its native receptor [79]. However, such a mutation also increased TSH clearance and this decreased in vivo activity [79]. In addition, the oligosaccharide chains on the TSH subunits are critically important for signal transduction [64,256]. In this regard, the α-subunit oligosaccharides are important for all the pathways activated by the receptor, whereas the TSH β-subunit oligosaccharide only influences the adenylate cyclase pathway [257]. The mechanism by which the oligosaccharides influence signal transduction is not known. A model for the action of the glycoprotein hormones has been proposed that suggests a role for the oligosaccharides indirectly modulating the influx of calcium into the target cell [258]. The ability of CG to bind to the TSH receptor was demonstrated in rat thyroid cells [259] and confirmed in studies using recombinant human TSH receptor [260,261]. The activity of CG was estimated to be less than 0.1% compared to TSH. LH was found to have a 10-fold higher potency for activation of the TSH receptor when compared to CG, but a mutant of CG that lacks the carboxyl-terminal region of CGβ from amino acid residues 115 to 145 showed a potency equivalent to that of LH [261]. This truncated form of CG is one of the forms in the heterogeneous population of CG molecules produced in normal pregnancy and in trophoblastic tumors and may be present in amounts sufficient to cause significant thyroid gland stimulation [262,263]. The occurrence of gestational hyperthyroidism due to a mutation in the TSH receptor that increases its sensitivity to CG has also been described [264].

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Signal Transduction at the TSH Receptor The three intracytoplasmic loops of the transmembrane domain appear to be important for signal transduction [265]. The TSH receptor is coupled to the Gs protein cascade probably through the carboxyl terminus of the third cytoplasmic loop [266]. Binding of Gs is dependent on TSH receptor cleavage [267] by the metalloprotease ADAM 10 [268]. Thus, binding of TSH activates adenylate cyclase to produce cAMP [269,270]. The Gq/phospholipase C/inositol phosphate/Ca21 pathway is also activated and appears to play a role in TSH synthesis, particularly in regulating iodination [271], but this pathway is slower and requires a higher concentration of TSH [269]. Specific amino acids in the third cytoplasmic loop have been identified that are important for the phosphatidylinositol pathway but do not appear to play a role in the adenylate cyclase pathway [270]. TSH is also able to signal through the JAK/ STAT [272] and mTOR/S6K1 [273] pathways, with important roles in thyroid cell growth. The unliganded TSH receptor has been found to have significant constitutive activity [274,275], suggesting that regulation may involve the release of an inhibitory restraint. This would explain the relatively high frequency of activating mutations of the TSH receptor compared to inactivating mutations. In cases of congenital hyperthyroidism [274,276,277], the mutations were located in the extracellular domain and the second, fourth, fifth and sixth transmembrane domains, while in hyperfunctioning adenomas the mutations were found to localize to the carboxyl terminus of the third cytoplasmic loop and adjacent sixth transmembrane domain. Recently, an activating mutation was described that localized to the intracellular C-terminal region [278]. All these mutations resulted in constitutive activation of adenylate cyclase [279]. Germline mutations of the cytoplasmatic tail of the TSH receptor have been described in 33.3% of patients with toxic multinodular goiter and 16.3% with Graves’ disease, and this mutation was found to result in an exaggerated cAMP response to TSH [280].

TSH actions TSH action on the receptor results in activation of the adenylate cyclase pathway and to some extent the phosphatidylinositol pathway, as described above, and leads to the activation of multiple proteins, including JAK/STAT [272], mTOR/S6K1 [273], and cell cyclerelated proteins [281]. Proteins phosphorylated by the protein kinase C pathway appear to be different from those phosphorylated by protein kinase A [282]. In addition, phosphoprotein phosphatases are activated and lead to the dephosphorylation of another set of

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proteins [283]. The effects of TSH on the thyroid gland include changes in thyroid gland growth, cell morphology, iodine metabolism and synthesis of thyroid hormone. Effects of TSH on Thyroid Gland Development and Growth Embryologic development of the thyroid gland appears to be independent of TSH, as shown by experiments using knockout mice deficient in TSH and TSH receptor in which the size and follicular structure, as well as thyroid-specific transcription factor expression and thyroglobulin production, were similar to wildtype [282]. However, expression of thyroperoxidase and sodium/iodine symporter and maintenance of thyroid gland architecture after birth is severely affected in these mice. In the adult thyroid gland, TSH is the main regulator of thyroid gland growth. After long-term stimulation by TSH, the thyroid gland enlarges as a result of hyperplasia and hypertrophy. Acutely, TSH has a rapid mitogenic effect on the thyroid gland that is evident within 5 minutes [284]. It increases DNA synthesis through the adenylate cyclase pathway [285], specifically through activation of protein kinase A type I [286]. TSH may also regulate growth by cAMPindependent pathways [287], such as the mTOR/S6K1 pathway [273], and interactions with the action of the growth factors EGF and insulin-like growth factor-1 (IGF-1) [288,289]. It has been found that TSH increases the transcription of specific immediate early genes in rat thyroid cells [290]. TSH also inhibits apoptosis [291], perhaps by regulating p53 and bcl-2, as shown for gonadotrophins [292,293]. Mutations within the α33 44 region were found to reduce growth stimulation but not affect cAMP production [294], although a clear dissociation of the various actions by TSH analogues has not yet been achieved. Effects of TSH on Thyroid Cell Morphology TSH causes dramatic changes in the morphology of the thyroid [295]. The initial response to TSH is the incorporation of exocytotic vesicles into the cell membrane at the apical pole of the follicular cells that is quickly followed by formation of cytoplasmatic projections and microvilli. The number of cytoplasmatic projections has been correlated with the level of TSH [296]. After stimulation, the follicular cells become columnar and filled with colloid droplets and luminal colloid is nearly depleted, collapsing the follicles. Lysosomes migrate from the basal pole toward the apical pole where they fuse with the colloid droplets and then migrate toward the basal pole, becoming smaller and denser. The cytoskeletal system, that includes

myosin, actin, tropomyosin, calmodulin, profilin, and tubulin, has been implicated in this process [295,297]. Effects of TSH on Iodine Metabolism As stated above, TSH is necessary for sodium/ iodide symporter and thyroid peroxidase expression both during embryogenesis and after birth [282]. TSH primarily regulates post-transcriptional activation of the sodium iodide symporter via the adenylate cyclase pathway [298]. Thyroid peroxidase transcription and mRNA stability are increased by TSH also through the adenylate cyclase pathway [299]. However, generation of peroxide and iodide organification appear to be mediated by a phosphatidylinositol pathway independent of protein kinase C [300]. Effects of TSH on the Synthesis of Thyroid Hormone The end-point of TSH action is the production of thyroid hormone by the thyroid gland. The process begins with thyroglobulin gene transcription, which in itself is able to occur independently of TSH [301]. However, the transcriptional rate and possibly the mRNA stability are increased by TSH [302]. TSH regulates the expression and activation of Rab5a and Rab7, which are rate-limiting catalysts of thyroglobulin internalization and transfer to lysosomes [303]. TSH stimulates iodide uptake and organification, as described above. TSH then acts on the iodinated thyroglobulin stored in the luminal colloid and stimulates its hydrolysis resulting in the release of the constituent amino acids, including the iodothyronines T3 and T4. TSH-Induced Receptor Desensitization The phenomenon of desensitization, whereby prior TSH stimulation leads to a decrease in the subsequent cAMP response to TSH stimulation, is mediated by cAMP [231]. Studies using recombinant TSH receptor have shown that desensitization does not occur when the receptor is expressed in nonthyroidal cells, suggesting that this phenomenon requires a cell-specific factor [304]. Extrathyroidal Actions of TSH The occurrence of precocious puberty in cases of severe juvenile primary hypothyroidism has suggested that high levels of TSH are able to cross-activate the gonadotrophin receptors. This interaction has now been demonstrated using recombinant human TSH, which has been found to be capable of activating the FSH [305] but not the CG/LH receptor [306]. Expression of thyrotrophin receptor has been reported in the brain [307] and pituitary gland [308]. In the brain, both astrocytes and neuronal cells were found

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to express TSH receptor mRNA and protein [307], and stimulated arachidonic acid release and type II 5’-iodothyronine-deiodinase activity [309]. In the pituitary gland, the TSH receptor was localized to folliculo-stellate cells and may be involved in paracrine feedback inhibition of TSH secretion that may also occur in response to TSH receptor autoantibodies [308,310]. Expression of both TSH and its receptor has been reported in lymphocytes [311], erythrocytes [312], adipose tissue [313], bone [314], hair follicle [315], liver [316], ovary [317], and thyroid C cells [318]. In bone marrow myeloid and lymphoid cells, there is a predominant expression of TSHβv, a variant of TSHβ coded for by exon 3 of the human TSHβ gene [319]. An ancestral glycoprotein hormone, thyrostimulin, has been found to be produced in a variety of tissues and to be able to activate the TSH receptor [320,321], and this may suggest a paracrine mechanism of regulation, which has been identified in bone formation [322]. More studies are needed to determine the physiological significance of the extrathyroidal effects mediated by the TSH receptor, as this may impact the safety of future treatment modalities for thyroid cancer that may attempt to target radioisotopes to the TSH receptor [323].

TSH MEASUREMENTS Accurate and specific measurements of serum TSH concentrations have become the most important method for diagnosing and treating the vast majority of thyroid disorders. Initially, the radioimmunoassays were very insensitive and could only detect high levels seen in primary hypothyroidism [324]. Modifications subsequently led to improved sensitivity and specificity, enabling detection of TSH levels as low as 0.5 1.0 mU/L. These were called “first-generation assays,” and were in use between 1965 and 1985. One hundred percent of primary hypothyroid subjects had elevated TSH levels but these “first-generation assays” could not accurately quantitate values within the normal range and there was considerable overlap with the values found in euthyroid and hyperthyroid subjects. The subsequent development of monoclonal antibody technology allowed two or more antibodies with precise epitope specificity to be used in sandwich-type assays starting in the mid-1980s that were called immunometric assays [325,326]. One or more of the monoclonal antibodies are labeled and are called the “signal antibodies.” The signal may be isotopic, chemiluminescent, or enzymatic. Another monoclonal antibody with completely different epitope specificity is attached to a solid support and is called the “capture

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antibody.” All antibodies are used in excess and therefore all TSH molecules in a sample are captured and the signal generated is directly proportional to the level of TSH. These modifications in the measurement of TSH resulted in important changes. First, the assays were highly specific with no crossreaction to the other human glycoprotein hormones. Second, 100% of euthyroid controls have detectable and quantifiable levels of TSH. Third, there is little or no overlap in TSH values in patients with hyperthyroidism compared to euthyroid controls. The degree to which a given assay can separate undetectable TSH levels found in hyperthyroid subjects from normal values in euthyroid controls has improved steadily [324]. These improvements have resulted in progressively lower functional detection limits, defined as the lowest TSH value detected with an interassay coefficient of variation # 20%. Thus, first-generation assays (usually radioimmunoassays) have functional detection limits of 0.5 1.0 mU/L, second-generation assays 0.1 0.2 mU/L, third-generation assays 0.01 0.02 mU/L, and fourth-generation assays 0.001 0.002 mU/L. At the present time, the most sensitive commercially available TSH assays are third-generation immunometric assays performed on automated immunoassay platforms. These assay improvements have established TSH as the first-line biochemical test to assess thyroid function in most clinical situations, as there is a log-linear relationship between circulating TSH and free T4 concentrations [327,328]. In commercially available TSH assays, the normal range is typically reported as between approximately 0.3 and 4.0 5.0 mU/L. Recent data cast doubts on this broad normal range, suggesting that the upper normal range is skewed by the inclusion of subjects with incipient thyroid dysfunction [329,330]. This leads to the conclusion that the true normal range is narrower, with an upper limit of normal of 3.0 4.0 mU/L. However, this assumption is controversial, with other data suggesting that TSH levels rise with age, thereby explaining the skewed upper limit of normal, and leading some experts to recommend age-specific reference ranges for serum TSH levels [331 334]. A relevant clinical question is whether variations in the TSH reference range correlate with important health outcomes. In this respect, longitudinal studies have clearly shown that TSH concentrations across the reference range predict the eventual development of thyroid dysfunction [335,336]. TSH levels above 2.0 mU/L correlate with an increased risk of incident hypothyroidism, while TSH levels below 0.4 mU/L are correlated with an increased risk of incident hyperthyroidism, compared to levels between 0.4 and 2.0 mU/L.

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A number of observational studies have emerged since 2005 indicating that variation in TSH levels within the euthyroid range are associated with other negative health outcomes [337]. In individual studies, highnormal TSH levels correlated with adverse cardiovascular, metabolic, and pregnancy effects, although a meta-analysis of individual participant data from 14 cohorts did not confirm an association between TSH levels within the reference range and coronary heart disease events or cardiovascular mortality [338]. It is also clear that low-normal TSH levels correlate with development of atrial fibrillation, osteoporosis, and fractures. Only one study simultaneously investigated a range of health outcomes across the TSH reference range in a single large population, concluding that high-normal TSH levels are associated with lower risk of multiple adverse events in older adults, including mortality [339]. No large prospective studies have yet demonstrated beneficial effects of treating patients with TSH levels at the lower or upper limits of the reference range [340]. Although the population normal range for serum TSH levels is relatively broad, within an individual subject TSH levels are more tightly regulated around an endogenous set-point. In a recent study of monthly sampling over a year in healthy euthyroid subjects, the significant difference in serum TSH levels on repeated testing was only 0.75 mU/L, far less than the population normal range [341]. It is not clear what determines this individual set-point, although studies of monozygotic and dizygotic twins suggest that it is primarily genetically determined [342]. Genetic analysis has revealed a number of significant linkage peaks, but no single gene appears to have a major regulatory influence, and the regulation of the TSH set-point is likely polygenic [205,343,344]. The main environmental factor that affects TSH levels in healthy euthyroid subjects appears to be iodine intake [345,346].

pituitary tumors [349], choriocarcinoma [350], and in a variety of nonpituitary and nonplacental malignancies including cancers of the lung, pancreas, stomach, prostate, and ovary [351,352].

Provocative Testing of TSH TRH directly stimulates TSH biosynthesis and secretion. Given intravenously, intramuscularly, or orally, TRH causes a reproducible rise in serum TSH levels in euthyroid subjects [178]. In euthyroid subjects, there is an immediate release of TSH rising to peak levels approximately 20 30 minutes after TRH injection, usually reaching values 5 10-fold higher than basal (Fig. 6.10). In hyperthyroid subjects, undetectable basal serum TSH levels correlate with absent TSH responses to TRH. Patients with low basal serum TSH levels secondary to pituitary or hypothalamic insufficiency have absent or attenuated TSH responses to TRH [353]. Patients with elevated TSH levels due to primary hypothyroidism have exuberant responses to TRH stimulation, while elevated TSH levels in patients with pituitary TSH-secreting tumors respond less than twofold to TRH stimulation.

Drugs and TSH Levels Among the most common causes of abnormal TSH levels are pharmacological interventions which alter TSH production. These can be divided into those that directly affect hypothalamic pituitary function, those

Free TSH β- and α-Subunit Measurements TSH β- and α-subunits were purified in 1974 from human TSH and specific antibodies to them developed [347]. Radioimmunoassays were first developed and then immunometric assays for the free α-subunit. In general, free TSHβ levels are detectable only in primary hypothyroidism and therefore are of limited utility. Free α-subunit levels have been useful in the evaluation of pituitary and placental disease. Free α-subunit is detectable and measurable in both euthyroid and eugonadal human subjects [348]. Elevated values of free α-subunit are found in the sera of patients with TSH-secreting or gonadotrophin-secreting

FIGURE 6.10 Schematic representation of thyrotrophin-releasing hormone (TRH) stimulation tests in patients with a variety of thyroid disorders. TRH was administered at time 0. Serum samples of TSH were collected at baseline and every 30 min for 3 h. Subjects with different disorders are indicated on the right. Source: Adapted from De Groot LJ, Jameson JL, editors. Endocrinology. 6th ed. Chapter 73, Thyroidstimulating hormone: physiology and secretion. Elsevier.

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that affect thyroid gland function, and those that alter the distribution of thyroid hormones between the free and protein-bound thyroid hormones in plasma. Only the drugs that directly affect TSH synthesis and/or secretion will be considered further here.

Drugs that Decrease Serum TSH Levels Clinically, the most important drug that results in decreased serum TSH levels is exogenously administered thyroid hormone. Twenty to thirty percent of patients treated with thyroid hormone have low serum TSH levels, most fitting the diagnostic criteria for subclinical hyperthyroidism. Thyroid hormone analogues such as TRIAC have the same effect in decreasing TSH secretion [354]. RXR analogues such as bexarotene, used in the treatment of cutaneous lymphoma, can decrease TSHβ transcription, serum TSH, and T4 levels with resultant central hypothyroidism [123]. Exogenous glucocorticoids, somatostatin and its analogues, and dopamine and its analogues all directly lower TSH production, as discussed in more detail in previous sections. Interestingly, although these drugs acutely decrease TSH production, chronic administration usually results in compensatory mechanisms that prevent clinical hypothyroidism from developing. Growth hormone administration stimulating IGF-1 production may decrease TSH levels by stimulation of endogenous hypothalamic somatostatin production [187]. Cytokine administration (interferon and interleukins) commonly suppresses TSH levels, which has been thought to be mediated through stimulation of endogenous glucocorticoids. However, a novel alternative mechanism postulates that cytokines stimulate hypothalamic NFκB production and this protein directly increases deiodinase 2 gene transcription in astrocytes leading to increased T4 to T3 conversion, TRH suppression, and central hypothyroidism [222 224]. A small study in patients with adrenal cortical carcinoma suggests that the adrenolytic agent mitotane may also impair TSH section [355]. Drugs affecting the serotonin pathway (the serotonin receptor antagonist cyproheptadine and the serotonin reuptake inhibitors sertraline and fluoxetine) have been reported to decrease TSH production in animal studies, but in human studies these drugs have not been shown to have a significant effect on TSH levels [356,357]. A similar lack of effect on the TSH level was found after administration of histamine receptor blockers (cimetidine and ranitidine) and benzodiazepines [358,359]. In contrast, the α-adrenergic blocker thymoxamine, used topically as an ophthalmologic agent, has been found to decrease TSH secretion when administered systemically [360].

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Drugs That Increase Serum TSH Levels A sustained increase in TSH production by direct stimulation of either the hypothalamus or pituitary is very unusual. TRH administration is the most potent but can be completely attenuated by subsequent rises in circulating thyroid hormones. The opioid class of drugs, including morphine, apomorphine, heroin, buprenorphine, and pentazocine, have all been associated with increases in TSH levels in humans, in contrast to decreased TSH levels seen in animal models [200,361]. Theophylline and amphetamines may directly stimulate hypothalamic TRH or pituitary TSH production [362,363]. The dopamine receptor antagonist metochlopramide increases TSH release by decreasing dopaminergic tone and thereby inhibiting tonic suppression of TSH by endogenous dopamine [190]. Exogenous leptin administration can stimulate hypothalamic TRH production resulting in higher TSH levels [213,216]. Certain neuroleptics, such as chlorpromazine, have been reported to increase TSH levels, although the circulating thyroid hormone levels are lower, suggesting a secondary effect [364].

DISORDERS OF TSH PRODUCTION Acquired TSH Deficiency TSH deficiency resulting in hypothyroidism (“central hypothyroidism”) can occur due to destructive processes in the anterior pituitary (“secondary hypothyroidism”) or hypothalamus (“tertiary hypothyroidism”) (Table 6.1). These destructive processes include infiltrative or infectious disorders, compressive neoplastic processes, and ischemic or hemorrhagic processes. The most common causes of acquired pituitary TSH deficiency are compression of normal anterior pituitary cells by a pituitary neoplasm, craniopharyngioma, or metastatic tumor [365]. These processes can also extend into the hypothalamus and interrupt normal TRH production. Multiple pituitary deficiencies are present, including LH, FSH, GH, and usually ACTH deficiency. Central hypothyroidism is manifested by low serum free T4 and T3 levels, generally in association with a low or normal basal TSH level, although in tertiary hypothyroidism the TSH may be minimally elevated [353], in which case the circulating TSH is biologically defective [366]. The 24-hour secretory profile of TSH in patients with tertiary hypothyroidism is also abnormal [169]. The frequency of the TSH pulses is the same as euthyroid controls, but the amplitude of the pulses is decreased, particularly at nighttime, resulting in a loss of the normal nocturnal surge (Fig. 6.7, bottom panel).

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Congenital TSH Deficiency Congenital causes of TSH deficiency (Table 6.1) include developmental abnormalities, such as midline defects and Rathke’s pouch cysts, which will not be discussed in this chapter, and genetic mutations. The latter may result in isolated TSH deficiency or may affect other pituitary hormones. Isolated TSH deficiency is generally inherited as an autosomal recessive disorder, and individuals affected with congenital hypothyroidism have severe mental and growth retardation. The molecular basis for isolated TSH deficiency has usually involved mutations in the TSHβ gene (Table 6.2). For example, a single base substitution in one family at nucleotide position 85 of the TSHβ gene altered the CAGYC region [367], a critically important contact point for the noncovalent combination of the TSH β- and α-subunits. In other kindreds, a single base substitution introduced a premature stop codon resulting in a truncated TSH β-subunit which included only the first 11 [391] amino acids. Another type of mutation involves a nonsense 25-amino-acid protein resulting from mutation of a donor splice site and a

TABLE 6.1 Causes of Central Hypothyroidism NEOPLASTIC Pituitary adenoma Craniopharyngioma Metastatic tumor Dysgerminoma Meningioma INFILTRATIVE Sarcoidosis Histiocytosis X Eosinophilic granuloma TRAUMATIC Radiation Head injury Postsurgical INFECTIOUS Tuberculosis Fungus Virus VASCULAR Stalk interruption Necrosis CONGENITAL Midline defects Rathke’s pouch cysts Genetic mutations

new out-of-frame translational start point [385,389]. In other cases, the disorder involves the production of biologically inactive TSH with loss of cysteine105 that disrupts the disulfide bridge formation important in the “seat-belt” stability [379,382], and is perhaps the most common of the TSH β mutations. There is also a less common mutation at cysteine85 that disrupts the cysteine knot that is important for heterodimer formation and TSH receptor binding [371,385], resulting in a similar phenotype, except that in some of these cases circulating TSH was detectable. TRH receptor mutations have also been described [392]. A more common cause of congenital TSH deficiency arises not as a result of a mutation in the TSHβ gene, but defective production of a key transcription factor necessary for TSHβ gene expression. This occurs in the syndrome known as CPHD. There are several types of CPHD. The first was described in subjects with congenital hypothyroidism and growth retardation secondary to TSH and GH deficiencies [393,394]. Mutations in the coding region of the Pou1F1 (Pit-1) gene alter the function of the Pou1F1 protein or completely disrupt its structure. The absence of Pou1F1 prevents normal pituitary development, resulting in hypoplasia of the pituitary and deficiency of TSH, GH, and prolactin that are dependent on the pituitary-specific transcription factor Pou1F1 for their expression. In heterozygotes, where a normal allele is present, the abnormal Pou1F1 protein can bind to DNA but is not able to effect transactivation, interfering with the function of the normal Pou1F1 (dominant negative mechanism). Interestingly, a similar combined hormone deficiency syndrome has been reported in two murine models in which the POU1F1 gene is defective: a point mutation found in the Snell dwarf (dw) [11] and a major deletion in the Jackson dwarf (dwJ) [395]. The second and more frequent type of CPHD is associated to mutations in the pituitaryspecific transcription factor called “prophet of Pou1F1” (PROP-1) [396,397]. Mutation of this paired-like homeodomain protein in the murine species causes the Ames dwarf (df) mouse phenotype [16]. Over 50% of families with CPHD have been shown to contain mutations in the PROP-1 gene [15], exceeding the prevalence of mutations in the POU1F1 gene. The mutations are all found in the homeodomain region of the molecule. Interestingly, the phenotype of patients with PROP-1 mutations includes deficiencies not only of GH, prolactin, and TSH, but also of LH and FSH. Furthermore, the hormone deficiencies may not be present at birth but rather progressively occur up to the age of adolescence. ACTH deficiency has also been reported as a late consequence in patients with PROP1 mutations [398]. Finally, mutations in other early developmental genes, including HESX1, SOX2, SOX3,

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TABLE 6.2 Congenital Hypothyroidism: Isolated TSH β Defects

Inheritance Syndrome

Serum Serum T4 TSH

TSH response to TRH

Nucleotide change

Protein defect

Reported cases

1

Autosomal recessive

Cretinism

k

None None detected detected

Exon 2, missense, G85A

G29R altered CAGYC region, no combination with α

Five families in Japan [83,367 369]

2

Autosomal recessive

Cretinism

k

None None detected detected

Exon 2, nonsense, G34T

E12 X premature stop (βL1 loop region) truncated TSHβ (11 amino acids)

Two families in Greece [370]

3

Autosomal recessive

Cretinism

k

Normal, Blunted k or or none none detected detected

Exon 3, deletion, T313del

C105Vfs114X altered seat-belt region and frameshift with premature stop codon (113 amino acids)

Over 10 families, in Brazil [255], Germany [371 377], Belgium [378], Switzerland [379], Argentina [380], Portugal [372], Francea [379], Poland [381], and the USA [382,383]

4

Autosomal recessive

Cretinism

k

k or Blunted none or none detected detected

Exon 3, nonsense, C145T

Q49X truncated TSHβ (48 amino acids)

Families in Egypt [384] Turkey [385], Greece [385,381], and Francea [372]

5

Autosomal recessive

Phenotypic variability, with moderate delay

k

k

Intron 2 donor splice site variant IVS2 1 5 G-A, leading to skipped exon 2 in mRNA

Silent change or nonsense protein of 25 amino acids, due to alternate exon 3 translation start site

Three families in Turkey [384,386,387]

6

Autosomal recessive

Cretinism

k

k or Blunted none detected

Exon 3, nonsense, C145T

C85R unstable or no combination with α

One case in Greece [385]

7

Autosomal recessive

Cretinism

k

None None detected detected

Exon 3, deletion, T169del

fs57X62, frameshift with One case in the USa [388] premature stop codon (62 amino acids)

8

Autosomal recessive

Phenotypic variability, with moderate delay

k

k

Blunted

Exon 2, 5’ donor splice site, c162 G-A, leading to skipped exon 2 in mRNA

Silent change (R34R), or One case in Argentina [389] nonsense protein of 25 amino acids, due to alternate exon 3 translation start site

9

Autosomal recessive

Cretinism

k

k

Blunted

Exon 3, Missense, G263A

C88Y Unstable or no combination with α

10 Autosomal recessive

Cretinism

k

None None detected detected

Blunted

Exon 1,2,3 and 5’ Absent TSH β protein untranslated region deletion

One case in Argentinaa [389] One case in Canada of Turkish origin [390]

a

Compound heterozygosity with T313del (C105Vfs114X).

LHX3, LHX4, and OTX2 [399,400] have also been associated with the CPHD syndrome.

Acquired TSH Excess Most cases of elevated serum TSH levels result from primary thyroid disease rather than primary pituitary disease. However, an important, although uncommon, cause is the TSH-secreting pituitary tumor. TSHomas comprise less than 1% of all pituitary tumors [401]. The patients have high levels of thyroid hormones in

association with normal or high levels of TSH. The tumor cells are quite differentiated but synthesize the α-subunit in excess of the TSH β-subunit [402], so that the molar ratio of α-subunit: TSH (ng/mL of α-subunit divided by μU/mL of TSH multiplied by 10) of greater than 1 supports the diagnosis of a TSH-secreting pituitary tumor when found in a hyperthyroid and eugonadal patient. This ratio is not accurate in menopausal women, who have high gonadotrophins and high free α-subunit levels. TSH-secreting tumors fail to respond to TRH stimulation and suppression by dopamine (Fig. 6.11). Another characteristic of these tumors is

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FIGURE 6.11 Thyroid-stimulating hormone (TSH) responses to various stimulation and suppression tests in a patient with a TSH pituitary tumor. Thyrotrophinreleasing hormone (TRH) (500 μg intravenously) gave no response, dopamine (4 μg/min for 4 h) resulted in no suppression, somatostatin (500 μg bolus followed by 250 μg/min for 4 h) resulted in significant suppression of serum TSH levels. Source: Adapted from De Groot LJ, Jameson JL, editors. Endocrinology. 6th ed. Chapter 73, Thyroid-stimulating hormone: physiology and secretion. Elsevier.

their failure to respond to thyroid hormone by the normal negative feedback of thyroid hormone on TSH production. In contrast, inhibition of TSH release in response to somatostatin is preserved in these tumors (Fig. 6.11), and somatostatin analogues have been used to control thyroid hormone excess and reduce tumor size in some patients [360,403].

Congenital TSH Excess Two interesting disorders resulting in elevated levels of serum TSH are resistance to thyroid hormone (RTH) [404,405] and resistance to TSH (RTSH) [406]. In 1967, Refetoff et al. [407] were the first to describe RTH in three siblings who were clinically euthyroid or hypothyroid with goiters, stippled epiphyses, and deaf mutism. Each of the children had elevated levels of protein-bound iodide which were subsequently shown to be associated with high serum total and free thyroid hormone levels, elevated TSH levels, and peripheral tissue responses that were refractory to not only the endogenous high levels of thyroid hormone, but also to exogenously administered supraphysiological levels of thyroid hormone [404]. RTH was found to be linked to the TRβ gene locus on chromosome 3, and was then localized to point mutations in the ninth and tenth exons of the TRβ gene which encode for the T3 binding and adjacent hinge domains. These mutations usually disrupt normal T3 binding without altering DNAbinding. Most cases of RTH are heterozygotes and inherited as autosomal dominant traits, with only half of the TRβ receptors being abnormal. The overwhelming majority of mutations are single-nucleotide substitutions which change a single amino acid or introduce a stop codon. Since 1967 over 3000 cases of RTH due to TRβ mutations, belonging to more than 450 families, have been identified [113]. However, only 171 different mutations have been identified, and many of these

have been proven to have developed independently in many unrelated families. In about 8% of the families with RTH, a TRβ mutation has not been identified, which may lead to difficulty in distinguishing RTH from TSHomas [408]. TRα gene mutations causing RTH were first reported in 2012 and 2013 with the description of four patients [409 411]. The phenotype is characterized by severe skeletal dysplasia and growth retardation unresponsive to T4 administration. TSH levels are normal with low/normal T4 and high/normal T3 concentrations. Mutations in the TRα gene in these subjects involve heterozygous nonsense or frameshift mutations resulting in expression of truncated TRα1 proteins. RTSH was first described [406] in three siblings with very high TSH levels, normal T4 and T3 levels, and thyroid glands of normal size. After excluding RTH with a normal response to T3 administration, sequencing of the TSH receptor revealed compound heterozygous mutations, with a different abnormal allele from each parent. Other mutations in the TSH receptor have since been found [271,412 414], with a prevalence of 29% in a group of 38 children with nonautoimmune subclinical hypothyroidism who were normal at neonatal screening [415].

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[364] Martinos A, Rinieris P, Papachristou DN, Souvatzoglou A, Koutras DA, Stefanis C. Effects of six weeks’ neuroleptic treatment on the pituitary thyroid axis in schizophrenic patients. Neuropsychobiology 1986;16:72 7. [365] Persani L. Central hypothyroidism: pathogenic, diagnostic, and therapeutic challenges. L Clin Endocrinol Metab 2012;97:3068 78. [366] Beck-Peccoz P, Amr S, Menezes-Ferreira NM, Faglia G, Weintraub BD. Decreased receptor binding of biologically inactive thyrotropin in central hypothyroidism: effect of treatment with thyrotropin-releasing hormone. N Engl J Med 1985;312:1085 90. [367] Hayashizaki Y, Hiraoka Y, Tatsumi K, Hashimoto T, Furuyama J, Miyai K, et al. DNA analyses of five families with familial inherited thyroid stimulating hormone (TSH) deficiency. J Clin Endocrinol Metab 1990;71:792 6. [368] Miyai K, Endo Y, Iijima Y, Kabutomori O, Hayashizaki Y. Serum free thyrotropin subunit in congenital isolated thyrotropin deficiency. Endocrinol Jpn 1988;35:517 21. [369] Mori R, Sawai T, Kinoshita E, Baba T, Matsumoto T, Yoshimoto M, et al. Rapid detection of a point mutation in thyroid-stimulating hormone beta-subunit gene causing congenital isolated thyroid-stimulating hormone deficiency. Jinrui Idengaku Zasshi 1991;36:313 16. [370] Dacou-Voutetakis C, Feltquate DM, Drakopoulou M, Kourides IA, Dracopoli NC. Familial hypothyroidism caused by a nonsense mutation in the thyroid-stimulating hormone betasubunit gene. Am J Hum Genet 1990;46:988 93. [371] Vuissoz J-M, Deladoey J, Buyukgebiz A, Cemeroglu GG, Gallati S, Mullis PE. New autosomal recessive mutation of the TSH-beta subunit gene causing central isolated hypothyroidism. J Clin Endocrinol Metab 2001;86:4468 71. [372] Karges B, LeHeup B, Schoenle E, Castro-Correia C, Fontoura M, Pfaffle R, et al. Compound heterozygous and homozygous mutations of the TSH beta gene as a cause of congenital central hypothyroidism in Europe. Horm Res 2004;62:149 55. [373] Doeker BM, Pfaffle RW, Pohlenz J, Andler W. Congenital central hypothyroidism due to a homozygous mutation in the thyrotropin beta-subunit gene follows an autosomal recessive inheritance. J Clin Endocrinol Metab 1998;83:1762 5. [374] Biebermann H, Liesenko¨tter KP, Emeis M, Oblanden M, Gru¨ters A. Severe congenital hypothyroidism due to a homozygous mutation of the betaTSH gene. Pediatr Res 1999;46:170 3. [375] Gru¨nert SC, Schmidts M, Pohlenz J, Kopp MV, Uhl M, Schwab KO. Congenital central hypothyroidism due to a homozygous mutation in the TSHβ subunit gene. Case Rep Pediatr 2011;2011:1 4. [376] Brumm H, Pfeufer A, Biebermann H, Schnabel D, Deiss D, Gruters A. Congenital central hypothyroidism due to homozygous thyrotropin beta 313deltaT mutation is caused by a founder effect. J Clin Endocrinol Metab 2002;87:4811 16. [377] Partsch CJ, Riepe FG, Krone N, Sippell WG, Pohlenz J. Initially elevated TSH and congenital central hypothyroidism due to a homozygous mutation of the TSH beta subunit gene: case report and review of the literature. Exp Clin Endocrinol Diabetes 2006;114:227 34. [378] Heinrichs C, Parma J, Scherberg NH, Delange F, Van Vliet G, Duprez L, et al. Congenital central hypothyroidism caused by a homozygous mutation in the TSH-beta subunit gene. Thyroid 2000;10:387 91. [379] Deladoey J, Vuissoz J-M, Domene HM, Malik N, GruneiroPapendieck L, Chiesa A, et al. Congenital secondary hypothyroidism due to a mutation C105Vfs114X thyrotropin-beta mutation: genetic study of five unrelated families from Switzerland and Argentina. Thyroid 2003;13:553 9.

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[380] Domene HM, Gruneiro-Papendieck L, Chiesa A, Iocansky S, Herzovich VC, Papazian R, et al. The C105fs114X is the prevalet thyrotropin beta-subunit gene mutation in Argentinean patients with congenital central hypothyroidism. Horm Res 2004;61:41 6. [381] Ramos HE, Labedan I, Carre´ A, Castanet M, Guemas I, Tron E, et al. New cases of isolated congenital central hypothyroidism due to homozygous thyrotropin beta gene mutations: a pitfall to neonatal screening. Thyroid 2010;20:639 45. [382] McDermott MT, Haugen BR, Black JN, Wood WM, Gordon DF, Ridgway EC. Congenital isolated central hypothyroidism caused by a “hot spot” mutation in the thyrotropin-beta gene. Thyroid 2002;12:1141 6. [383] Felner EI, Dickson BA, White PC. Hypothyroidism in siblings due to a homozygous mutation of the TSH-beta subunit gene. J Pediatr Endocrinol Metab 2004;17:669 72. [384] Bonomi M, Proverbio MC, Weber G, Chiumello G, BeckPeccoz P, Persani L. Hyperplastic pituitary gland, high serum glycoprotein hormone alpha-subunit, and variable circulating thyrotropin (TSH) levels as hallmark of central hypothyroidism due to mutations of the TSH beta gene. J Clin Endocrinol Metab 2001;86:1600 4. [385] Sertedaki A, Papadimitriou A, Voutetakis A, Dracopoulou M, Maniati-Christidi M, Dacou-Voutetakis C. Low TSH congenital hypothyroidism: identification of a novel mutation of the TSH beta-subunit gene in one sporadic case (C85R) and of mutation Q49stop in two siblings with congenital hypothyroidism. Pediatr Res 2002;52:935 40. [386] Pohlenz J, Dumitrescu A, Aumann U, Koch G, Melchior R, Prawitt D, et al. Congenital secondary hypothyroidism caused by exon skipping due to a homozygous donor splice site mutation in the TSH beta-subunit gene. J Clin Endocrinol Metab 2002;87:336 9. [387] Borck G, Topaloglu AK, Korsch E, Martine´ U, Wildhardt G, Onenli-Mungan N, et al. Four new cases of congenital secondary hypothyroidism due to a splice site mutation in the thyrotropin-beta gene: phenotypic variability and founder effect. J Clin Endocrinol Metab 2004;89:4136 41. [388] Morales AE, Shi JD, Wang CY, She JX, Muir A. Novel TSHbeta subunit gene mutation causing congenital central hypothyroidism in a newborn male. J Pediatr Endocrinol Metab 2004;17:355 9. [389] Baquedano MS, Ciaccio M, Dujovne N, Herzovich V, Longueira Y, Warman DM, et al. Two novel mutations of the TSH-beta subunit gene underlying congenital central hypothyroidism undetectable in neonatal TSH screening. J Clin Endocrinol Metab 2010;95:E98 103. [390] Hermanns P, Couch R, Leonard N, Klotz C, Pohlenz J. A novel deletion in the thyrotropin Beta-subunit gene identified by array comparative genomic hybridization analysis causes central congenital hypothyroidism in a boy originating from Turkey. Horm Res Paediatr 2014;82:201 5. [391] Borck G, Topaloglu AK, Korsch E, Martine U, Wildhardt G, Onenli-Mungan N, et al. Four new cases of congenital secondary hypothyroidism due to a splice site mutation in the thyrotropin-beta gene: phenotypic variability and founder effect. J Clin Endocrinol Metab 2004;89:4136 41. [392] Collu R, Tang J, Castagne´ J, Lagace´ G, Masson N, Huot C, et al. A novel mechanism for isolated central hypothyroidism: Inactivating mutations in the thyrotropin-releasing hormone receptor gene. J Clin Endocrinol Metab 1997;82:1561 5. [393] Rogol AD, Kahn CR. Congenital hypothyroidism in a young man with growth hormone, thyrotropin, and prolactin deficiencies. J Clin Endocrinol Metab 1976;39:356 63.

[394] Wit JM, Drayer NM, Jansen M, Walenkamp MJ, Hackeng WHL, Thijssen JHH, et al. Total deficiency of GH and prolactin and partial deficiency of thyroid stimulating hormone in two Dutch families: a new variant of hereditary pituitary deficiency. Horm Res 1989;32:170 7. [395] Behringer RR, Mathews LS, Palmiter RD. Dwarf mice produced by genetic ablation of growth hormone expressing cells. Genes Dev 1988;2:453 61. [396] Wu W, Cogan JD, Pfaffle RW, Dasen JS, Frisch H, O’Connell SM, et al. Mutations in PROP-1 cause familial combined pituitary hormone deficiency. Nat Genet 1998;18:147 9. [397] Fluck C, Deladoey J, Rutishauser K, Eble A, Mrti U, Wu W, et al. Phenotypic variability in familial combined pituitary hormone deficiency caused by a PROP-1 gene mutation resulting in the substitution of Arg . Cys at codon 120 (R120C). J Clin Endocrinol Metab 1998;83:3727 34. [398] Lamesch C, Neumann S, Pfa¨ffle R, Kiess W, Paschke R. Adrenocorticotrope deficiency with clinical evidence for late onset in combined pituitary hormone deficiency caused by a homozygous 301 302delAG mutation of the PROP1 gene. Pituitary 2002;5:163 8. [399] Kelberman D, Dattani MT. Hypopituitarism oddities: congenital causes. Horm Res 2007;68:138 44. [400] Diaczok D, Romero C, Zunich J, Marshall I, Radovick S. A novel dominant negative mutation of OTX2 associated with combined pituitary hormone deficiency. J Clin Endocrinol Metab 2008;93(11):4351 9. [401] Beck-Peccoz P, Persani L. Thyrotropinomas. Endocrinol Metab Clin North Am 2008;37:123 34. [402] Kourides IA, Ridgway EC, Weintraub BD, Bigos ST, Gershengorn MC, Maloof F. Thyrotropin-induced hyperthyroidism: use of alpha and beta-subunit levels to identify patients with pituitary tumors. J Clin Endocrinol Metab 1977;45:534 43. [403] Yamada S, Fukuhara N, Horiguchi K, Yamaguchi-Okada M, Nishioka H, Takeshita A, et al. Clinicopathological characteristics and therapeutic outcomes in thyrotropin-secreting pituitary adenomas: a single-center study of 90 cases. J Neurosurg 2014;121:1462 73. [404] Refetoff S, Weiss RE, Usala SJ. The syndromes of resistance to thyroid hormone. Endocr Rev 1993;14:348 99. [405] Refetoff S. Resistance to thyroid hormone: one of several defects causing reduced sensitivity to thyroid hormone. Nat Clin Pract Endocrinol Metab 2008;4:1. [406] Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S. Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 1995;332:155 60. [407] Refetoff S, DeWind LT, DeGroot LJ. Familial syndrome combining deaf-mutism, stippled epiphyses, goiter, and abnormally high PBI: possible target organ refractoriness to thyroid hormone. J Clin Endocrinol Metab 1967;27:279 94. [408] Macchia E, Lombardi M, Raffaelli V, Piaggi P, Macchia L, et al. Clinical and genetic characteristics of a large monocentric series of patients affected by thyroid hormone (Th) resistance and suggestions for differential diagnosis in patients without mutation of Th receptor β. Clin Endocrinol (Oxf) 2014;81:921 8. [409] Bochukova E, Schoenmakers N, Agostini M, Schoenmakers E, Rajanayagam O, et al. A mutation in the thyroid hormone receptor α gene. N Engl J Med 2012;366:243 9. [410] van Mullem A, van Heerebeek R, Chrysis D, Visser E, Medici M, et al. Clinical phenotype and mutant TRα1. N Engl J Med 2012;366:1451 3. [411] Moran C, Schoenmakers N, Agostini M, Schoenmakers E, Offiah A, Kydd A, et al. An adult female with resistance to

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C H A P T E R

7 Gonadotrophin Hormones Ursula B. Kaiser

INTRODUCTION The pituitary gonadotrophin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are heterodimeric glycoprotein hormones that play an essential role in the mammalian reproductive process. Synthesized in the gonadotrophs of the anterior pituitary, LH and FSH are secreted into the systemic circulation and act on the ovaries and testes to direct steroidogenesis and the final steps of gametogenesis. As these hormones are responsible for sexual maturation and normal reproductive function, the regulation of their synthesis and secretion is essential for the preservation of a species. LH and FSH are each composed of two distinct carbohydratecontaining protein subunits in noncovalent association, a common alpha (α) subunit and a distinct beta (β) subunit that bestow biologic specificity (LHβ and FSHβ, for LH and FSH, respectively). Befitting their important roles in endocrine physiology, the synthesis and secretion of LH and FSH are under complex regulation by counterbalancing stimulatory hypothalamic inputs (e.g., gonadotrophin-releasing hormone (GnRH)) and negative feedback from gonadal sex steroid and peptide hormones (e.g., estrogens, testosterone, progesterone, inhibins), with further paracrine modulation by local factors produced within the pituitary gland itself (e.g., activins, and follistatin) [1]. The integration of these signals results in the coordinated control of subunit gene expression, protein synthesis, and gonadotrophin secretion to promote sexual maturation and control normal reproductive function.

DEVELOPMENT, EMBRYOLOGY, AND HISTOLOGY Gonadotrophic Cells in the Pituitary The anterior pituitary cell types responsible for the synthesis and secretion of LH and FSH are known as The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00007-6

gonadotrophs and first appear in the anterior pituitary gland during early fetal development. Gonadotrophs constitute about 7 15% of anterior pituitary cells. In the human pituitary, gonadotrophs are dispersed throughout the pars distalis. Gonadotrophs are oval or irregular in shape, with a prominent nucleus. Electron microscopy reveals a spherical and eccentric nucleus, prominent, rough endoplasmic reticulum and Golgi complexes, and electron-dense secretory granules of two sizes. In studies of rat pituitary cells fractionated by elutriation, most of the gonadotrophin-staining cells are among the largest cells [2]. However, a significant number of cells among the poorly granulated small cell fractions also secrete gonadotrophins. Small gonadotrophs may represent the immature population that later gives rise to the highly responsive large secretory cells. For details of the morphology of the gonadotroph, the reader is referred to several classical reviews [3,4]. Immunocytochemical studies have demonstrated the presence of both bihormonal and monohormonal groups of gonadotrophs. Around 70% of gonadotrophs in the adult male rat pituitary contain both LH and FSH, 15% contain LH alone, and 15% FSH alone. The distribution of these three populations is dynamic and shifts under different physiological conditions, such as castration or throughout the estrous cycle. These findings suggest that monohormonal cells represent a similar cell type in different secretory phases. On the other hand, monohormonal gonadotroph populations may underlie differential LH and FSH release under different physiological conditions. Gonadotrophs cannot be distinguished on morphologic grounds. Castration leads to an increase in size as well as number of gonadotrophs that become morphologically distinct. These “castration cells” are characterized by large vacuoles in the cytoplasm due to dilation of the endoplasmic reticulum. Gonadotroph hyperplasia and hypertrophy following removal of the gonads

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whereas those required for the differentiation of only one cell lineage are associated with a single hormone deficiency. The list of transcription factors implicated in gonadotroph development and differentiation is rapidly growing, and a few key factors will be mentioned here (Fig. 7.1; Table 7.1). During pituitary development, Notch signaling is active early in pituitary organogenesis, signaling through HES family members [5]. HESX1 is one of the earliest markers of the pituitary primordium and also contributes to the development of the forebrain, the ventral diencephalon, and the hypothalamus. In humans, mutations in HESX1 have been identified and are associated with variable septo-optic dysplasia and hypopituitarism, ranging from CPHD including gonadotrophins to isolated growth hormone (GH) deficiency [8,9]. Mutations in SOX2 and SOX3, members of the SRY-related high-mobility group box (SOX) genes, are also associated with septo-optic dysplasia and anterior pituitary hypoplasia and HH [10]. PITX1 and PITX2, members of the class of bicoid homeodomain proteins, show a high degree of homology and are expressed in an overlapping pattern during pituitary development [6,11]. PITX1 is expressed in all five anterior pituitary lineages in both the fetal and adult pituitary gland and is able to activate the expression of all six major anterior pituitary hormones, including LH and FSH, frequently acting in synergy with other pituitary transcription factors [12]. Mutations in PITX2 cause Rieger syndrome, characterized by defects in the eyes, teeth, and heart, as well as pituitary hormone deficiencies [13]. FGF8 activates two key regulatory genes, LHX3 and LHX4, two LIM-type homeodomain transcription factors essential for pituitary development. Targeted deletion of either of these two genes in mice results in the failure of pituitary gland morphogenesis and an

may lead to an increase in the size of the sella. On the other hand, studies in patients with idiopathic hypogonadotrophic hypogonadism (HH) reveal only few poorly developed gonadotrophs.

Molecular Basis of Gonadotroph Development Pituitary development is discussed in detail in Chapter 1, Pituitary Development. The focus here will be on gonadotroph development and differentiation. Advances in genetic and molecular techniques have greatly increased our understanding of the mechanisms underlying pituitary development, and genetic analyses of mutations associated with developmental disorders of the pituitary in humans have begun to reveal the molecular mechanisms of pituitary development and cell lineage determination [5,6]. The anterior pituitary gland arises from midline cells in the anterior neural ridge, which form Rathke’s pouch, an oral ectodermal invagination, by the fourth to fifth weeks of gestation in response to inductive signaling from the ventral diencephalon. Coordinated spatiotemporal regulation of cell lineage-specific transcription factors expressed in pluripotential pituitary stem cells together with dynamic gradients of locally acting extrinsic signals regulate progenitor cell proliferation, lineage commitment, and terminal differentiation. Neuroectodermal signals important for pituitary morphogenesis include bone morphogenetic protein-4 (BMP-4), FGF8/10/18, and WNT5. Ventral developmental patterning is dictated by BMP2 and sonic hedgehog (SHH). Coordinated, temporal expression of a number of transcription factors directs the embryological development of the differentiated cell types. Mutations in transcription factors required for Rathke’s pouch formation, cell proliferation, or the differentiation of multiple lineages are associated with combined pituitary hormone deficiency (CPHD), Tbx19 LSD1

Melanotrophs

ER LSD1

Pitx1, 2 Lhx3, 4

TR

FGF10 Shh

-Catenin Prop1

Pit1+

Pitx2 Isl1 Lhx3, 4 Notch

Lactotrophs

FIGURE 7.1 Model for development of the human anterior pituitary gland and cell lineage determination, including gonadotroph differentiation. Source: From Zhu X, Wang J, Ju BG, Rosenfeld MG. Signaling and epigenetic regulation of pituitary development. Curr Opin Cell Biol 2007;19(6):605 11 [7].

Somatotrophs

Math3 LSD1 Gata2 LSD1 Thyrotrophs

Hes1 Pitx2

Tbx19 NeuroD1

SF1

Egr1 LSD1

Gonadotrophs

Corticotrophs

LSD1

Tbx19

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205

TABLE 7.1 Clinical Features Associated With Mutations in Genes Involved in Gonadotroph Development Transcription factor

Chromosome Inheritance Hormone deficiencies

Other features

HESX1

3p21

AR, AD

CPHD, IGHD

Septo-optic dysplasia

PITX2

4q25

AD

CPHD

Rieger’s syndrome

SOX2

3q27

AD (de novo)

HH, variable GHD

Anophthalmia/microphthalmia, esophageal atresia, genital tract abnormalities, hypothalamic hamartoma, sensorineural hearing loss, diplegia

SOX3

Xq26

XL

CPHD, IGHD

Mental retardation

LHX3

9q34

AD

GH, TSH, LH, FSH, PRL, ACTH Limited neck rotation, short cervical spine, sensorineural may be deficient deafness

LHX4

1q25

AD

CPHD (GH, TSH, ACTH deficiencies; variable gonadotrophin deficiency)

Cerebellar abnormalities

PROP1

5q35

AR

GH, TSH, LH, FSH, PRL Evolving ACTH deficiency

May show transient AP hyperplasia

OTX2

14q21

AD

IGHD or CPHD (GH, TSH, PRL, Bilateral anophthalmia, bilateral severe microphthalmia LH, FSH)

SIX6

14q22

AD

CPHD

Brachio-otorenal and oculoauriculo-vertebral syndromes

NR5A1

9q33

AD/AR

FSH, LH

Adrenal insufficiency, gonadal defects, XY sex reversal

NR0B1

Xp21.3

X-linked

FSH, LH

Adrenal hypoplasia congenital, gonadal defects, XY sex reversal

AR, autosomal recessive; AD, autosomal dominant; XL, X-linked; CPHD, combined pituitary hormone deficiency; IGHD, isolated growth hormone deficiency; HH, hypogonadotrophic hypogonadism; AP, anterior pituitary.

aplastic or hypoplastic pituitary with reduced numbers of all cell types. Mutations in LHX3 and LHX4 have been identified in patients with CPHD including impaired gonadotrophin release [6,8,10]. Mutations in PROP1 (Prophet of Pit1) are the most common genetic cause of CPHD [5]. PROP1 is a member of the paired-like family of homeodomain transcription factors, expressed early in Rathke’s pouch. Gonadotroph differentiation is also impaired, and homozygous females and most males are infertile. Most patients with PROP1 mutations develop GH and thyroid-stimulating hormone (TSH) deficiency in childhood, whereas the reproductive phenotype is more variable, with some presenting as pubertal failure in adolescence and others developing hypogonadism later in life. Corticotroph insufficiency can also develop later in life in some patients. In contrast to PROP1 mutations, mutations in PIT1, also a member of the POU homeodomain family, have pituitary deficiencies limited to GH, prolactin, and TSH [6]. Steroidogenic factor-1 (SF-1) is a member of the nuclear receptor family that is expressed throughout the reproductive axis (hypothalamus, pituitary, and gonads) and in the adrenal gland. It is a key transcriptional regulator of many genes involved in sexual differentiation, steroidogenesis, and reproduction, including the pituitary

gonadotrophin α-subunit, LHβ, FSHβ, and GnRHR genes [14]. Patients with mutations in NR5A1, the gene encoding SF-1, have been described with varying degrees of XY sex reversal, testicular dysgenesis, ovarian insufficiency, adrenal failure, and impaired pubertal maturation. DAX1 is a related nuclear receptor transcription factor with a similar distribution pattern of expression. Mutations in this X-linked gene cause primary adrenal insufficiency and HH [14]. A role for DAX-1 in gonadotroph development has not been established; the major role for DAX-1 appears to be, paradoxically, as a repressor of SF-1mediated transcription. In addition to the genes discussed here, the list of transcription factors implicated in gonadotroph development and differentiation and in control of gonadotrophin gene expression, such as GATA2, is growing. The importance of epigenetic modification of DNA and histones in cellular differentiation during development is becoming increasingly recognized. Distinct cell types, arising from common progenitors, share an identical genetic composition, yet establish unique profiles of gene expression and cellular function [7]. The field of epigenetics has advanced greatly in recent years, with the identification of enzymes and protein complexes that catalyze DNA methylation and histone modifications, including acetylation, methylation,

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phosphorylation, and ubiquitination [15]. LSD1, a histone demethylase important for chromatin modification and transcriptional regulation, is expressed in the pituitary throughout development and has been implicated in pituitary and gonadotroph development [16]. LSD1 interacts with PIT1 to regulate activation of PIT1 target genes and also attenuates Notch1 signaling, thereby establishing LSD1 as a functional component of both coactivator and corepressor complexes to regulate activation and repression programs important for terminal differentiation. The targeted deletion of LSD1 from the pituitary resulted in reduced levels of GH, TSHβ, LHβ, and POMC, markers of somatotroph, thyrotroph, gonadotroph, and corticotroph differentiation, respectively, and implicating LSD1 in the terminal differentiation of all pituitary cell types. Stem cells and other progenitor cells were long thought to be restricted to embryonic tissues in most cell types. However, during the last two decades, stem cells are being identified in an ever-increasing number of adult organs and tissues, including those long considered to be postmitotic with negligible regenerative potential. Plasticity such as lactotroph hyperplasia during pregnancy or thyrotroph hyperplasia during primary hypothyroidism support regenerative properties of the pituitary, including gonadotroph. Evidence has emerged supporting Sox2, Sox9, Oct4, and nestin as markers of pituitary progenitors with properties for multipotent pituitary cell differentiation [17,18]. Expression of potential stem-cell markers such as nestin in the marginal zone around Rathke’s cleft has suggested that the stem-cell population may exist in this area. Cell lineage tracing analysis has demonstrated that Sox2- and Sox9-expressing progenitors can selfrenew and give rise to pituitary endocrine cells in vivo, supporting the model of these as tissue stem cells. Moreover, these cells can become mobilized and differentiate toward a specific cell fate in response to physiological stress [19]. These concepts open new avenues for the application of pluripotent stem cells to treat hypopituitarism [20,21]; stem cells may also play a role in the pathogenesis of pituitary adenomas.

BIOCHEMICAL STRUCTURE AND MOLECULAR BIOLOGY OF LH AND FSH Hormone Structure The gonadotrophins belong to a family of dimeric glycoprotein hormones that includes LH, FSH, TSH, and placental chorionic gonadotrophin (CG). Each of these hormones is heterodimeric, consisting of two different noncovalently associated subunits, an α- and a β-subunit (Fig. 7.2). The α-subunit is common to all

FIGURE 7.2 Schematic depiction of the subunit structure and glycosylation sites of the four human glycoprotein hormones and their subunits. Asn-linked oligosaccharides are depicted as and Ser/Thr-linked oligosaccharides as . Source: From Baenziger J. The asparagine-linked oligosaccharides of the glycoprotein hormones. In: Chin WW, Boime I, editors. Glycoprotein hormones Norwell: Serono Symposia; 1990. p. 1 8 [22].

members of this family, whereas each β-subunit has a different amino acid sequence and confers biologic specificity [23]. The α- and β-subunits are encoded by different genes located on separate chromosomes. The common α-subunit contains 92 amino acids, while the β-subunits of FSH, LH, and CG contain 111, 121, and 145 amino acids, respectively (Table 7.2) [24]. Both subunits are glycosylated at specific residues. Significant homology among subunits suggests that they evolved from a common ancestral gene. The individual subunits have no known biological activity; heterodimerization is essential to confer hormonal bioactivity. The β-subunit is the rate-limiting factor in the biosynthesis of LH and FSH, and the α-subunit is present in excess. As a result, intact dimeric glycoprotein hormones and free α-subunits are present in the circulation, but free β-subunits are rarely found. Each subunit is cysteine-rich and highly linked internally by disulfide bonds. The location of the cysteines, in large part, confers the three-dimensional structure of the glycoprotein by determining the folding [24]. The two subunits are noncovalently associated, dependent on a unique “seat-belt” arrangement provided by the disulfide pairing between highly conserved cysteine residues within each subunit. The regions of similarity between different β-subunits are felt to be involved in binding to the α-subunits and the variable regions in specific receptor binding. Each subunit also bears carbohydrate moieties that contribute to biologic activity and metabolic fate of the glycoprotein hormones.

The α-Subunit The human α-subunit gene (GNAS) is located on the short arm of chromosome 6 and consists of four exons and three introns (Fig. 7.3; Table 7.2). The gene

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FIGURE 7.3 Schematic representation of the human gonadotrophin subunit genes. The top part of each scheme depicts the gene structure. Open bars indicate noncoding sequences; solid bars indicate coding sequences. The bottom part of each scheme shows the protein structure. Signal peptides are shaded, while the mature peptide is depicted by an open bar. The positions of N-linked glycosylation sites are depicted by triangles, and O-linked glycosylation by circles. Amino acid positions are depicted by numbers. Source: From Themmen APN, Huhtaniemi IT. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev 2000;21(5):551 83.

TABLE 7.2 Structures of Glycoprotein Hormone α, Thyroid-Stimulating Hormone β, Luteinizing Hormone β, Human Chorionic Gonadotropin β, and Follicle-Stimulating Hormone β Gene length (kb)

Number of exons

mRNA length (kb)

Number of amino acids

Common α 6p21.1 23

9.4

4

0.8

92

TSHβ

1p22

4.9

3

0.7

118c

1 (N: 23)

LHβ

19q13.3

1.5

3

0.7

121

1 (N: 30)

CGβ

19q13.3

1.9

3

1.0

145

6 (N: 13, 30; S: 121, 127, 132, 138)

FSHβ

11p13

3.9

3

1.8

117

2 (N: 7, 24)

Subunit

Gene locus

a

Number of glycosylation sites (location)a 2 (N: 52, 78)b

Oligosaccharide chains are attached either to asparagine (N) (N-linked) or to serine (S) (O-linked). N or S residues are numbered according to their position in the respective sequence. Free α-subunit may also contain an additional site of O-glycosylation at threonine (T) 39. c 118-amino-acid coding region; six amino acids can be cleaved at the C-terminal end. b

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encodes for an mRNA 0.8 kilobase (kb) in length. This mRNA encodes a precursor peptide with a molecular mass of 14 kD, comprised of a 24-amino-acid signal peptide followed by a 92-amino-acid protein in humans. The gene is expressed in gonadotrophs and thyrotrophs of the anterior pituitary gland and in the placenta. A striking feature of the mature α-subunit is the presence of 10 highly conserved cysteine residues, which form five disulfide bonds critical to the tertiary “cystine knot” structure of the mature protein. This cystine knot motif identifies the glycoprotein hormones as members of the cystine knot growth factor superfamily, which also includes the nerve growth factor, platelet-derived growth factor, and transforming growth factor (TGF)-β families. The resultant tertiary structure enables heterodimerization with β-subunit partners as well as ligand receptor interaction. In addition to the cysteines, the mature α-subunit also bears two asparagine-linked oligosaccharides, at amino acid residues 56 and 83 [22].

The LHβ Subunit The LH/CGβ subunit genes (LHB, CGB) form a cluster with a complex organization [24]. The human LHβ subunit is encoded by a member of a cluster of seven genes, which also includes the gene coding for the CGβ subunit. This gene cluster evolved by gene duplication, and the other five copies are thought to be pseudogenes. The LHB gene is expressed in the pituitary gonadotroph with high specificity, whereas the CGB gene is expressed primarily in the placenta, limited to certain mammalian species such as humans, nonhuman primates, and horses. The LHB gene is relatively small in size, only B1.5 kb. The general organization of the LHB gene, with three exons and two introns, is similar to other glycoprotein hormone β-subunit genes (Table 7.2; Fig. 7.3). Several distinctions are noticeable between the LHB and CGB genes. First, the CGB genes are present only in primate and equine species, whereas LHB genes are present in all vertebrates examined. Second, the LHB gene encodes a 145-amino-acid precursor protein, cleaved to produce a 24-amino-acid signal peptide followed by a 121-amino-acid mature LHβ subunit. In contrast, the mature CGβ protein is 145 amino acids in length and does not include a signal peptide, but rather contains a 24-amino-acid carboxyterminal extension [24]. Third, the LHB and CGB genes have different transcriptional start sites and utilize different promoters, accounting for their different tissue distribution patterns of expression. Nonetheless, like all members of the glycoprotein hormone β-subunit family, the LHβ subunit has a highly conserved backbone of 12

cysteine residues to form six disulfide bonds. The LHβ subunit also contains one glycosylation site, whereas the CGβ subunit contains two. The CGβ subunit contains four additional serine O-linked oligosaccharide units in its carboxyterminal extension, which influence processing of the subunit and are important for maintaining the longer biologic half-life of human chorionic gonadotrophin (hCG). The amino acid sequences of the human LHβ and CGβ subunits share 82% homology, and when associated with the α-subunit, these two hormones activate the same receptor.

The FSHβ Subunit The human FSHβ subunit is encoded by a single gene located on the short arm of chromosome 11. The organization of the FSHB gene is similar to that of other glycoprotein hormone β genes in that it has three exons and two introns [24]. The first exon contains only 5’-untranslated sequences, while the second and third exons contain the entire coding sequence (Table 7.2; Fig. 7.3). The third exon also contains a relatively long (1 1.5 kb) 3’-untranslated sequence. The human FSHβ subunit includes a signal peptide of 19 amino acids followed by a mature peptide of 111 amino acids, with two glycosylation sites (Asn7 and Asn24). These N-glycans on the FSHβ subunit are essential for efficient dimerization with the α-subunit and for FSH secretion [25]. Like LHβ, the FSHB gene is expressed only in pituitary gonadotrophs. Interestingly, differences in the LHβ and FSHβ peptide sequence seem to influence the pathway and pattern of hormone secretion. A gonadotroph-specific sorting determinant on LH was identified that, if added to the FSHβ peptide, diverts FSH to the LH secretory pathway and enhances ovarian responses in vivo [26].

Synthesis and Posttranslational Processing of LH and FSH The subunit precursors are processed by enzymatic removal of amino terminal leader peptides and also by addition of carbohydrates. The α-subunit contains two asparagine-linked carbohydrate chains, while the β-subunit chains contain one or two. In addition, O-linked oligosaccharides are added to serine or threonine residues of the free α-subunit and the carboxyterminal extension of the CGβ subunit (see Figs. 7.2 and 7.3). Tissue- and protein-specific differences occur in the processing of glycoprotein hormones, and variations can occur even for a given hormone. As a result, several isomeric forms of LH and FSH can be produced. For example, shifts to more biologically active isoforms of circulating

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gonadotrophins occur at the onset of puberty or at specific phases of the menstrual cycle. These complex oligosaccharide side chains influence hormone metabolic clearance rates and bioactivity [23,27]. Predominantly sulfated oligosaccharides have shorter plasma halflives than those with sialylated oligosaccharides, likely contributing to the longer circulating half-life of FSH (1 4 hours) compared to LH (10 50 minutes). The carboxyterminal sequence of the CGβ subunit with its O-linked glycosylation prolongs the half-life and in vivo bioactivity of hCG compared to other glycoprotein hormones. Fusion of this sequence to the other subunits, such as LHβ or FSHβ, in a region that is not important for receptor binding or signal transduction, does not interfere with subunit folding or assembly, secretion, receptor binding, or in vitro bioactivity of the dimers. Nonetheless, the presence of this sequence significantly increases in vivo bioactivity and half-lives of the engineered chimeras [28].

ONTOGENY AND PHYSIOLOGY OF LH AND FSH SECRETION Fetal Life GnRH is present in the fetal hypothalamus as early as 6 weeks of gestation [29]. The fetal pituitary contains measurable amounts of LH and FSH by 10 weeks, and gonadotrophins are first detectable in the human fetal circulation by weeks 12 14 [30]. Their biosynthesis at this early stage of development appears to be at least partially dependent on GnRH, as reflected by the absence of LHβ and FSHβ in the pituitary glands of anencephalic infants. Serum levels of LH and FSH rise gradually to a peak at about 20 weeks. In the second half of pregnancy, serum LH and FSH levels in the fetus decline progressively, likely the result of the rise in sex steroid secretion by the fetal gonad, rising maternal estrogen levels, and the development of negative-feedback mechanisms [29,30]. Placental hCG plays a significant role in stimulating androgen production by the fetal testis in early pregnancy. High androgen levels are required for differentiation of Wolffian structures in the male. In addition, FSH stimulates differentiation and development of seminiferous tubules. These data are consistent with observations that patients with HH have normal differentiation of Wolffian structures and external genitalia because the placental hCG drives the fetal testis to produce sufficient androgen even in the absence of pituitary LH and FSH. However, because of FSH deficiency, these patients have impaired development of seminiferous tubules. On the other hand, testicular descent is partially dependent on androgen levels

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during the later period of fetal life, which are maintained by LH derived from the fetal pituitary. As a result, patients with HH often have associated cryptorchidism, or undescended testes [31]. Thus a thorough understanding of the ontogenetic changes in gonadotrophin secretion can be helpful in defining the pathophysiology of the disorders of sexual differentiation.

Postnatal Life and Childhood Years After birth, serum LH and FSH levels rise again, and in the first 6 months of postnatal life, LH and FSH are measurable in blood. Serum LH and FSH levels peak around 2 3 months of age and then decline to prepubertal levels by about 6 months of age in boys and 1 2 years of age in girls [31,32]. This brief period of postnatal life thus provides a unique, albeit narrow, window in which the integrity of the hypothalamic pituitary gonadal axis can be assessed before gonadotrophin and sex steroid levels fall to the low range of childhood. During childhood, the hypothalamic pituitary gonadal axis remains quiescent until the onset of puberty [29 32]. While the pituitary and the testes retain the ability to respond to GnRH and LH, respectively, the response of the prepubertal pituitary to a GnRH stimulus is relatively dampened. In addition, the GnRH-induced rise in serum FSH in prepubertal children is greater than that in LH, in contrast to an adult in whom a single dose of GnRH causes a greater rise in LH. FSH levels tend to exceed LH before puberty. During pubertal maturation, serum LH and FSH levels rise progressively, with substantial overlap among the various pubertal stages [33]. Nocturnal, sleep-entrained pulsatile secretion of LH is characteristic of early stages of puberty. Reactivation of the hypothalamic GnRH pulse generator at the time of puberty results in progressive increases in the amplitude and frequency of gonadotrophin pulses, initially nocturnally and then during the daytime as puberty progresses. The quiescence of the GnRH pulse generator and of circulating LH and FSH levels during childhood and their reactivation at the time of puberty are independent of gonadal function, as reflected in the patterns observed in agonadal humans and monkeys (Fig. 7.4) [34]. The preservation of this developmental pattern in agonadal monkeys and human subjects led to the generation of a model of central inhibition or restraint to account for the prepubertal reduction in pulsatile GnRH release, followed by a pubertal increase in GnRH resulting from either removal of an inhibitory input or development of a stimulatory signal to the GnRH neuronal network, or a combination of both [35].

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FIGURE 7.4 The on off on pattern of gonadotrophin-releasing hormone pulse generator activity during postnatal development in agonadal male (stippled area) and female (closed data points 6 error bars) rhesus monkeys as reflected by circulating mean LH (top panel) and FSH (bottom panel) concentrations from birth to 142 166 weeks of age. Note that in females, the intensity and duration of the prepubertal hiatus in the secretion of FSH, and to a lesser extent LH, is truncated in comparison to males. Source: From Plant TM. Hypothalamic control of the pituitary-gonadal axis in higher primates: key advances over the last two decades. J Neuroendocrinol 2008;20(6):719 26.

Gonadotrophin Secretion During the Menstrual Cycle Both males and females secrete gonadotrophins in a pulsatile fashion during adulthood, but in very different patterns. In the adult male, wide variations in LH interpulse intervals have been reported, with an average 2-hour frequency [30]. In the female, the reproductive axis is under more dynamic regulation, with a cyclical pattern of intricate changes in gonadotrophin secretion, ovarian sex steroid secretion, and responses of the endometrium and reproductive tract to these hormonal changes constituting the menstrual cycle (Fig. 7.5). Menses serve as a useful clinical marker and, by tradition, the first day of the bleeding is designated day 1 of the human menstrual cycle. The cycle length is usually between 25 and 30 days. Extensive reviews of the hormonal and histologic changes during the menstrual cycle have been published. This section will focus primarily on changes in the gonadotrophins. During the early follicular phase, serum FSH levels are relatively higher but LH, estradiol, and progesterone levels are low. This relative preponderance of FSH in the early part of the cycle is felt to be important in

FIGURE 7.5 The menstrual cycle. Changes in (A) hypothalamic pulsatile GnRH release lead to (B) changes in serum levels of LH and FSH, inducing (C) ovarian follicle maturation and (D) changes in estradiol and progesterone secretion, in turn leading to changes in the endometrial lining during the menstrual cycle. Source: From http://www.soc.ucsb.edu/sexinfo/article/the-menstrual-cycle.

the recruitment and maturation of a cohort of ovarian follicles, one of which will eventually ovulate [36]. Suppression of plasma FSH during the early follicular phase delays the development of the dominant follicle and prolongs the follicular phase, whereas supraphysiologic doses of FSH can lead to simultaneous development of several follicles. The mechanisms that lead to selection of the dominant follicle and atresia of all others are not fully understood. FSH promotes follicular growth and estradiol production and induces LH receptors on granulosa cells [24]. Serum estradiol levels gradually rise as the follicular phase progresses and follicular growth occurs. Increasing estradiol levels suppress serum FSH levels. In the late follicular phase, serum estradiol levels begin to rise rapidly. The positive-feedback effects of the high late-follicular-phase estradiol levels result in the mid-cycle LH surge [37]. The mid-cycle FSH peak is of a smaller magnitude than the LH peak. After ovulation, the FSH levels decrease and remain low during

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ONTOGENY AND PHYSIOLOGY OF LH AND FSH SECRETION

the luteal phase. In contrast, LH is essential for maintaining corpus luteum function; LH stimulates the production of progesterone and estradiol by the luteinized follicle [38]. In the absence of fertilization, corpus luteum function declines, as reflected by decreasing progesterone and estradiol levels during the latter part of the luteal phase. Serum FSH levels then begin to rise, partly in response to a decrease in serum levels of estradiol and progesterone levels, initiating events for the next cycle. The pattern of LH pulses has been well characterized during different phases of the menstrual cycle and these data have been used to derive inferences about the hypothalamic GnRH secretion. LH pulses have an interpulse interval of 1 2 hours during the early follicular phase. The LH pulse frequency increases during the late follicular phase, but then the pulse generator slows markedly during the luteal phase; the interpulse interval may range from 2 to 6 hours [30]. Changes in the expression of gonadotrophin subunit genes have been examined during the rat estrous cycle [1]. In the female rat, the estrous cycle has an average length of 4 days. Serum LH and FSH concentrations are low throughout the cycle except for the surge on the late afternoon and evening of proestrus. LHβ- and α-subunit mRNAs change little on the day of metestrus, but increase twofold during diestrus. LHβ mRNA levels increase threefold before the preovulatory rise in serum LH, but α mRNA levels remain unchanged during the proestrus gonadotrophin surges. FSHβ mRNA levels increase during the metestrus morning, falling to basal levels by metestrus evening. On the afternoon of proestrus, FSHβ mRNA concentrations increase but the maximal expression of FSHβ mRNA is seen 2 hours after the proestrus FSH surge. Thus, the changes in the expression of the three gonadotrophin subunit genes do not tightly correlate with the circulating LH and FSH levels.

Aging and Gonadotrophins Serum testosterone levels decline progressively in men with advancing age (Fig. 7.6) [39]. Furthermore, the sex hormone-binding globulin (SHBG) levels increase with age, resulting in a greater decrease in free and bioavailable testosterone than total testosterone. The diurnal rhythm of testosterone secretion, observed in younger men, may be attenuated or lost in older men. There is also an increase in circulating estradiol and estrone levels with age due, in part, to the increased peripheral aromatization of androgen to estrogen. Aging-associated declines in testosterone levels occur due to defects at all levels of the hypothalamic pituitary gonadal axis. Androgen secretion by

211

the testis of elderly men is decreased due to primary abnormalities at the gonadal level. In addition, secondary defects may exist at the hypothalamic pituitary level, with more irregular secretion of LH and less synchronicity between LH and testosterone secretion than younger men. Therefore, aging is associated with abnormalities of the normal feedback control mechanisms that control the flow of information between different components of the hypothalamic pituitary testicular network, and a disruption of the orderly pattern of pulsatile hormonal secretion [40]. Aging also has dramatic effects on the reproductive system in women [41,42]. The reproductive system is one of the first biological systems to show age-related decline. Certainly the most notable changes in the hypothalamic pituitary gonadal axis arise from the decline in ovarian function, and thus the loss of negative-feedback effects on the hypothalamus and pituitary. Reproductive aging in women is related to the depletion of a fixed number of germ cells within the ovary, reflected in a decline in serum antimu¨llerian hormone levels, a marker of ovarian reserve [43]. A progressive decrease in inhibins also occurs and results in an early increase in FSH, which initially maintains folliculogenesis and estradiol secretion. Over time, regular ovulatory cycles give way to inconsistent folliculogenesis and ovulation, fluctuations in estradiol and gonadotrophin levels, and irregular cycles. Most of the decrease in estradiol and increase in FSH associated with menopause occurs during the late menopausal transition. While depletion of ovarian follicles clearly relates to the end of reproductive function in females, evidence is accumulating that neuroendocrine changes can precede the decline in ovarian function and a hypothalamic defect is critical in the transition from cyclicity to acyclicity [44]. The central nervous system (CNS) does not respond normally to estrogen and fails to produce preovulatory LH surges in women in the early transition. Changes in the sensitivity to estrogen positive feedback for the ovulatory LH surge may contribute to cycle disruption. Decreases in hypothalamic dopamine, norepinephrine, glutamate, vasoactive intestinal polypeptide, and insulin-like growth factor-1 (IGF-1) have been reported [45]. It has been postulated in humans that repercussions of anovulatory menstrual cycles, precipitated by loss of hypothalamic responsiveness to estradiol (E2) during perimenopause, establish the dysregulation of ovarian trophic support by FSH, activin, and inhibin that cascades into accelerated ovarian decline and menopause. Kisspeptin expressed in neurons in the hypothalamic anteroventral periventricular (AVPV) nucleus has been implicated as a mediator of E2positive feedback [46]. Indeed, the age-related decline

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FIGURE 7.6 Age-related changes in testosterone secretion. (A) Effects of age on serum T, SHBG, and free T index. Individual data points

for T (upper panel), SHBG (middle panel), and free T index (lower panel) against age. Best-fit regression lines, r2 and p values are shown. Total T concentrations and free T index values decreased linearly with increasing age, whereas SHBG exhibited a curvilinear increase with age, rising at a slightly greater rate in the older, than in younger, men. (B) Longitudinal effects of aging on T and free T index. Linear segment plots for total T and free T index versus age are shown for men with T and SHBG values on at least two visits. Numbers in parentheses represent the number of men in each cohort. With the exception of free T index in the ninth decade, segments show significant downward progression at every age, with no significant change in slopes for T or free T index over the entire age range. Source: From Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab 2001;86(2):724 31.

of the LH surge corresponds to the decreased sensitivity of AVPV kisspeptin neurons to E2-positive feedback [47]. Blunting of pituitary expression of the GnRH receptor and the gonadotrophin subunits in middle-aged rats has also been reported, suggesting that age-related changes in pituitary physiology may also contribute to reproductive senescence [48]. Studies in older postmenopausal women also indicate that changes in the neuroendocrine axis occur with aging that are independent of the changing ovarian hormonal milieu of the menopausal transition. LH and FSH decrease progressively after the menopause, as does GnRH pulse frequency [41]. Clinically, it is important to appreciate that the entire reproductive system, not just the ovary, undergoes change across the transition [49].

BIOLOGIC FUNCTIONS OF LH AND FSH Roles of LH and FSH in the Male The target organs of LH and FSH are the gonads. In the male, the primary role of LH is to stimulate testosterone biosynthesis by Leydig cells (Fig. 7.7). LH stimulates the activity of steroidogenic enzymes, including CYP cholesterol-side-chain cleavage enzyme and CYP 17α-hydroxylase in Leydig cells, which are required for testosterone synthesis [52]. LH is required for maintaining high intratesticular levels of testosterone, essential for spermatogenesis. Circulating testosterone is also essential for maintaining sexual function, secondary sexual characteristics, and other androgendependent physiologic processes such as bone,

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BIOLOGIC FUNCTIONS OF LH AND FSH

FIGURE 7.7 Functions of LH and FSH in the male and female. Source: Modified from Richards JS, Pangas SA. The ovary: basic biology and clinical implications. J Clin Invest 2010;120(4):963 72 [50] and Layman LC. Genetics of human hypogonadotropic hypogonadism. Am J Med Genet 1999;89(4):240 8 [51].

mineral, and protein metabolism and muscle mass and function [24]. FSH in the male is responsible for the initiation of spermatogenesis (Fig. 7.7). FSH binds to specific receptors on Sertoli cells and stimulates the production of inhibins, transferrin, androgen-binding protein, androgen receptor (AR), and a glutamyl transpeptidase. FSH is felt to be essential for spermiogenesis, i.e., the maturation process by which spermatids develop into mature spermatozoa, through effects mediated on Sertoli cells. In rats and primates, testosterone alone can maintain spermatogenesis when administered shortly after hypophysectomy or stalk resection. However, if testosterone is given after a lapse of several weeks to months, it is much less effective in re-initiating spermatogenesis. FSH is required for initiating the spermatogenic process but once this has occurred, testosterone in high doses can maintain spermatogenesis. Even though FSH and testosterone have independent roles in spermatogenesis, they also act in a cooperative manner to promote quantitative spermatogenesis. Both FSH and testosterone act in a stage-dependent manner and act at different cellular sites during spermatogenesis in order to optimize the spermatogenic process. Thus, the combination of testosterone and FSH is more effective than testosterone alone in spermatogenesis [53].

The Roles of LH and FSH in the Female Although many of the early stages of follicle growth occur independent of pituitary gonadotrophins, FSH is required for granulosa cell differentiation and follicular growth (Fig. 7.7) [50]. In the ovary, granulosa cells

213

are the only target cells of FSH action. FSH receptors are acquired by granulosa cells in the early stages of their differentiation and FSH is important in the differentiation of granulosa cells. FSH plays a critical role in follicle growth and is responsible for the development of a mature follicle, although a number of growth factors also play important roles in stimulating granulosa cell mitosis. The initiation of follicular growth can occur independent of gonadotrophin stimulation, after which further maturation requires FSH. FSH receptor is only expressed from the primary follicle onward, and in the absence of FSH follicular growth can occur only up to the stage of secondary recruitment. Only at more advanced stages of development do follicles become responsive to FSH and obtain the capacity to convert the theca-cell-derived substrate androstenedione to E2 by the induction of the aromatase activity [54]. FSH is, therefore, critical in regulating estrogen production in the ovary. During the later stages of follicular growth, activins and estrogen enhance the actions of FSH [55,56]. FSH also controls granulosa cell production of inhibins during the follicular phase and also modulates LH receptor expression in granulosa cells. FSH not only promotes the development of the dominant follicle, but it also initiates the recruitment of the next generation of follicles that will enlarge during subsequent cycles [24,50]. LH is a major regulator of ovarian steroid synthesis. The mid-cycle LH surge stimulates resumption of oocyte meiosis and maturation in the preovulatory follicle, initiates the rupture of the ovulatory follicle and ovulation, oocyte meiosis, expansion of the cumulus cell oocyte complex, and conversion of the follicle wall into the corpus luteum (luteinization) [50,54]. In the ovary, LH stimulates estrogen production by promoting synthesis of androgen precursors in theca cells, which then diffuse into neighboring granulosa cells where they are aromatized into estrogens under the control of FSH [24]. LH causes rapid increases in the amount of cholesterol available for steroidogenesis. The transfer of cholesterol from the outer to the inner membrane where it becomes available for steroidogenesis is mediated by StAR protein [57]. The StAR protein regulates this rate-limiting step in the steroidogenic process and is in turn regulated by LH. In addition, LH causes an increase in the activity of the side-chain cleavage enzyme to convert cholesterol into pregnenolone. The long-term effects of LH include stimulation of the gene expression and synthesis of a number of key enzymes in the steroid biosynthetic pathway, including not only the side-chain cleavage enzyme, but also 3-β-hydroxysteroid dehydrogenase, 17-α-hydroxylase, and 17,20-lyase. LH stimulates the expression of progesterone receptors (PRs) on the granulosa cells of the dominant follicle, which

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promotes luteinization. In addition, LH helps to sustain luteinization of the ruptured ovarian follicle to form a corpus luteum. Taken together, LH and FSH play different but equally important roles in follicular development. FSH is important for early granulosa cell maturation, including the expression of LH receptors. LH, acting initially on theca cells, promotes androgen production to supply substrate for aromatization to estrogen. Although only FSH is required in early folliculogenesis, full ovarian steroidogenesis requires LH as well.

Gonadotrophin Receptors Gonadotrophins stimulate cyclic adenosine monophosphate (cAMP) production through their cognate G protein-coupled receptors in the plasma membranes of their target cells. The interaction of the receptor with its dimeric hormone partner induces a conformational change in the receptor, which in turn activates a membrane-associated, G-protein-coupled signaling system. The LH/hCG receptor (LHR) can bind both LH and hCG and this binding stimulates adenylate cyclase activity and intracellular cAMP production

that leads to testosterone production. Activation of the LHR also stimulates other intracellular second messenger systems including phospholipase pathways. Both the LHR and FSHR consist of a large extracellular N-terminus domain, followed by the classical seven-transmembrane α-helical domains and a small intracytoplasmic carboxyterminal domain, sharing homology with other members of this family such as rhodopsin, adrenergic, muscarinic, and serotonin receptors, as well as with the TSH receptor and with each other (Fig. 7.8). Their large extracellular hormonebinding domain at the N-terminus distinguishes this receptor subgroup among the larger family of Gprotein-coupled receptors [24]. FSH binds specifically to the FSHR, while LH and hCG both bind to the same LHR. The LHR gene, located on human chromosome 2p21, encodes for a protein of up to 674 amino acids, including a 26-amino-acid signal peptide and a mature protein with a predicted molecular mass of 75 kDa. The purified LHR has an apparent molecular mass of 93 kDa, with the difference between the predicted and observed molecular weight likely due to receptor glycosylation [59]. In the case of the LHR, the N-terminal extracellular domain comprises 341 amino-acid residues, with six potential sites for N-linked glycosylation

FIGURE 7.8 (A) Structure of the LH receptor. Schematic representation of the human LH/hCG receptor (LHR) with known mutations.

Inactivating mutations are indicated by filled squares, whereas activating mutations are indicated by filled circles. ins 5 insertion; fs 5 frameshift. (B) Structure of the FSH receptor. Schematic model of the human FSH receptor (FSHR) with currently known mutations. Source: From Huhtaniemi I, Alevizaki M. Mutations along the hypothalamic-pituitary-gonadal axis affecting male reproduction. Reprod Biomed Online 2007;15(6): 622 32 [58].

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ASSAY SYSTEMS FOR THE MEASUREMENT OF GONADOTROPHINS

and several leucine-rich segments [24]. This extracellular domain has been shown to be both necessary and sufficient for ligand binding, and the leucine repeat motifs are thought to be involved in receptor activation after formation of the hormone receptor complex. The carboxyterminal cytoplasmic segment has several serines and threonines which likely serve as sites for phosphorylation. The FSH receptor gene, located on human chromosome 2p21 16, spans 85 kb and encodes a 678-aminoacid protein with 50% sequence homology to the LHR in their extracellular domains and 80% homology in the transmembrane segments. Like the LHR, the FSHR is also a G-protein-coupled receptor consisting of a large glycosylated N-terminal extracellular domain with a leucine-rich repeat region as well as four potential N-linked glycosylation sites. The extracellular domain again appears responsible for the recognition and binding of the hormone ligand. FSH action in stimulating E2 production by the granulosa cells is mediated via the cAMP pathway. Gonadotrophin receptor expression is classically thought to be restricted to gonadal cell populations. Indeed, FSHR is restricted to granulosa cells of the ovary and Sertoli cells of the testes [50,60]. In the testis, LHR is primarily expressed on Leydig cells. Within the ovary, LHR is expressed on differentiated granulosa, luteal, theca, and interstitial cells. Although LHR expression is nearly undetectable in granulosa cells of preantral follicles, these levels are markedly increased with development to the preovulatory stage [50,54]. Corpus luteum cells express high levels of LHR. Some human adrenal tumors ectopically express LHR and respond excessively to gonadotrophic stimulation [61]. Such tumors often develop when gonadotrophin secretion is chronically elevated, such as in postmenopausal women. Interestingly, the rare conditions of ACTHindependent and pregnancy-associated Cushing’s syndrome, both associated with cortisol excess, present with direct LH/hCG effects on the adrenal cortex.

ASSAY SYSTEMS FOR THE MEASUREMENT OF GONADOTROPHINS Two types of measurement systems have been developed for quantitation of LH and FSH in blood (Table 7.3). First are immunoassays which quantitate the mass of immunoreactive species; second are assays which measure biologic activity. However, because of their cumbersome nature, assays based on bioactivity are not widely used in clinical practice. Most laboratories today utilize fully automated, commercial assay systems for peptide hormones including LH and FSH.

TABLE 7.3 Methods for Measurement of LH and FSH in Serum or Plasma Method

Comments

MEASUREMENT OF MASS: IMMUNOASSAYS Traditional radioimmunoassay Sensitivity limited α-Subunit crossreactivity Immunofluorometric assay

Sensitivity much better No α-crossreactivity No radioactivity involved

Immunochemiluminescent assay

Sensitivity much better No α-crossreactivity No radioactivity involved

MEASUREMENT OF BIOACTIVITY Receptor-binding assays

Uses rat interstitial cells

Classical bioassays

Rat MA-1 cells

LH

Cumbersome Susceptible to serum effects

1. Rat interstitial cell testosterone assay 2. Mouse interstitial cell testosterone assay FSH

Sensitivity limited

1. Granulosa cell aromatase assay 2. Sertoli cell aromatase assay

Radioimmunoassays for LH and FSH The first immunoassays for measurements of serum LH and FSH were radioimmunoassays (RIAs), developed over 20 years ago. Antisera to hCG were initially used in assays for LH based on the close structural and immunologic similarities between these two hormones, but were eventually replaced with antisera to purified LH. RIAs using antisera against the hCGβ subunit or its unique carboxyterminal sequence are highly specific for the measurement of hCG, even in the presence of high levels of LH. Subunit antisera are also available for the measurement of glycoprotein hormone subunits present in the circulation. However, crossreactivity of the free α-subunit in the LH RIA is considerable. Another problem pertains to the microheterogeneity of circulating LH and FSH species. Posttranslational modifications may contribute to heterogeneity of circulating isoforms and may also result in altered biologic activity [27]. A third problem was that most of the existing reference preparations of LH and FSH are not homogeneous and have varying degrees of impurities, so that direct comparisons of data obtained by using different types of reference preparations may not always be valid. Fourth, the sensitivities of the conventional LH and FSH RIAs are

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216

7. GONADOTROPHIN HORMONES

limited so that the precision of the conventional RIAs was not optimum in the low range. This greatly limited their application in clinical and physiologic paradigms requiring measurements of low or suppressed levels, e.g., during the peripubertal period, in hypogonadotrophic disorders, or during GnRH analogueinduced gonadotrophin suppression.

Improvements in LH and FSH Immunoassays Commercially available LH and FSH assays today generally use two-site-directed nonisotopic methods, avoiding the use of radioactive tracers (e.g., immunofluorometric assay, or immunochemiluminescent assay). These assays achieve specificity by using a biotinylated monoclonal antibody to the α-subunit as a capture antibody, with a second monoclonal antibody to the β-subunit used as the indicator antibody. Most assays are calibrated in terms of IU/L of International Reference Preparations of highly purified human LH and FSH. These advances have overcome many of the limitations of RIAs. The sensitivity is much better, able to measure serum LH levels down to 0.1 mIU/mL [33]. Furthermore, crossreactivity is no longer a problem in these two-site-directed assays, and correlation with bioassays is much improved. These more sensitive LH and FSH measurement systems are useful in studying physiologic events characterized by low LH and FSH levels [33].

Bioassays for LH and FSH The first category of bioassays for LH and FSH is receptor binding assays. These have been used primarily in research, but have not attracted clinical applicability. The traditional assay uses 125I-hCG/125I-LH or 125 I-FSH as the ligand, and crude rat gonadal homogenates as the source of membrane receptors [62]. Other bioassays for LH are based on stimulation of testosterone secretion from dispersed Leydig cells. These include the mouse or rat interstitial cell testosterone assay. The free or unassociated subunits have no intrinsic biologic activity and, therefore, do not crossreact in the LH or FSH bioassay. Additional in vitro bioassays have been developed for FSH. These include the granulosa cell aromatase bioassay and the Sertoli cell aromatase bioassay. Although these assays allow measurement of bioassayable serum FSH down to about 2.5 mIU/mL, they are cumbersome, timeconsuming, and require difficult cell culture procedures. Nonetheless, the availability of bioassays for LH and FSH in combination with radioimmunoassay permit calculation of bioactive/immunoactive ratios, which can provide a useful index of qualitative

changes in the gonadotrophins, research purposes.

particularly

for

HYPOTHALAMIC REGULATION OF LH AND FSH The biosynthesis and secretion of LH and FSH are tightly regulated throughout development and across the reproductive cycle. Gonadotrophin expression and secretion are controlled by hypothalamic factors (primarily GnRH), paracrine intrapituitary factors (primarily activin and follistatin), and gonadal feedback (both gonadal sex steroids and gonadal peptide hormones) (Fig. 7.9). Hypothalamic control of gonadotrophin secretion occurs primarily through actions of GnRH, a neuropeptide encoded by a gene on human chromosome 8p. During early research on the hypothalamic control of gonadotrophin secretion, it was first postulated that there would be two distinctly different hypothalamic hypophysiotrophic factors regulating LH and FSH. While GnRH was originally identified as the LHreleasing hormone, it stimulates the release of both LH and FSH, and a bona fide distinct hypothalamic FSHreleasing hormone has yet to be identified. Insights into the mechanisms that regulate activation of GnRH secretion have been provided by the identification and study of genetic abnormalities in patients with pubertal disorders or infertility as well as using animal models. These pathways have been reviewed extensively recently and will be discussed only briefly here in light of our focus on the pituitary [32,63,64]. Molecular defects that manifest with altered timing of puberty can be classified as defects in GnRH

FIGURE 7.9 Schematic view of the hypothalamic pituitary gonadal axis. E2, estradiol; P4, progesterone; T, testosterone; 1 , stimulatory; 2 , inhibitory.

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HYPOTHALAMIC REGULATION OF LH AND FSH

FIGURE 7.10 Genetic and molecular basis of GnRH neuronal development and migration, GnRH secretion, and GnRH action. Source: Adapted from Bianco SD, Kaiser UB. The genetic and molecular basis of idiopathic hypogonadotropic hypogonadism. Nat Rev Endocrinol. 2009;5(10):569 76.

neuronal development, defects in control of GnRH secretion, and defects in GnRH action (Fig. 7.10; Table 7.4).

GnRH Neuronal Development During development, GnRH neurons originate in the region of the olfactory placode. These neurons migrate, along with the olfactory and vomeronasal nerves, into the forebrain and then into their final location in the medial basal hypothalamus (MBH). This orderly migration of GnRH neurons requires the coordinated action of direction-finding molecules, adhesion proteins, and enzymes that help guide the neuronal cells through intercellular matrix. Mutations of any of these proteins can arrest the migratory process and result in GnRH deficiency. In at least a subset of patients, HH, failure to undergo pubertal maturation, can be viewed as a developmental migratory disorder resulting from failure of the GnRH neurons to migrate into the hypothalamus. These patients frequently have an associated phenotype of anosmia, explained by the common embryonic origins and developmental pathways of GnRH and olfactory neurons. Impaired migration of GnRH and olfactory neurons is the underlying cause of Kallmann syndrome (HH associated with anosmia). Other associated neurological and somatic abnormalities, such as synkinesia, cerebellar ataxia, sensorineural deafness, mental retardation, unilateral renal agenesis, and cleft palate, may also coexist [63,65,66]. The first gene identified to play a role in GnRH neuronal migration was KAL1, located on the X chromosome and encoding an extracellular adhesion protein,

217

anosmin-1, essential for axonal guidance and migration of olfactory and GnRH neurons from the nasal placode to their final location in the brain. Anosmin-1 colocalizes with basic fibroblast growth factor receptor 1 (FGFR1) in the olfactory bulb during development and is involved in FGFR1 signaling [67]. In the presence of heparan sulfate, FGFs bind FGFR1 with high affinity, activating downstream signaling to regulate neuronal migration, differentiation, and survival. Kallmann syndrome caused by mutations in FGFR1 is typically transmitted in an autosomal dominant fashion and can be associated with failed morphogenesis of the olfactory bulbs, cleft palate, and dental agenesis. The severity of the hypogonadism and the presence of associated phenotypes have variable expressivity with incomplete penetrance, and FGFR1 mutations have been reported in patients with normosmic IHH [68]. Mutations in FGF8 have also been associated with Kallmann syndrome, suggesting that FGF8 is the FGFR1 ligand responsible for GnRH neuronal development and migration [69]. Other genes implicated in Kallmann syndrome through involvement in the FGF signaling pathway include HS6ST1, FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 [32,64]. The inactivation of the prokineticin 2 system also leads to defective olfactory morphogenesis and hypogonadism in mice and humans [63,70]. Mice deficient in prokineticin 2 (Prok2) or its cognate G-protein-coupled receptor, prokineticin receptor 2 (Prokr2), have olfactory hypoplasia, HH, and an absence of GnRH neurons in the hypothalamus [71,72]. The phenotypic similarities between Prokr22/2 mice and Kallmann syndrome in humans inspired the hypothesis that inactivating mutations in the prokineticin system could lead to anosmia and HH in humans. Indeed, human mutations in PROK2 and PROKR2 have been found in the heterozygous, homozygous, or compound heterozygous state, suggesting a complex mode of inheritance [70,72,73]. Interestingly, PROK2 is expressed in a rhythmic manner in the suprachiasmatic nucleus, PROKR2 is expressed in most primary target areas of the suprachiasmatic nucleus, and PROK2 expression/release is controlled by core clock genes, suggesting a role for the prokineticin 2 system in circadian rhythms. Indeed, Prok2 and Prokr2 null mice exhibit disruption of circadian rhythms, with reduced rhythmicity of locomotor activity, body temperature, and sleep [74]. Mutations in SEMA3A, encoding a protein which interacts with neuropilin and is involved in axonal pathfinding, have also been identified in patients with Kallmann syndrome. Like the prokineticin system, the identification of this genetic cause of Kallmann syndrome followed the description of the phenotype in mice [75,76]. Mutations in SEMA3E, also encoding a semaphorin involved in GnRH neuronal development, have also been identified as a genetic

I. HYPOTHALAMIC PITUITARY FUNCTION

TABLE 7.4 Genetic and Acquired Causes of Hypogonadotrophic Hypogonadism Genetic causes gene

Locus

Protein

Inheritance

Phenotype

GNRHR

4q13.2

GnRH receptor

Autosomal recessive

HH

KISS1

1q32.1

Kisspeptin 1

Autosomal recessive

HH

KISS1R

19p13.2

Kisspeptin 1 receptor

Autosomal recessive

HH

TAC3

12q13.3

Neurokinin B

Autosomal recessive

HH

TACR3

4q24

Neurokinin B receptor

Autosomal recessive

HH

GNRH1

8p21.2

GnRH

Autosomal recessive

HH

PCSK1

5q15

Prohormone convertase 1

Autosomal recessive

HH, obesity, hypoglycemia, hypocortisolemia

LEP

7q31.3

Leptin

Autosomal recessive

HH, obesity

LEPR

1p31

Leptin receptor

Autosomal recessive

HH, obesity

KAL1

Xp22.31

Anosmin

X-linked

KS

FGFR1

8p11.2

Fibroblast growth factor receptor 1

Autosomal dominant

KS/HH

FGF8

10q24.32

Fibroblast growth factor 8

Autosomal dominant

KS/HH

PROK2

3p13

Prokineticin 2

Autosomal recessive

KS/HH

PROKR2

20p12.3

Prokineticin receptor 2

Autosomal recessive

KS/HH

CHD7

8p12.1

Chromodomain helicase DNA binding protein 7

Autosomal recessive

KS/HH

SEMA3A, SEMA3E

7q12.1, 7q21.1

Semaphorins 3A, 3E

Autosomal dominant

KS

WDR11

10q26.12

WD repeat-containing protein 11

Autosomal recessive

KS/HH

HS6ST1

1p36.12

Heparan sulfate 6-O-sulfotransferase 1 Autosomal dominant

KS/HH

Genes encoding components of FGF pathway

KS/HH

FGF17, IL17RD, DUSP6, SPRY4, and FLRT3

Autosomal dominant

Prader Willi syndrome Laurence Moon Biedl syndrome Mutations in FSHB or LHB Mutations of pituitary transcription factors ACQUIRED DISORDERS Hypothalamic amenorrhea Chronic renal failure Hemochromatosis Hyperprolactinemia Neoplastic Inflammatory and infiltrative HH, hypogonadotropic hypogonadism; KS, Kallmann syndrome.

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HYPOTHALAMIC REGULATION OF LH AND FSH

cause of Kallmann syndrome; in this case, in murine studies the defect appears to be in GnRH neuron survival/apoptosis rather than axon guidance, possibly through interactions with CHD7 [77]. Mutations in the gene CHD7, encoding chromodomain-helicase-DNA-binding protein 7 and thought to be involved in chromatin remodeling, have been identified in patients with CHARGE syndrome, a multisystem disorder that may include HH. CHD7 is expressed in olfactory epithelium, the hypothalamus, and the pituitary. Mutations in CHD7 have also been identified in patients with normosmic HH or Kallmann syndrome without other manifestations of CHARGE syndrome [78]. Mutations in WDR11, encoding a protein with WD domains important for protein protein interactions, have also been found in patients with Kallmann syndrome; the function of WDR11 is not well understood but suggests a possible connection between SHH signaling and pubertal development [79].

GnRH Secretion In primates, the majority of the GnRH neuronal cell bodies are located in the arcuate nucleus of the MBH, with additional cells in the preoptic area of the anterior hypothalamus. GnRH produced by these neurons is transported through axons in the tuberoinfundibular tract to the median eminence where it is released into the hypophyseal portal circulation and delivered to the anterior pituitary for action on gonadotrophs via a highly specific, G-protein-coupled GnRH receptor. GnRH neurons also project to other CNS regions including the limbic system, amygdale, hippocampus, and periaqueductal gray matter, where GnRH may influence reproductive behavior. However, only the

GnRH neuronal projections to the median eminence significantly control gonadotrophin secretion. The hallmark of the hypothalamic secretion of GnRH is its pulsatile rather than continuous release into the hypophyseal portal circulation, resulting in episodic stimulation of the gonadotroph. In a series of pioneering studies, Knobil and colleagues in the late 1970s demonstrated the absolute requirement for such a pulsatile stimulus to sustain LH and FSH secretion, whereas continuous infusion of GnRH was not effective (Fig. 7.11) [80]. This observation has been confirmed in humans by the restoration of gonadotrophin secretion in GnRH-deficient subjects after exogenous pulsatile GnRH treatment. Conversely, after a transient stimulatory response, continuous GnRH exposure suppresses gonadotrophin secretion. This inhibitory effect of continuous GnRH has been exploited in the treatment of sex steroid-dependent disorders including precocious puberty, endometriosis, prostate cancer, and breast cancer, as well as in infertility therapies [81]. Because of the short half-life of GnRH and the large dilutional effect, levels of GnRH in the systemic circulation are too low to measure reliably. Measurement of hypophyseal portal GnRH concentrations has been performed in sheep and monkeys, confirming that GnRH secretion is pulsatile [82,83]. In humans, of course, such measurements are not feasible. Nonetheless, circulating LH levels have been shown to be highly correlated with GnRH release into the hypophyseal portal system, with each pulse of GnRH being followed by an LH pulse. Therefore, frequent blood sampling (i.e., every 10 minutes) for measurement of LH pulses can be used as an accurate indicator of GnRH secretion patterns. While FSH secretion is also correlated with GnRH secretion, it is less useful as a marker because of its longer half-life. FIGURE 7.11 Knobil’s experiments: pulsatile GnRH administration is essential for physiologic LH and FSH secretion. Source: From Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E. Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone. Science 1978;202(4368):631 3.

999 Pulsatile

Continuous

Pulsatile

200

15

150

10

100

5

50

0

10

5

0

5

10

15

20

25

30

35

FSH (ng/mL)

LH (ng/mL)

20

219

0

Days

I. HYPOTHALAMIC PITUITARY FUNCTION

220

7. GONADOTROPHIN HORMONES

Many neurotransmitter systems directly or indirectly modulate GnRH secretion. These include norepinephrine, dopamine, serotonin, γ-aminobutyric acid (GABA), glutamate, opiate peptides, neuropeptide Y (NPY), and galanin, among others. Glutamate and norepinephrine provide stimulatory drive to the reproductive axis, whereas GABA and opioid peptides are inhibitory. NPY acts at both hypothalamic and pituitary levels, by increasing GnRH release from the median eminence in the presence of gonadal steroids and by facilitating GnRH-induced LH release at the level of the gonadotroph. Pituitary adenylate cyclaseactivating polypeptide has also been implicated in the upregulation of LH secretion, acting both as a hypothalamic hypophysiotrophic factor and a pituitary paracrine factor [84]. New insights into pathways that regulate GnRH secretion have been provided by the identification and study of genetic abnormalities in patients with HH. The roles of kisspeptin and its G-protein-coupled receptor, KISS1R (also known as GPR54), were revealed in 2003, when inactivating mutations in KISS1R were found in members of families with a history of normosmic IHH [85 87]. Subsequent studies have shown that both kisspeptin and its receptor are expressed in areas of the hypothalamus involved in the control of reproduction. Centrally or peripherally injected kisspeptin results in a robust stimulation of GnRH and gonadotrophin secretion and accelerates puberty in prepubertal rodents [63]. In rodents, the hypothalamic regions with the highest levels of kisspeptin-expressing neurons are the arcuate (ARC) and the AVPV nuclei, each differentially regulated by gonadal steroids, acting through ERα and likely AR. Kisspeptin expression increases in the ARC following gonadectomy and decreases with gonadal steroid administration. Thus, ARC kisspeptin neurons are predicted to play an important role in sex steroid negative-feedback regulation of the reproductive axis. Kisspeptin expression in the AVPV is sexually dimorphic, with 10 20-fold higher levels in females than males, and increases in response to gonadal steroids. AVPV kisspeptin expression has also been shown to vary during the estrous cycle, culminating with the highest levels of expression coincident with the ovulatory LH surge. These findings suggest a role for the AVPV kisspeptin neurons as effectors of the estrogen positive-feedback mechanism leading to the gonadotrophin surge in females [32,63,88]. The key role of kisspeptin signaling in the regulation of the onset of puberty was reinforced when the first identifiable genetic cause of central precocious puberty (CPP) was recognized as a gain-of-function mutation in KISS1R, the result of a reduced rate of desensitization of this

G-protein-coupled receptor [89]. Mutations in the KISS1 gene encoding the ligand have also been reported in patients with CPP [90]. Studies of kisspeptin administration in humans have demonstrated its ability to stimulate pulsatile LH release and support its potential use in treatment of infertility [91,92]. Interestingly, neurokinin B (NKB), a member of the substance P-related tachykinin family encoded by TAC3, is coexpressed with kisspeptin in the ARC, as is its G-protein-coupled receptor, NK3R, encoded by TACR3. Loss-of-function mutations have been identified in both TAC3 and TACR3 in patients with normosmic HH [32,64,93]. Additional genetic mutations that cause impaired GnRH secretion include mutations in the gene encoding GnRH itself [94,95] and mutations in prohormone convertases that process the GnRH prohormone to the mature secreted biologically active decapeptide. Mutations in PCSK1 have been reported as a cause of normosmic HH in association with obesity, hypoglycemia, hypocortisolemia, and evidence of impaired processing of POMC and proinsulin [96]. Another link between obesity and HH occurs in patients with mutations in the gene encoding leptin or its receptor. Leptin is a fat-derived hormone that regulates food intake, energy expenditure, and reproduction mediated through hypothalamic pathways. The central role of leptin in these cases is highlighted by the recovery of gonadotrophin secretion and menstrual cyclicity following treatment with recombinant leptin in females with amenorrhea due to congenital leptin deficiency or hypothalamic amenorrhea (HA) [96]. Kisspeptin has been implicated as a mediator of the effects of leptin on GnRH secretion [97]. The first gene with an inhibitory effect on GnRH secretion with loss-of-function mutations in humans was identified in patients with familial CPP [98]. The MKRN3 gene is located in the Prader Willi syndrome critical region on chromosome 15; prior to the identification of its role in puberty, the few reports in the literature about this gene came from research into Prader Willi syndrome [98 100]. The maternal MKRN3 allele is imprinted and only the paternal allele is expressed, the first imprinted gene shown to play a role in the timing of puberty. The mechanism by which MKRN3 regulates puberty initiation is not yet completely understood, but expression in the arcuate nucleus of mice, a region implicated in puberty and where the kisspeptin and NKB systems act, is high prepubertally and decreases before puberty initiation to very low levels in adult life. MKRN3 belongs to the Makorin family, a family of E3 ubiquitin ligases. These factors have led to speculation that MKRN3 may act to inhibit stimulators of GnRH secretion (Fig. 7.12) [32].

I. HYPOTHALAMIC PITUITARY FUNCTION

HYPOTHALAMIC REGULATION OF LH AND FSH

FIGURE 7.12 Mechanisms of action of factors involved in the neuroendocrine control of puberty onset. Essential regulators of GnRH secretion in which mutations have been identified in humans with pubertal disorders. Dashed lines represent proposed pathways. GnRHR, gonadotrophin-releasing hormone receptor; KISS1, kisspeptin; KISS1R, kisspeptin receptor; MKRN3, makorin ring finger protein 3; NKB, neurokinin B; NK3R, neurokinin B receptor; 1 , stimulatory effect; , inhibitory effect; ?, proposed mechanism. Source: From Abreu AP, Kaiser UB. Pubertal development and regulation. Lancet Diabetes Endocrinol 2016;4:254 64.

GnRH Action The pattern of GnRH signal is important in determining the quantity and quality of gonadotrophins secreted [101]. GnRH signaling begins with recognition by its receptor, GnRHR. The GnRHR is expressed in the pituitary as well as in additional minor sites of expression including hypothalamic GnRH neurons, testicular Leydig cells, ovarian granulosa and luteal cells, placental cells, and in endometrial, breast, and prostate cancers and cancer cell lines. The GnRHR was first cloned from the murine gonadotroph-derived αT3-1 cell line and subsequently from multiple other species, all composed of three exons and two introns and with a high degree of homology within their coding regions [37]. The GnRHR (encoded by GNRHR) is

221

a 328-amino-acid protein that belongs to the rhodopsin family of G-protein-coupled receptors. Activation of the GnRHR increases calcium mobilization and stimulates influx of extracellular calcium. This increase in intracellular calcium induces pituitary LH and FSH secretion [102]. Mutations in GNRHR were among the first genetic mutations identified in patients with IHH [32,64]. Since then, more than 20 additional loss-of-function mutations in the GnRHR have been identified in patients with IHH (Fig. 7.13) [103,104]. The cellular and molecular phenotypes of each mutation have been studied extensively in vitro, and mutations can be classified as partial or complete loss-of-function mutations. Amino acid substitutions have been identified in virtually every section of the receptor. Most known GNRHR mutations are missense, leading to single amino acid substitutions. These mutations impair GnRH signaling due to loss of receptor expression, ligand binding, G protein coupling, and/or abnormal intracellular trafficking of the receptor. Large-scale screening indicates that GNRHR mutations account for 3.5 16% of sporadic cases of normosmic HH and up to 40% of familial cases of normosmic HH [63,103]. Inheritance is autosomal recessive and most patients have compound heterozygous GNRHR mutations [63]. Functional responses and structure function relationships of GnRH receptor mutations have been described in detail [103,104]. Before being trafficked to the cell membrane, receptors are first modified and folded at the site of synthesis within the endoplasmic reticulum. At this level, defective and misfolded proteins may be routed to pathways of degradation. Recent evidence suggests that the human GnRHR is prone to such misrouting, and even a proportion of wild-type receptors never reach the plasma membrane [104,105]. Misfolding is not inevitable as cells naturally rely on molecular chaperones to help properly arrange nascent proteins. This observation has led to the development of “pharmacoperones” (pharmacological compounds acting as chaperones). Pharmacoperones are small molecules that enter cells, bind specifically to misfolded mutant proteins, correct their folding, and allow correct routing. Frequently, such molecules are identified as peptidomimetic antagonists from high-throughput screens [104]. Of interest, a nonpeptidic cell permeant GnRHR antagonist, IN3, has been shown to partially restore the function of several naturally occurring mutant human receptors [104]. As this compound was also able to enhance cell surface expression of the wildtype human receptor, it was proposed that IN3 could promote and/or rescue proper GnRHR folding. Thus the development of pharmacological chaperones to overcome receptor misrouting has the potential to lead

I. HYPOTHALAMIC PITUITARY FUNCTION

222

7. GONADOTROPHIN HORMONES

Membrane

K11 K10 Q M L P I S N N I A S C H N Q N Q E P S A S N A M –NH2 G Extracellular domain N Y200 L H C S R106 C108 F Q W P T V S Y Q K T V A V F T G Q W N R L 104 I L T E C S L G W S L M I32 T I Q L A D S S H D L E L N C H P Q P S W K I A V D G M F K V M N F Y N G R R H I L W F L V S I F W Y D Y F F F T T P L Y I F 21 R T V M K L Q F G L S 7 I V F F L C V L F P A 90 17 K L F L S G A T F L Y F F E T Y M F I P Y C28 N L X31 1 168 R L A N129 I P L V S T 4 4 L S L P A L P W C S A A N F L F V C D F F T L M I M I T P L A N T V M W L S F Y27 I L L32 H F A V L I T N Y 9 L S L K L I M G A C A A G 0 L K L L D S S V V F Y L K T K R Q F Q S I M G M S K I H,C139 L R K V L –COOH W A F S K L R26 T I T 6 L S T Q T L K N K K R T K S K R P R L L G K A E K A L V R Q26 L P Intracellular domain 2 H Q I E H P D N L Q N L K N Q S

FIGURE 7.13 Schematic representation of the human GnRHR with reported mutations. This two-dimensional diagram shows the human GnRHR embedded in the cell membrane with its seven-transmembrane domains connected by three extra- and intracellular loops. Naturally occurring mutations are highlighted by shaded circles with the resulting “mutant” residue and its relative position listed next to each. Source: From Bedecarrats GY, Kaiser UB. Mutations in the human gonadotropin-releasing hormone receptor: insights into receptor biology and function. Semin Reprod Med 2007;25(5):368 78.

to new therapeutic approaches for patients with IHH due to GnRHR mutations. Similarly, a small-molecule LHR agonist has been shown to functionally rescue intracellularly retained mutant LHR by facilitating cell surface expression [106]. These strategies may have applications for the treatment of infertility. The phenotypic spectrum of HH due to mutations in the GnRHR ranges from complete absence of any sexual maturation to partial phenotypes with some evidence of pubertal development, the fertile eunuch variant, and delayed puberty [63]. The clinical phenotype generally correlates with biochemical LH pulsatility profiles and responses to exogenous GnRH. Complete HH is typically associated with low levels of apulsatile LH, whereas some spontaneous LH pulses, albeit reduced in amplitude, may be seen in patients with partial phenotypes. While patients with complete HH due to inactive GnRHR variants are characterized by an absent response to exogenous GnRH, some patients with GnRHR mutations respond to exogenous GnRH administration, albeit with a reduced sensitivity to GnRH. This partial resistance to GnRH can be overcome, as is evident from the successful use of pulsatile GnRH for ovulation induction and pregnancy in a patient with a compound heterozygous mutation in GnRHR [107]. Phenotype genotype correlation studies indicate that the clinical phenotype is, in most cases,

well correlated with the functional alteration of the GnRHR in vitro, with the phenotype and response to exogenous GnRH administration correlating best with the GnRHR variant with the less severe loss of function in compound heterozygotes [108]. Phenotypic variability has been observed in affected related subjects bearing the same mutations, suggesting that other factors may influence the phenotype. Potential modifying factors include gender (e.g., via sex steroid hormone effects), environmental factors, or modifier genes. The identification of digenic mutations in some pedigrees with IHH and GnRHR mutations may account for at least some such phenotypic heterogeneity [109].

Influence of Patterns of Pulsatile GnRH The amplitude, frequency, and contour of GnRH pulses can all vary and each of these characteristics can influence gonadotroph responses, providing a mechanism for the differential synthesis and secretion of the two gonadotrophins, LH and FSH. GnRH pulse pattern varies across the female menstrual cycle. Studies have estimated that GnRH pulses occur every 94 minutes on average in the early follicular phase, increasing to every 71 minutes due to effects of increasing circulating levels of estradiol in the late

I. HYPOTHALAMIC PITUITARY FUNCTION

FEEDBACK REGULATION OF LH AND FSH SECRETION

994 1 pulse/3 h

1 pulse/h

50

500

45

450

40

400

35

350

30

300

25

250

20

200

15

150

10

100

5

50

0

20

15

10

5

0

5

10 Days

15

20

25

follicular phase, and slowing to every 216 minutes under the influence of elevated levels of progesterone in the luteal phase [110]. In an extension of Knobil’s pioneering studies, it was demonstrated that more rapid GnRH pulse frequencies favor LH secretion, whereas slower pulse frequencies favor FSH (Fig. 7.14) [111]. These and other studies established that the pulsatile release of GnRH at an optimum pulse frequency and amplitude is integral to optimum gonadotroph function, and variations in GnRH pulse frequency markedly influence both the absolute levels and the ratio of LH and FSH release [112]. In parallel rat models as well as in in vitro systems, decreased frequency of GnRH pulses results in a differential increase in FSHβ transcription and FSH biosynthesis over LH [112,113]. Thus, it is hypothesized that this leads to greater intracellular stores of FSH to allow for the greater FSH secretion. These alterations in GnRH pulse frequency are one mechanism by which two functionally distinct gonadotrophins can be differentially regulated by a single hypothalamic-releasing hormone. The molecular mechanisms by which these differential regulatory effects occur have been the subject of considerable investigation (see section on Molecular Biology of Gonadotrophin Subunit Genes).

FEEDBACK REGULATION OF LH AND FSH SECRETION Estrogens The gonadal steroid hormones include estrogens, progesterones, and androgens. Effects on gonadotrophins occur both directly at the level of the

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223 FIGURE 7.14 Knobil’s experiments: the frequency of pulsatile GnRH administration has differential effects on gonadotrophin secretion: more rapid GnRH pulse frequencies favor LH secretion, whereas slower pulse frequencies favor FSH. Source: From Wildt L, Hausler A, Marshall G, Hutchison JS, Plant TM, Belchetz PE, et al. Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey. Endocrinology 1981;109(2):376 85.

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gonadotroph and indirectly via effects at the hypothalamus that modulate GnRH secretion. Estrogen, androgen, and PRs have been identified in gonadotrophs, consistent with direct actions. Within the hypothalamus, receptors for sex steroids have been identified in multiple neuronal cell types, suggesting that alterations in GnRH release can also occur indirectly through modulation of neuronal systems known to impinge on GnRH neurons [114]. The complexity of estradiol effects on gonadotrophin secretion has been well reviewed [114 116]. In women, estrogens can exert dual feedback effects on gonadotrophin secretion, depending on the reproductive state. The negative- feedback effects of estrogens are clearly demonstrated by the elevation of LH and FSH levels that occur following ovariectomy or after menopause, which reverse with estrogen replacement. On the other hand, during the late follicular phase of the menstrual cycle, the feedback effects of estrogens shift from negative to positive, triggering the midcycle ovulatory surge of LH and FSH secretion. Estrogen receptors (ERs) are present in many subcellular locations and signal through multiple pathways (Fig. 7.15). In the classical genomic pathway, estradiol binds to ERs, and then the ER binds to estrogen response elements (EREs), resulting in changes in gene transcription. An alternate mechanism is the nonclassical genomic pathway, in which estradiol binds with ERα, which then binds to a transcription factor, potentially resulting in transcription of other genes. There is also a nonclassical, nongenomic pathway, in which membrane receptors or receptors in other locations signal through protein kinase cascades, resulting in phosphorylation of transcription factors that then may result in transcription of yet other genes [114]. In addition to

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FIGURE 7.15 Estradiol signals through ERα via multiple pathways: (1) classical genomic pathway; (2) nonclassical genomic pathway; (3) nonclassical, nongenomic pathway. E2, estradiol; ERα, estrogen receptor alpha; TF, transcription factor; ERE, estrogen response element; RE, response element. Source: From Blaustein JD. The year in neuroendocrinology. Mol Endocrinol 2010;24(1):252 60.

the predominant ER, ERα, there is also a second nuclear receptor for estrogen, ERβ, and a putative G-proteincoupled receptor for estrogen, GPR30. Negative-feedback effects of estrogens are observed at the level of α-subunit, LHβ, and FSHβ subunit mRNA levels through effects on gene transcription in addition to effects on LH and FSH secretion. Estrogen’s negative-feedback effects are thought to be mediated in part directly at the level of the pituitary gland. Estrogen decreases GnRH-stimulated LH secretion in vitro in cultured pituitary cells as well as in vivo in monkeys. Estrogen also likely has negativefeedback effects at the levels of the hypothalamic pulse generator, as estradiol inhibits LH pulse amplitude in normal men and in GnRH-deficient men maintained on GnRH [117]. Whether ERα and/or ERβ are expressed and functional in GnRH neurons has been controversial [116]. Increasing evidence suggests that kisspeptin neurons in the arcuate nucleus could be responsible for mediating the negative-feedback effects of estrogen on GnRH secretion. These neurons contain ERα and gonadectomy increases arcuate kisspeptin expression, whereas estrogen replacement restores expression to that observed in intact, untreated animals. The suppression of kisspeptin activity by sex steroids in the arcuate nucleus appears to be mediated by ERα in the female. These findings are consistent with a role for kisspeptins in mediating the

negative-feedback effects of gonadal steroids on GnRH secretion [46,118] (Fig. 7.16). Positive-feedback effects of estrogen are mediated in large part at the level of the hypothalamus. Direct positive effects of estrogens at the pituitary level may also contribute. In support of a pituitary site of action, estrogen increases LHβ mRNA synthesis in vitro in pituitary cultures [113]. Nonetheless, the major site of positive feedback appears to be at the hypothalamus, mediated through kisspeptin neurons in the AVPV of the rodent. Kisspeptin expression in the AVPV is positively regulated by sex steroids. Targeted disruption of the ERα gene blocks the ability of estradiol to induce the expression of kisspeptin in the AVPV [46,118]. The AVPV is thought to play a vital role in the generation of the preovulatory gonadotrophin surge, so the current working model proposes that kisspeptin neurons in this region mediate positive-feedback effects of estrogens on gonadotrophin secretion (Fig. 7.16). Evidence demonstrating that a blockade of kisspeptin signaling in the female rat abolishes the preovulatory LH surge is consonant with a role in positive feedback for kisspeptin in the female [120]. Moreover, based on studies of the NERKI (nonclassical ER knock-in) mouse, which has a mutant ERα allele that does not bind to DNA and can signal only through membrane-initiated or ERE-independent genomic pathways, it appears that classical, ERE-dependent

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PRKO mice have demonstrated involvement of PRs in the generation of preovulatory gonadotrophin surges. The relatively rapid effects of P4 on gonadotrophin secretion are suggestive of additional mechanisms that do not involve changes in gene transcription. A study in PRKO mice demonstrated that progesterone can exert inhibitory effects on GnRH release that are manifest even in the absence of PRs and that these effects may be mediated by membrane receptors coupled to Gi protein involving the inhibition of intracellular cAMP formation [123].

Androgens FIGURE 7.16 Proposed model for classical and nonclassical ERα signaling in estrogen positive and negative feedback. Negativefeedback actions of estrogen on GnRH/LH secretion are mediated in part by nonclassical (ERE-independent) ERα signaling mechanisms. In contrast, positive-feedback actions of estrogen on GnRH/LH surges are mediated by classical (ERE-dependent) ERα signaling mechanisms. These classical and nonclassical mechanisms appear to be mediated at least in part by kisspeptin neurons. Source: Adapted from McDevitt MA, Glidewell-Kenney C, Jimenez MA, Ahearn PC, Weiss J, Jameson JL, et al. New insights into the classical and non-classical actions of estrogen: evidence from estrogen receptor knock-out and knock-in mice. Mol Cell Endocrinol 2008;290(1 2):24 30 [119].

pathways mediate estradiol action on the LH surge mechanism in female mice, whereas nonclassical pathways are responsible for the effect of estradiol on negative feedback of LH [114,119,121]. The rescue of negative feedback in NERKI females appears to also reflect rapid, nongenotrophic actions of E2 originating at the plasma membrane [122].

Progesterone The principal effect of progesterone is to decrease the frequency of gonadotrophin pulses, presumably mediated by hypothalamic effects on GnRH pulse frequency. During the luteal phase of the human menstrual cycle, when progesterone concentrations are the highest, LH pulse frequency markedly slowed [30]. An additional direct pituitary effect of progesterone cannot be excluded, and progesterone can also augment the stimulatory effects of estrogen on LH secretion in the late follicular phase, when serum progesterone levels are rising along with estrogens, contributing to the ovulatory LH surge. The majority of progesterone actions are currently believed to be dependent upon the binding and activation of its cognate nuclear receptors PRs. Bound PRs recruit coactivator proteins and function as ligandactivated transcription factors that regulate transcription of target genes. Neuroendocrine assessments of

Testosterone and its aromatized derivative estradiol are the two steroid hormones that exert negativefeedback effects on gonadotrophin secretion in the male. Serum LH and FSH levels and α, LHβ, and FSHβ mRNA levels rise after castration in a number of experimental animals. The effects of testosterone on FSH synthesis and secretion are complex. The net in vivo effect of testosterone administration to normal men is inhibition of serum FSH levels. It is, however, clear that the direct effects of testosterone on FSH output at the pituitary level are stimulatory. In isolated pituitary cell cultures, testosterone increased FSHβ mRNA levels and FSH release [124]. The differential effects of testosterone on LHβ and FSHβ expression can be attributed to opposing effects on FSHβ between the hypothalamus and the pituitary. Testosterone inhibits LH secretion when given to normal men. Much like the effects of estrogen, these inhibitory effects are felt largely to be at the hypothalamic level [117]. The available evidence suggests that 5-α reduction of testosterone is not essential for the inhibitory effects of testosterone on LH. Administration of a potent 5-α reductase inhibitor, finasteride, to normal men did not result in elevated LH and FSH levels, consistent with direct inhibitory effects on LH by testosterone without obligatory 5-α reduction [125]. Much as the negative-feedback effects of estrogen on GnRH secretion are mediated by kisspeptin neurons in the arcuate nucleus, these neurons also contain AR and the suppression of kisspeptin activity by testosterone in the arcuate nucleus appears to be mediated by both AR and ERα. These findings indicate a role for kisspeptins in mediating the negative-feedback effects of gonadal steroids on GnRH secretion in the male as well [46,118].

Inhibins, Activins, and Follistatins The hypothesis that a peptide of gonadal origin selectively regulates FSH secretion dates back to at

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least 1932 [126]; it took over 50 years to isolate and characterize the structure of inhibin and its related peptides [127]. Inhibins are dimeric proteins covalently linked by a disulfide bridge and consisting of a common α-subunit and one of two highly homologous β-subunits, βA or βB (Fig. 7.17A). The heterodimers of α:βA are called inhibin A and of α:βB heterodimers, inhibin B. Whether inhibin A and inhibin B play similar or distinct roles in physiological settings is yet to be resolved, although some functional differences have been suggested. In addition, βA subunits can form homodimers called activin A, βB subunits can form homodimers called activin B, or heterodimers of the two β-subunits can form activin AB. Activins stimulate FSH synthesis and secretion [127]. To date, five β-subunits have been identified (βA to βE), although only dimers of the βA- and βB-subunits have been shown to have an effect on FSH secretion. A structurally unrelated glycosylated monomeric polypeptide hormone, follistatin, has also been identified based on its ability to inhibit FSH [127]. Effects on LHβ gene expression and LH secretion are modest, and these three peptides (inhibins, activins, and follistatin) are considered to be relatively selective for FSH in terms of their effects on gonadotrophins. Inhibin-related peptides are widely distributed in organ systems and have significant homology with members of the TGFβ family of proteins that also includes Mu¨llerian-inhibiting substance, bone morphogenetic proteins (BMPs), and other growth and differentiation factors. While inhibins appear to act primarily as classic circulating endocrine hormones, activins play an important role as regulators of growth and differentiation in diverse tissues, acting locally as autocrine/paracrine factors. For example, activin has been shown to act in the ovary to regulate luteolysis, in the testis to stimulate Sertoli cell proliferation and spermatogenesis, and in the pancreas to enhance β-cell proliferation and insulin secretion [127]. Just as activin is a member of the TGFβ family, the activin receptor and signaling system is similar to the TGFβ receptor system. Activin receptors are heteromeric complexes comprising type I (Act-RI) and type II (Act-RII) serine threonine kinase receptors (Fig. 7.17B). Activin binds to the type II receptors, thereby increasing association with the type I receptor and stimulating its trans-phosphorylation, which in turn results in the activation by phosphorylation of intracellular signaling proteins, receptor-regulated Smad (R-Smad) proteins. Activins, TGFβs, and growth differentiation factor 9 signal via Smad2 and Smad3, whereas BMPs generally signal via Smads 1, 5, and 8. These activated receptor Smads then interact with the common Smad4, resulting in translocation of the Smad complex to the nucleus, where it binds to gene

FIGURE 7.17 Activins and inhibins and their mechanism of action. (A) Inhibin and activins are dimeric proteins made up of two subunits. Inhibins are made up of an α-subunit linked to one of two β-subunits, whereas activins are made up by dimerization of two β-subunits. (B) Activin signaling pathway. Activins bind to specific sets of serine threonine kinase type I and type II receptors on the cell surface. Upon ligand binding, the type II receptor phosphorylates and thereby activates the type I receptor, which in turn phosphorylates downstream signaling molecules, the receptor-regulated Smads (R-Smads). Once phosphorylated, the R-Smads associate with the common co-Smad (Smad4) and translocate to the nucleus where, in combination with cell type-specific binding partners, they bind to the promoter sequences of target genes to regulate gene transcription and cellular function. (C) Inhibins bind to activin type II receptors and block the recruitment of type I receptors, thereby blocking RSmad activation and activin signaling. The presence of TGFBR3, a TGFβ superfamily accessory receptor also known as betaglycan, enhances the binding of inhibins to type II receptors, thereby enhancing the antagonistic actions of inhibins. Source: From Stenvers KL, Findlay JK. Inhibins: from reproductive hormones to tumor suppressors. Trends Endocrinol Metab 2010;21(3):174 80 [128].

regulatory elements and interacts with other transcription factors and coactivators to regulate gene transcription, thereby influencing cell fate and function [128]. Activin is typically produced locally in multiple tissues where it acts as an autocrine/paracrine factor. Activin biosynthesis itself does not appear to be modulated to a significant degree. Local activity of activin is modulated through multiple modalities. Follistatin and inhibin act as extracellular modulators of activin through distinct mechanisms. These two extracellular proteins modulate activin signaling before activin binds its receptor (follistatin), or before signal

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transduction is fully activated (inhibin), thereby providing the first of several levels for regulating activin action. Follistatin serves as an activin-binding protein and inhibits activin action by interfering with activin’s ability to bind to its receptor. The high affinity of the activin follistatin interaction approaches irreversibility due to its slow dissociation rate, rendering the bound activin unavailable for binding to its own receptor. The bound complex consists of two follistatin molecules for each activin β/β homodimer; the low affinity of inhibin for follistatin may be due to the availability of only a single β-subunit in the α/β heterodimer [127]. Inhibins compete for binding to type II activin and BMP receptors through their β-subunits. In this manner, inhibins can antagonize the actions of activins, BMPs, and potentially other TGFβ superfamily members that utilize these type II receptors by preventing the recruitment of the respective type I receptors, thereby blocking R-Smad activation (Fig. 7.17C). Since inhibins have significantly lower affinity for the activin and BMP type II receptors compared with the agonists themselves, additional inhibin-binding proteins that increase the affinity of inhibin for the type II receptors are thought to be involved in the antagonistic actions of inhibins. The type III TGFβ superfamily accessory receptor, TGFBR3 (also known as betaglycan), increases the binding affinities of inhibins to the activin and BMP type II receptors, thereby enhancing the antagonistic actions of inhibins [128]. At the intracellular level, the activin signal is modulated by inhibitory Smads. Inhibitory Smads form stable associations with type I receptors but cannot be activated, thereby preventing activation of the receptor-activated Smads. Smad 7 inhibits signaling in the activin/TGFβ pathway by blocking phosphorylation of Smads 2 and 3. Smad 6, on the other hand, represses the BMP pathway. Another level of regulation occurs through membrane-bound modifiers like BMP and activin receptor membrane-bound inhibitor (BAMBI) and Cripto, a GPI-anchored membrane protein, which inhibits activin signaling by forming an active complex with activin and ActRIIA [129]. At the intracellular level, another mechanism of regulation of activin signaling is through a protein named SMAD anchor for receptor activation (SARA). SARA binds to unphosphorylated SMAD2 to mask its nuclear localization signal and maintain a cytoplasmic distribution. Following receptor activation, SARA interacts with the type I receptor to facilitate SMAD2 phosphorylation and activation. As a result, SMAD2 dissociates from SARA and complexes with SMAD4, after which the complex translocates to the nucleus [129]. While inhibin A does not seem to be a major endocrine regulator of FSH in the human male, inhibin B is

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stimulated by gonadotrophins and circulates systemically to provide feedback inhibition of FSH. In women, inhibin A is secreted by dominant follicles and corpora lutea, contributing to the high circulating levels during the late follicular and luteal phases. Inhibin B is reciprocally elevated during the late luteal and early follicular phases of the cycle [129,130]. Circulating inhibin levels drop abruptly after castration in animal models, supporting a primarily gonadal origin. The correlation between circulating inhibin levels and FSH secretion is consistent with a role for inhibin in the feedback regulation of FSH. Activin is detectable in the serum, but levels are low, do not change across the menstrual cycle, and are highly protein-bound to follistatin and α2-macroglobulin. Therefore, while inhibin is thought to circulate in the bloodstream and act in a classical endocrine fashion, both activin and follistatin can be produced by extragonadal sources and may exert their effects via paracrine/autocrine mechanisms at or near their site of production [131]. Within the pituitary, the gonadotrophs and other cell types produce the inhibin α- and β-subunits as well as follistatin. Activin B is produced locally and supports FSH production, as evidenced by a decrease in FSH secretion following treatment of pituitary cultures with an activin-blocking antibody. GnRH and gonadal steroid hormones have been shown to modulate inhibin subunit and follistatin gene expression, and in this way may modulate gonadotrophin gene expression and secretion directly as well as via modulation of activin action [132]. Activin not only increases FSH secretion, but also induces FSHβ gene expression in gonadotrophs. Smad 3 appears to be the principal regulator of activin-induced FSHβ transcription through interactions with the winged-helix/forkhead transcription factor, forkhead box L2 [1,133]. Additional details of the molecular mechanisms underlying regulation of FSHβ transcription by activin are described below in the section on Molecular Biology of LH and FSH Subunit Genes.

MOLECULAR BIOLOGY OF LH AND FSH SUBUNIT GENES α-Subunit To date, the promoter regions of mouse and human α-subunit genes have been studied the most among species, and the cis-elements and trans-factors necessary for gonadotroph-specific gene expression are quite similar in these two species. The mouse α-subunit gene contains at least three types of sequences for transcription factor interaction within the proximal 5’-flanking region: cell type-specific

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FIGURE 7.18 Schematic diagram of 5’-flanking regions of the rodent α-subunit, LHβ, and FSHβ genes which contain cis-acting elements and transcription factors important for cell-specific and GnRH-regulated expression of each gene. Note that this illustration is simplified for clarity. AP-1, activating protein-1; CArG, CC (A/T)6GG factor; CRE, cAMP response element; CREB, CRE binding protein; DARE, downstream activin regulatory element; Egr-1, early growth response protein-1; ER, estrogen receptor; ERE, estrogen response element; FoxL2, forkhead/ winged-helix family protein; GnRH-RE, GnRH-response element; GRAS, GnRHR activating sequence; GSE, gonadotroph-specific element; Lhx, LIM-homeodomain protein; NF-Y, nuclear transcription factor-Y; Oct-1, octamer-1; Otx, orthodenticle; PGBE, pituitary glycoprotein hormone basal element; Pitx, pituitary homeobox; SBE, SMAD binding element; SF-1, steroidogenic factor-1; SMAD, Sma/Mothers against decapentaplegic homolog; Sp-1, specificity protein-1; SURG-1, sequence underlying responsiveness to GnRH.

(gonadotrophs vs thyrotrophs and/or trophoblasts), basal, and hormone-responsive cis-elements. These include a GnRH-response element (GnRH-RE) at positions 406 to 399, a pituitary homeobox-1 (Pitx-1) binding site at 398 to 385, a pituitary glycoprotein hormone basal element (PGBE) at 344 to 300, and a gonadotroph-specific element (GSE) at 215 to 207, relative to the major transcriptional start site α-subunit [134] (Fig. 7.18). The element in the mouse α-subunit promoter that has been best characterized as a basal, tissue-specific enhancer is the GSE. The GSE sequence is highly conserved among species and is bound by SF-1. An orphan nuclear receptor, SF-1, was first identified by its ability to coordinately regulate the expression of genes encoding enzymes in the corticosteroid biosynthetic pathway. It is expressed in the ventromedial hypothalamus, pituitary gonadotrophs, adrenal cortex, and gonadal tissues and is considered as a defining factor for pituitary-specific expression of α-subunit gene. SF-1 deletion in mice precludes adrenal and gonadal development and also results in the selective loss of expression of gonadotroph-specific markers, including LHβ, FSHβ, and GnRHR, and a reduction in α-subunit levels, and a mutation in human SF-1 causes similar defects [135,136]. An additional putative basal enhancer of the mouse α-subunit gene is the PGBE [134]. A member of the LIM (lin-11, isl-1, mec-3, and Lmx-1)-homeodomain (HD) family of transcription factors, Lhx2, was shown to bind to this element. Lhx homeodomain transcription factors are important for pituitary development

and differentiation as well as for transcriptional activation of the gonadotrophin subunit genes. Lhx3 is expressed in the mouse pituitary throughout development and in the adult and can also bind to the PGBE. Targeted disruption of the Lhx3 gene in mice leads to failure of growth and differentiation of the anterior and intermediate lobes of the pituitary, and the development of all pituitary cell lineages, except the corticotrophs, is affected. Similar phenotypes have been reported in human patients with CPHD due to Lhx3 gene mutations [137]. A pan-pituitary bicoid-related HD protein, Pitx-1, has been shown to interact with a Pitx-1 binding site and has been implicated in cooperative function specifically with Lhx3 for the mouse α-subunit gene expression through protein protein interaction [134]. Pitx-1deficient mice exhibit a diminished number of gonadotrophs and reduced expression of gonadotroph-specific genes, including the α-subunit. Pitx-2 and Hesx1 have also been implicated in regulation of the α-subunit gene through the Pitx-1 binding site. Like the mouse α-subunit gene, the human α-subunit gene also contains an array of cis-elements within the proximal 5’-flanking region. A PGBE is located at positions 329 to 320, α-basal element 1 and 2 (αBE1 and 2) at 316 to 302 and 296 to 285, respectively, a GSE at 219 to 211, an α-activating element at 161 to 141, two cAMP response elements (CREs) at 146 to 111, a Pitx-1 responsive element at 80 to 65, and two E boxes at 51 to 45 and 21 to 16. These sequences serve as gonadotroph-specific cis-elements or as common cis-elements for expression

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in gonadotrophs, thyrotrophs, and/or trophoblasts [134]. Two CREs within the 5’-flanking region of the human α-subunit gene are the most important contributors to human promoter activity. These tandem CREs have been shown to regulate α-subunit gene transcription in a synergistic manner [134]. These two CREs play a role not only in regulation by cAMP, but also in basal expression and tissue specificity. Unlike the human, the α-subunit genes of lower primates contain a single CRE, and the α-subunit genes of other mammalian species, including the mouse, also have a single CRE but with a one-basepair substitution from the primate CRE (TGATGTCA). This one-basepair substitution in these species has been shown to decrease the binding affinity for CREB, while heterodimers of cJun and ATF-2 bind to this nonprimate variant CRE with higher affinity. Thus, α-subunit gene responsiveness to cAMP has diverged between primate and nonprimate species. In primates, CREB appears to play a central role, whereas in nonprimates, other nuclear proteins may mediate both tissue specificity and responsiveness to cAMP. The mouse α-subunit gene promoter confers robust GnRH-induced activity compared with that of other species. An upstream element of the mouse α-subunit gene, which binds an Ets factor, confers responsiveness to GnRH, as well as to PMA and to cAMP. GnRH responsiveness requires the cooperative interaction of this GnRH-RE and the PGBE. The need for a complex response unit for the mediation of GnRH stimulation may provide a mechanism for the maintenance of appropriate, tissue-specific expression regulation [134]. The nuclear AR has an important role in modulation of gonadotrophin subunit gene expression, mediating feedback inhibition by testosterone [134]. Previous studies have shown that AR represses human α-subunit gene promoter activity by either directly or indirectly binding to the CRE and αBE region in a ligand-dependent manner, suggesting that activated AR may interfere with the binding of cognate factors, thereby leading to an attenuation in transcription of human α-subunit gene. In contrast, estrogen and ER do not have this effect. Subsequent studies have suggested that AR-mediated suppression of human α-subunit gene expression occurs through protein protein interaction with c-Jun and ATF-2. Thus, in the presence of testosterone, both human and mouse α-subunit gene expression appear to be under repression due to the prevention of binding of cognate factors to the CREs, the PGBE, and/or αBEs by activated AR, thereby interfering with functional and combinatorial interactions through a common coactivator or adapter complex. Moreover, GnRH stimulation, or the absence of testosterone, releases the AR bound to the c-Jun/ATF-2

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heterodimer to allow interactions between these protein complexes and result in synergistic activation of α-subunit gene transcription. Dominant negative mutant forms of Ras, ERK1, and ERK2 reduce expression of the human α-subunit gene, suggesting an involvement of the mitogen-activated protein kinase cascade in the regulation. A mouse with a pituitary-specific deletion of ERK1/2 had reduced α-subunit gene expression and failure to induce expression in response to GnRH, supporting a key role for this signaling pathway in the regulation of α-subunit gene expression [138].

LHβ Subunit Almost all the regulatory cis-elements identified from each species to date as important for LHB gene expression are located in the 5’-flanking region, within 500 bp of the transcriptional start site. Within this region, the proximal 140 bp are conserved across species, implying their functional significance. The rat Lhb gene contains two specificity protein-1 (Sp-1) binding sites located at positions 450 to 434 and 366 to 354, a CC(A/T)6GG factor (CArG) binding site at 443 to 434, two GSEs at 127 to 119 and 58 to 50, two early growth response protein-1 (Egr-1) binding sites at 112 to 104 and 49 to 41, a Pitx-1 binding site at 99 to 94, and a TATA box at 30 to 26 [134]. Among them, five elements, where transcription factors Pitx-1, SF-1, and Egr-1 bind, form a proximal promoter core cassette. Two Sp1 and the CArG binding sites, where the GC box-binding protein Sp1, a three-zinc-finger transcription factor, and a serum response factor-related protein CArG bind, respectively, form a distal enhancer subunit (Fig. 7.18). Proximal 5’-flanking regions of mouse and human LHB genes contain a similar set of proximal elements [1]. The zinc finger protein Egr-1 is an immediate-early serum response gene product expressed in various tissues in response to a range of physiological states. Egr1 mRNA and protein levels are stimulated by GnRH in gonadotroph cell lines and are critical in mediating GnRH-induced Lhb gene expression by binding to its cognate binding sites. Disruption of Egr-1 in mice causes defects in reproductive function, as well as in growth [1]. In the pituitary, gonadotrophs are normal in numbers, but specifically fail to express the LHβ subunit, whereas FSHβ production is evident. These findings suggest that Egr-1 influences reproductive capacity through its regulation of LHB gene transcription. On the other hand, SF-1 is critical for gonadotroph-specific or basal, but not for GnRHinduced, expression of the LHB gene. Both Egr-1 and SF-1 interact with Pitx-1, forming a tripartite protein

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complex to synergistically activate the LHB gene promoter. This functional synergism suggests the importance of interactions among these factors, directly and cooperatively, for both gonadotroph-specific and GnRH-induced expression of the LHB gene. The upstream Sp1 binding sites are also required for GnRH responsiveness. Mutations of these elements alone, preventing Sp1 binding, can reduce GnRHinduced and/or SF-1/Egr-1-mediated rat LHβ gene promoter activity, and combined mutations of the Sp-1, GSE, and Egr-1 binding sites further eliminate GnRH responsiveness [1,112,113]. A transcriptional coactivator, small nuclear RING finger protein (SNURF), interacts with Sp1 and SF-1 to mediate interactions between the distal and proximal GnRH-response regions of the LHB promoter to stimulate transcription. An Egr-1 binding protein (Nab1) is stimulated by GnRH. Nab1 suppresses Egr-1 activation of Lhb gene expression. Nab1 is activated by low-frequency pulsatile GnRH stimulation, whereas Egr-1 is induced to a greater extent by high-frequency pulsatile GnRH stimulation, implicating the balance of Egr-1/Nab1 in the frequency-dependent differential regulation of Lhb by pulsatile GnRH to favor Lhb activation at higher GnRH pulse frequencies [37,139] (Fig. 7.19). The rat Lhb gene promoter region harbors an ERE between 1173 and 1159, which binds to ER and confers a direct stimulatory response to estradiol. This ERE contains a 15-base imperfect palindromic sequence, and ER binds to this region with high affinity. The rat Lhb gene promoter also confers an inhibitory response to testosterone. This inhibitory response to testosterone suppresses GnRH stimulation of Lhb gene promoter activity, and the AR-mediated suppression appears to occur by disrupting the interaction of SNURF with the distal and proximal stimulatory elements of the Lhb promoter [140]. A PKC-mediated stimulatory effect of GnRH on Lhb gene transcription has been well characterized. ERK1 and -2, JNK, and p38 all have been shown to be involved in mediating GnRH-induced Lhb gene expression, downstream of PKC, suggesting an involvement of a multitude of diverse and complex signaling pathways which may eventually participate in a complicated crosstalk network to maximize the signaling amplification. In support of the central role of ERK in mediating Lhb stimulation, targeted pituitary deficiency of ERK1/2 resulted in female infertility due to markedly reduced Lhb expression and LH biosynthesis [138].

FSHβ Subunit GnRH stimulates FSHB expression in gonadotrophs. Along with GnRH, activin from paracrine/autocrine

sources is considered as a major regulator of FSHB gene expression. Activin acts independent of GnRH and can induce FSH secretion in GnRH-desensitized pituitary cells. Activin also enhances GnRH responsiveness in gonadotroph cells and synergizes with GnRH in the stimulation of FSHβ gene transcriptional activity. Rat Fshb gene promoter cis-elements identified to date include a Pitx1/2 binding site in the proximal promoter ( 54/ 48 in rat, 53/ 49 in mouse). This element is important not only for basal expression, but also for synergy with GnRH in the activation of Fshb transcription [141]. Lhx3 expression in gonadotroph cell lines (albeit at low levels) and binding of the protein to the proximal murine Fshb promoter suggests a role in basal, cell-specific expression as well [142]. Given that Pitx-1 can interact with Lhx3 and that the rat Fshb gene promoter contains both Pitx-1 and Lhx3 binding sites, a logical analogy is that these factors may interact with each other to synergize in the tissuespecific expression of the Fshb gene. SF-1 and NF-Y are important basal regulators in many systems. Despite the observation that SF-1 alone has only a minimal effect on mouse Fshb gene promoter activity, NF-YA and SF-1 functionally interact to regulate the mouse Fshb gene expression in a cell type-specific manner, which requires intact GSE and NF-Y binding sites. It is possible that SF-1 and NF-Y maintain and regulate basal and gonadotroph-specific expression of the mouse Fshb gene [1,142] (Fig. 7.18). A major GnRH-responsive element within the proximal FSHB promoter, which contains a partial CRE/ AP1 site, has been characterized by several groups in the rat and mouse gene [1,112,142,143]. This GnRHresponsive element is conserved in the human. This site appears promiscuous with the ability to bind the bZIP transcription family member, CREB, as well as members of the AP1 family, such as c-Fos and c-Jun. A mechanism by which GnRH stimulates FSHβ transcription is by inducing phosphorylation of promoterbound CREB, leading to the recruitment of the histone acetyltransferase CREB binding protein [144]. The transcriptional repressor, inducible cAMP early repressor (ICER), is induced by pulsatile GnRH. High GnRH pulse frequencies preferentially induce ICER, which binds to the CRE/AP1 site to antagonize CREBmediated FSHβ stimulation, thereby attenuating FSHB expression [112]. These data suggest that ICER production antagonizes the stimulatory action of CREB to attenuate FSHB transcription at high GnRH pulse frequencies, thereby playing a critical role in regulating cyclic reproductive function (Fig. 7.19). In contrast to the αGSU and LHβ genes, androgen has a stimulatory effect on the ovine FSHβ gene promoter activity [1]. This effect is mediated by direct

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Low GnRH pulse frequency

In the case of Fshb, much of this specificity is derived from interaction with FoxL2, a winged-helix/forkhead transcription factor, which binds to DNA and is essential for Fshb expression and activin responsiveness, both in vitro and in vivo in mice [133,145]. The TALE homeodomain proteins, Pbx1 and Prep1, have also been shown to interact with Smad proteins to mediate activin induction of Fshb transcription [146].

High GnRH pulse frequency

CBP

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DIAGNOSTIC TESTS

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P300 SP1 SP1

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FIGURE 7.19 Low and high GnRH pulse frequencies differentially regulate FSHβ and LHβ transcription through induction or modification of transcription factors and recruitment of coactivators. CREB, cAMP response element binding protein; CBP, CREB binding protein; SF-1, steroidogenic factor-1; Egr-1, early growth response protein-1; SP-1, specificity protein-1; Nab, NGFI-A binding protein; P300, transcriptional coactivator. Source: From Ciccone NA, Kaiser UB. The biology of gonadotroph regulation. Curr Opin Endocrinol Diabetes Obes 2009;16(4):321 7.

interaction of the AR with the proximal FSHB promoter. Progesterone has similar activating effects on FSHB, mediated by the PR. In contrast, estrogen did not appear to have direct positive or negative effects on FSHB gene expression [1]. Activin is a potent inducer of Fshb transcription. Intracellular signaling molecules SMAD2, -3, and -4 have all been shown to mediate activin-stimulated activity of the Fshb promoter as well as FSHβ mRNA synthesis, although the role of SMAD2 is less clear [133,145]. Three activin response elements have been identified in the Fshb promoter. SMAD3 and -4 bind to the distal SBE ( 266/ 259) of the rat Fshb gene promoter, and deletion of this SBE abolishes activin-mediated Fshb gene transcriptional activity. Pitx-2 has been shown to upregulate basal and activin-mediated rat Fshb gene promoter activity in a dose-dependent manner, and elimination of the Pitx-2 binding site results in a loss of activin-regulated Fshb gene promoter activity. Activin-regulated Fshb gene expression is highly specific and selective compared with other gonadotrophin genes. This signal specificity and selectivity is likely to be achieved by the interaction of SMADs with other tissue- and cell-specific partners or coregulators.

The effects of graded single doses of GnRH (25 100 μg) given intravenously on serum LH and FSH secretion in normal men and women have been extensively studied [33]. Rapid and dose-dependent increases in serum LH and FSH levels are seen with peak levels of both hormones within 20 30 minutes. Increases in serum LH are greater than those in serum FSH levels. The utility of a 100-μg GnRH bolus as a diagnostic test in classifying 155 patients with disorders of the hypothalamic pituitary gonadal axis was assessed [147]. A wide range of LH responses was seen, with peak values ranging from 8 to 34 mIU/mL. Patients with HH have diminished LH responses and often exhibit a greater FSH than LH response. A similar prepubertal pattern of diminished LH and FSH response and reversal of the usual LH/FSH ratios after GnRH administration can be seen in patients with anorexia nervosa. After repetitive administration of GnRH pulses, LH and FSH responses are normalized in both groups of patients, indicating the intactness of the pituitary. The GnRH stimulation test has some utility in the diagnosis of pubertal disorders. An alternative test now more commonly used, particularly in the diagnosis of CPP, is the measurement of LH 2 3 hours after administration of a GnRH agonist such as leuprolide [148]. However, the LH and FSH responses to GnRH also vary with the sex, age, degree of sexual maturation, and the phase of menstrual cycle. Thus, the GnRH stimulation test has had a limited usefulness in the diagnosis of hypothalamic pituitary disorders in adults. In most instances, careful evaluation of baseline hormone levels (LH, FSH, and testosterone) in the appropriate context of historical and radiologic data is sufficient to arrive at the correct diagnosis. The GnRH stimulation test has also not generally been useful in differentiating delayed puberty from IHH.

Clomiphene Test Clomiphene is an antiestrogen, but acts as a partial agonist in that it also has weak estrogenic activity at

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high doses. In adult men and women, its antiestrogenic action predominates in vivo, resulting in increased secretion of GnRH and thereby LH and FSH. In prepubertal children with very low or negligible amounts of estrogen, it acts as an estrogen and inhibits LH and FSH. The usual protocol is to administer 100 mg of clomiphene orally each day for 5 7 days. Serum LH levels generally increase by 100% or more, while FSH levels show about a 50% increase over baseline [149]. A normal response to clomiphene indicates normality of the hypothalamic pituitary axis. However, an abnormal/absent response does not distinguish hypothalamic from pituitary disease. Clinical indications for the clomiphene test are limited.

Detection and Characterization of Gonadotrophin Pulse Patterns Soon after the introduction of LH and FSH RIAs, it became apparent that these hormones are secreted into the circulation in a pulsatile pattern. The ability to measure small amounts of hormones in serum samples led to recognition that secretion of many, if not all, hormones is episodic [150]. In light of this episodic hormone secretion, there was an interest in the development of discrete pulse-detection algorithms. Signal-detection methods, including spectral analysis, cross-spectral analyses, and other procedures for smoothing and filtering data to enhance detection of a signal of interest have existed for a considerable time, but have limitations for the detection of biological hormone pulses. Several difficulties inherent in the biologic data confounded efforts to develop a single, ideal pulse-detection algorithm. As opposed to the highly episodic physical or mathematical events, LH pulse patterns in humans are characterized by a lack of absolute regularity in the frequency and amplitude of LH pulses, limitations of assay precision, and dependence of false-positive and false-negative rates on sampling intensity [150]. Santen and Bardin developed the first algorithm, which defined a peak as a 20% increase in the hormone concentration in a single sample over the preceding sample [151]. Subsequent modifications of the method include selection of a chosen multiple (e.g., threefold) of the assay coefficient of variation to define the pulse. The simplicity of use and apparent freedom from assumptions have made this a widely used program, and modified versions are still commonly used [86]. A number of refinements and alternative algorithms have been developed [150]. Such refinements have enhanced the validity and applicability of pulsedetection algorithms. The development of highly sensitive and specific immunoassays for quantitation of LH

and FSH have helped to clarify the nature and significance of low-amplitude pulses. Developments of conditional probability modeling methods and the adaptation and validation of cross-correlation methods for demonstrating coincidence of two or more hormone pulses have shown a high degree of concordance between LH, FSH, free α-subunit, and testosterone pulses [150]. Application of deconvolution to pulse analysis has made it possible to determine the instantaneous secretory rates of hormones. Deconvolution techniques resolve the hormone series into appearance and disappearance curves. Current deconvolution models permit determination of: (1) identity and characterization of all secretory episodes; (2) production rates of the hormone; and (3) estimation of half-life of hormone disappearance. The recent development of a highly sensitive enzyme-linked immunosorbent assay for assessment of mouse LH concentrations in small volumes of whole blood have now opened the door for applying these techniques to research studies in animal models to gain more insights into the control of gonadotrophin pulse patterns [152].

CLINICAL DISORDERS AFFECTING THE GONADOTROPH Disorders affecting gonadotrophin secretion and action can be broadly classified into two categories: first, hypogonadotrophic disorders, or those associated with decreased LH and/or FSH secretion; and second, hypergonadotrophic disorders, or those characterized by excessive or physiologically inappropriate secretion of LH and/or FSH.

Hypogonadotrophic Disorders Because LH and FSH are trophic hormones for the testes and ovaries, impaired secretion of these gonadotrophins (hypogonadotropism) results in hypogonadism. Clinically, patients with HH may present with symptoms and signs of sex steroid deficiency and/or infertility. The symptoms and signs of androgen deficiency depend on the time of onset and the degree of gonadotrophin deficiency. Androgen deficiency during fetal life may result in failure of the Wolffian structures to develop, ambiguity of external genitalia due to failure of fusion, hypospadias, microphallus, or a combination of these. In patients with isolated HH, placental hCG stimulates the fetal testis to produce sufficient androgen in early fetal life. Therefore, most patients with congenital GnRH deficiency have normal Wolffian structures and external genitalia. However, during the second half of pregnancy, the fetal gonad is under the

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control of fetal pituitary-derived LH and FSH. Therefore, severe LH and FSH deficiency during this period may result in undescended testes and microphallus, since testicular descent is partly androgendependent. In addition, because of FSH deficiency, these patients have impaired development of seminiferous tubules [31]. The ovaries have little functional activity during fetal development, so LH and FSH deficiency during fetal development in females results in no significant clinical manifestation. If androgen or estrogen deficiency occurs after birth but before puberty, sexual development is delayed or arrested; these children present with delayed or absent puberty. Other androgen- and estrogen-dependent events that occur in the peripubertal period, such as the epiphyseal fusion of long bones and calcification of laryngeal cartilages, are also delayed. Delay in the fusion of the epiphyses results in continued growth of long bones causing increased height and reversal of the upper segment to lower segment ratios, with eunuchoidal proportions (span greater than height by more than 2 cm). Androgen deficiency acquired after completion of puberty is characterized by regression of the secondary sexual characteristics, impairment of libido and sexual function, loss of muscle mass, increased fat mass, and infertility. However, these changes often occur insidiously so that many years may elapse before these patients seek medical attention. This may partly explain why men with hypogonadism resulting from prolactin-secreting pituitary adenomas usually have large tumors (macroadenomas) at the time of initial presentation. Early interruption of the menstrual cycle by hyperprolactinemia in women, on the other hand, alerts them to seek medical advice earlier, leading to an earlier diagnosis of their pituitary adenoma and detection when the tumor is still small (microadenomas). Disorders associated with HH can be classified into congenital and acquired disorders (Table 7.4). Acquired disorders are much more common than congenital disorders and may result from functional abnormalities in GnRH or gonadotrophin secretion or from organic diseases such as neoplastic, inflammatory, or infiltrative diseases.

Congenital Hypogonadotrophic Disorders Heterogeneity of Pulsatile Gonadotrophin Secretion in Patients with Congenital HH There is considerable heterogeneity in the clinical presentation of HH. The phenotype, to a large degree, is determined by the severity of GnRH deficiency. Those with the most severe deficiency may present with complete absence of pubertal development,

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sexual infantilism, and in some cases with varying degrees of hypospadias and undescended testes. Male patients may have complete absence of secondary sex characteristics, infantile testes, and azoospermia, while female patients may present with primary amenorrhea. Patients with partial GnRH deficiency may have varying degrees of delay in sexual development in proportion to the severity of gonadotrophin deficiency. The term “fertile eunuch syndrome” has been used to describe patients with eunuchoidal proportions and delayed sexual development but who have normalsized testes. Such individuals appear to have sufficient gonadotrophins to stimulate high intratesticular testosterone levels and to initiate spermatogenesis, but not enough testosterone secretion into the blood to adequately virilize the peripheral tissues; they are, in fact, partially gonadotrophin-deficient [153]. Patients with HH are quite heterogeneous in their LH secretory profiles [86,154]. Patients with the most severe GnRH deficiency typically display no pulsatile LH secretion at all. Others may display low-amplitude pulses or reduced frequency. A subset is characterized by sleep-entrained pulses reminiscent of the pattern seen in early stages of puberty. Nonreproductive Phenotypes Associated with Kallmann Syndrome and HH Kallmann first described a syndrome characterized by delayed or arrested sexual development associated with anosmia [155]. These patients have selective gonadotrophin deficiency resulting from an isolated defect in GnRH secretion. The primary pathogenic defect in these patients is impaired migration of GnRH and olfactory neurons into the hypothalamus during development. Details of the genetics and pathogenesis of this syndrome were described earlier (see section on GnRH Neuronal Development); here we will focus on the distinctive clinical manifestations [31,64]. Although anosmia and hyposmia are the most well known and the first associations described in this syndrome, a spectrum of other somatic abnormalities has been recorded. Synkinesia is found in about 80% of patients with Kallmann syndrome due to mutations in the Xlinked KAL1 gene, and unilateral renal agenesis or horseshoe-shaped kidneys in approximately 30%. This defect is often asymptomatic and must be evaluated by ultrasound examination. Other manifestations include sensorineural hearing loss, high-arched palate, cleft lip and palate, color blindness, cryptorchidism, and optic atrophy. Nonreproductive phenotypes associated with Kallmann syndrome due to FGFR1 mutations include synkinesia in about 10% of affected individuals, cleft lip and/or palate, dental agenesis, digit malformations (brachydactyly, syndactyly), and

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agenesis of the corpus callosum, which can be seen on brain magnetic resonance imaging (MRI). Nonreproductive phenotypes associated with mutations in PROK2 or PROKR2 have been less well characterized, but some that have been reported include obesity, pectus excavatum, seizures, synkinesia, higharched palate, pes planus, hyperlaxity of digits, and hearing loss. Whether these manifestations are all truly linked to the genetic mutation remains to be determined. Mutations in CHD7 have also been identified in patients with Kallmann syndrome. This gene has been implicated in CHARGE syndrome, a multisystem disorder consisting of eye coloboma, heart defects, choanal atresia, retardation of growth and development, genitourinary anomalies, and ear abnormalities (vestibular and auditory). CHD7 mutations in patients with Kallmann syndrome without the full CHARGE syndrome are thought to represent milder allelic variants, but some of the features of CHARGE syndrome may be present. In particular, high-arched or cleft palate, dental agenesis, auricular dysplasia, deafness and hypoplasia of semicircular canals, coloboma, and short stature have been associated phenotypic features [78,156]. The genetic causes of HH due to defects in GnRH secretion (rather than developmental defects in GnRH neuronal migration) are not typically associated with anosmia and also usually have fewer nonreproductive phenotypes. These disorders are often termed isolated or normosmic HH. For example, patients with inactivating mutations in KISS1R have isolated HH with hypogonadism but no other identified distinctive phenotypic features. Some male patients with KISS1R mutations have been reported to have cryptorchidism [157]. Mutations in TAC3 or TACR3 are more commonly associated with microphallus in males and absent spontaneous thelarche in females, yet these patients had evidence for reversibility of their hypogonadotropism later in life. These phenotypic manifestations suggest that the NKB pathway plays an important role during early sexual development, but its importance in sustaining the integrity of the hypothalamic pituitary gonadal axis appears attenuated over time [154]. Mutations in the GnRH and GnRHR genes also cause isolated normosmic HH, with the clinical severity determined in large part by the degree of loss of function caused by the mutation [94,95,103]. Mutations in the genes encoding leptin or leptin receptor cause normosmic HH associated with morbid obesity, and mutations in PCSK1, a prohormone convertase that processes the GnRH prohormone to the mature secreted biologically active decapeptide, is also associated with obesity, as well as hypoglycemia and hypocortisolemia associated with impaired processing of POMC and proinsulin [96].

Mutations in the Genes Encoding LHβ and FSHβ Subunits Hypogonadism Associated with Mutations in the FSHβ Gene Inherited mutations of the FSHB gene are uncommon, but have been reported to produce hypogonadism and delayed puberty in both males and females. Four inactivating FSHB mutations have been characterized in women, which resulted in a similar phenotype of delayed puberty, absent or incomplete breast development, primary amenorrhea, and infertility, with low levels of E2 and P, high LH, and undetectable FSH [158 161]. A detailed study of ovarian function from one of these FSH-deficient women showed no effects of LH excess. Ovarian synthesis of sex steroids occurs according to a two-cell model involving collaborative actions of theca and granulosa cells. LH acts on theca cells to stimulate the synthesis of androgens, which diffuse into adjacent granulosa cells. FSH acts on granulosa cells to mediate follicular development and the expression of aromatase, converting androgens derived from theca cells to estrogens. In other conditions involving increased ratios of LH to FSH, such as polycystic ovary syndrome, hyperandrogenism occurs. In contrast, the patient with isolated FSH deficiency had a low serum testosterone concentration despite a high serum LH concentration, suggesting that FSH-induced follicular recruitment and development are necessary for increased androgen production. This finding is consistent with studies showing that FSH enhances the production of androgen by theca cells. These findings provide supportive evidence that FSH is not necessary for the development of small preantral follicles readily responsive to FSH. Female mice deficient in FSHβ are infertile with a block in folliculogenesis prior to antral follicle formation [162]. Four men with inactivating FSHβ mutations have been described. All were azoospermic, but two had normal puberty associated with normal to low-normal testosterone levels and high LH levels, whereas a third presented with a low testosterone concentration and absent puberty [158,160,161,163]. FSH-deficient male mice, on the other hand, are fertile although they have small testes and subnormal spermatogenesis, indicating that FSH is not absolutely essential for spermatogenesis in mice [162]. Hypogonadism Associated with Mutations of the LHβ Gene Mutations that abolish the activity of LH are rare; they have been reported in six men and one woman [161,164 166]. The phenotypes of the men suggest that

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LH is not required for male sexual differentiation but is critical to the proliferation and function of Leydig cells and to the induction of puberty. Infertility and very low levels of spermatogenesis persist in the affected men, despite long-term exposure to hCG, suggesting that the absence of perinatal exposure to LH alters Leydig cell proliferation and maturation, impairing the onset of normal spermatogenesis. The one case reported in a female revealed a phenotype characterized by normal pubertal development and menarche, secondary amenorrhea, and infertility.

Inactivating Mutations of LH and FSH Receptor Genes Inactivating Mutations of the LH Receptor Gene In genetic males, homozygous inactivating mutations of LHR cause 46,XY disorders of sexual development and differentiation, whereas mutations that cause only partial loss of function result in hypospadias and/or micropenis and hypogonadism [167] (Fig. 7.8A). hCG and LH bind to the same LHR on testicular Leydig cells. Early in gestation, hCG, acting through the LHR, is fundamental for secretion of testosterone and development of the male external and internal genitalia. Inactivating mutations of LHR result in reduced or absent testosterone secretion and consequent impairment of masculinization of the external genitalia. In the absence of androgen action during human fetal development, the external genitalia remain female, irrespective of chromosomal or gonadal sex. Interestingly, a deletion of exon 10 of LHR has been described, resulting in a phenotype of hypogonadism in a man with normal male external genitalia. In vitro, the LHR with this deletion responded normally to hCG, but not to LH, explaining the phenotype while revealing that exon 10 of the LHR is responsible for specificity of LH binding [167]. The clinical findings of LH-deficient hypogonadal men with LHB mutations contrast with the feminization of the external genitalia in 46,XY subjects harboring inactivating mutations in the LHR. Four unrelated women with different mutations in LHR have been reported [167]. All patients were identified because they were sisters of probands with 46,XY disorders of sexual development. All had female external genitalia, normal development of secondary sex characteristics, increased LH levels, primary or secondary amenorrhea, and infertility. In contrast to the 46,XY subjects, the phenotypes of 46,XX patients with LHB and LHR mutations were very similar, with the exception of the difference in circulating LH levels. Women with LHB mutations may be treated with exogenous LH or hCG, whereas women with LHR

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mutations are resistant to LH and, at present, no treatment is effective in rescuing their fertility. Inactivating Mutations of FSH Receptor Gene The first inactivating FSHR mutation in females was found in association with features of hypergonadotrophic hypogonadism—primary amenorrhea, variable pubertal development, high gonadotrophins, and low estrogen levels [167]. Ovaries were hypoplasic with impaired follicle growth. No responses were observed to treatment with high doses of recombinant FSH. This first identified mutation was only partially inactivating. Identification and characterization of a patient with a completely inactivating FSHR mutant showed delayed puberty, primary amenorrhea, elevated gonadotrophins, low estradiol and inhibin B levels, and testosterone levels in the lower/normal range. She had osteoporosis, a hypoplasic uterus and ovaries, and follicles were at the primordial stage with absent secondary follicles. Thus, FSH appears to be critical for follicular maturation beyond the primary stage in humans. FSH resistance is associated with the persistence of a large number of small follicles in the ovaries, probably due to low follicular recruitment, as observed in prepubertal ovaries (Fig. 7.8B). Male family members harboring the first identified inactivating FSHR mutation had normal pubertal development but moderately or slightly decreased testicular volume, and were fertile. They had normal plasma testosterone, normal to elevated LH levels, but high FSH. None of the patients had azoospermia, but showed variable spermatogenic abnormalities. Studies of these individuals showed that FSH action is required for a normal spermatogenesis but is not critical for male fertility [167].

Other Congenital Hypogonadotrophic Syndromes Prader Willi Syndrome Prader Willi syndrome is a multisystemic disorder associated with dysfunction of the hypothalamic pituitary axis. It is characterized by a range of mental and physical symptoms mostly related to hypothalamic deficiency, including short stature, muscular hypotonia, excessive appetite with progressive obesity, hypogonadism, GH deficiency, mental retardation, behavioral abnormalities, sleep disturbances, respiratory disease, and dysmorphic features [168]. Prader Willi syndrome arises from the lack of expression of paternally inherited imprinted genes on chromosome 15q11-q13. These genes are imprinted and silenced on the maternally inherited chromosome. Therefore, the deletion of the paternally derived copy of the normally active genes produces the

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disease. In 70 75% of affected individuals there is a deletion of that segment of the paternally derived chromosome 15; 20 25% of patients exhibit maternal disomy of the same region of chromosome 15 (inheritance of both copies of this region of chromosome 15 from the mother, rather than one from each parent); 2 5% have sporadic or inherited microdeletion in the imprinting center; and 1% have translocations. Three paternally derived intronless genes (MKRN3, MAGEL2, and NDN) have been identified in the 15q11-q13 region, as well as the small nuclear ribonucleoprotein polypeptide N gene encoding for SNURF [168]. Interestingly, as noted earlier, isolated deletion of the paternally inherited allele of MKRN3 causes isolated CPP, without other phenotypic features of Prader Willi syndrome [98]. Patients with Prader Willi syndrome more often have HH or hypergonadotrophic hypogonadism, suggesting that other genes in the imprinted Prader Willi syndrome critical region may also influence function of the hypothalamic pituitary gonadal axis, although some patients with Prader Willi syndrome do present with CPP [169]. Laurence Moon Biedl Syndrome Laurence Moon Biedl or Bardet Biedl syndrome (BBS) is an autosomal recessive disorder characterized by obesity, hypogonadism, mental retardation, polydactyly, and retinitis pigmentosa. Renal abnormalities are common, and speech disorders, brachydactyly, polyuria and polydipsia, ataxia, poor coordination/clumsiness, diabetes mellitus, left ventricular hypertrophy, and hepatic fibrosis can also occur. Twelve genes are known to be associated with Bardet Biedl syndrome. Molecular genetic testing is available on a clinical basis for some of the most common mutations. However, despite the identification of 12 BBS genes, the molecular basis of the disorder remains elusive. All of the known BBS proteins are components of the centrosome and/or basal body and have an impact on ciliary transport [170]. Miscellaneous Congenital Hypogonadotrophic Syndromes A large number of congenital defects and syndromes have been described in association with HH. An extensive list of these disorders can be found in textbooks of genetic disorders.

septo-optic dysplasia and hypopituitarism, ranging from CPHD including gonadotrophins to isolated GH deficiency [8 10]. Mutations in SOX2 and SOX3 are also associated with septo-optic dysplasia, anterior pituitary hypoplasia, and HH [10]. Mutations in PITX2 cause Rieger syndrome, characterized by defects in the eyes, teeth, and heart as well as pituitary hormone deficiencies [13]. Mutations in LHX3 and LHX4 have been identified in patients with CPHD including impaired gonadotrophin release [6,8,10,137]. Mutations in PROP1 are the most common genetic cause of CPHD [5]. Gonadotroph differentiation is also impaired, and homozygous females and most males are infertile. In contrast to PROP1 mutations, mutations in PIT1, also a member of the POU homeodomain family, have pituitary deficiencies limited to GH, prolactin, and TSH [6]. SF-1 is a member of the nuclear receptor family expressed throughout the reproductive axis (hypothalamus, pituitary, and gonads) and in the adrenal gland. It is a key transcriptional regulator of many genes involved in sexual differentiation, steroidogenesis, and reproduction, including the pituitary gonadotrophin α-subunit, LHβ, FSHβ, and GnRHR genes [14]. SF-1 null mice show complete adrenal and gonadal agenesis as well as impaired development of the ventromedial hypothalamus and of gonadotrophs. Patients with mutations in SF-1 have been described with varying degrees of XY sex reversal, testicular dysgenesis, ovarian insufficiency, adrenal failure, and impaired pubertal maturation. DAX-1 (dosage-sensitive sex-reversal adrenal hypoplasia critical region on the X chromosome protein 1) is a nuclear receptor transcription factor related to SF-1 with a similar distribution pattern of expression. Mutations in NR0B1, the X-linked gene encoding DAX-1, are associated with X-linked HH and adrenal hypoplasia congenita [14]. HH is often mild, with a mixed defect of hypothalamic and pituitary function, revealing itself as failure to undergo puberty or incomplete puberty. DAX-1 is a transcriptional repressor and paradoxically has been shown to inhibit SF-1-mediated transcription of an array of target genes, including LHβ. How the loss of function in these two opposing genes, SF-1 and DAX-1, results in similar phenotypes is not well understood.

ACQUIRED HYPOGONADOTROPHIC DISORDERS

Transcription Factor Mutations Mutations in pituitary transcription factors associated with HH were discussed earlier (see section on Molecular Basis of Pituitary Development) (Fig. 7.1, Table 7.1). For example, human mutations in HESX1 have been identified and are associated with variable

Hypothalamic Amenorrhea HA is a reversible disorder in which no anatomic or organic abnormalities of the hypothalamic pituitary ovarian axis can be identified [171,172]. It is

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the most common cause of secondary amenorrhea, responsible for approximately 35% of cases. HA is associated mainly with conditions of stress or energy deficits. Dieting, robust psychological stress, acute or chronic medical illness, or excessive exercise can lead to disruption of hypothalamic pituitary activity controlling ovarian function. All such stressors negatively affect the reproductive axis by acting on hypothalamic regulatory pathways. The key finding in HA is the reduction in central GnRH release from the MBH, leading to a reduction in GnRH pulse frequency, amplitude, or both, which in turn results in lower levels of LH and FSH secretion by the pituitary gland. The resulting gonadotrophin deficiency fails to provide adequate stimulation to the ovarian follicles so that the normal sequence of follicular growth, maturation, follicular selection, and ovulation becomes attenuated. As a result, ovarian estradiol production is low and endometrial growth is reduced, resulting in prolonged intervals of amenorrhea. The transition from normal menstrual cycles to anovulation and amenorrhea can take place gradually and may be characterized by inadequate luteal phases, irregular menses, and ultimately complete amenorrhea [171,172]. HA is of particular clinical importance as the associated hypoestrogenism has been correlated with decreased bone density. Diagnostic criteria for HA include amenorrhea for at least 6 months with low serum LH and FSH levels. HA is associated with eating disorders such as bulimia and anorexia nervosa and with exercise-associated amenorrhea [171,172]. There is a high prevalence of amenorrhea, anovulatory cycles, and other menstrual irregularities in adult female athletes, particularly long-distance runners, dancers, and swimmers. The athletes tend to weigh less and have a lower percentage of body fat. Serum estradiol levels and the frequency of LH pulses are lower in athletes who are amenorrheic than in nonexercising controls. If intense exercise or an eating disorder manifests prior to the onset of puberty, then the onset and progression of puberty can be delayed. Periods of rest or reduction in exercise intensity due to injury are associated with rapid sexual development and the occurrence of menses. Hypogonadism in critical illness is well documented. The degree of suppression of the hypothalamic pituitary gonadal axis correlates with the severity of illness. In men, serum testosterone levels fall at the onset of illness and recover during recuperation. Although the magnitude of gonadotrophin suppression is generally correlated to the severity of illness, there is considerable heterogeneity in serum gonadotrophin profiles in acutely ill patients. The pathophysiology of reproductive dysfunction that

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accompanies the course of acute illness is unknown. Malnutrition, cytokines, and other mediators and products of systemic inflammatory response, and drugs may all contribute to the suppression at multiple levels of the reproductive axis. Similarly, in many chronic illnesses, there is a high frequency of HA, suppression of gonadotrophins, and low testosterone levels. In addition to taking a history and excluding pregnancy, a thorough evaluation of individuals presenting with HA requires baseline studies of FSH, prolactin, estradiol, and an MRI study of the hypothalamic pituitary region to rule out other etiologies of amenorrhea. A progestin challenge test will usually result in scant or no menstrual bleeding; however, addition of combined estrogen with progestin will result in endometrial growth followed by menses because the uterine compartment remains functionally normal [171,172]. It has been hypothesized that maintenance of normal reproductive function in women requires a minimum fat-to-body-mass ratio. The circulating leptin level is a gauge for energy reserves and directs the CNS to adjust food intake and energy expenditure accordingly. Through leptin receptor-mediated pathways in the hypothalamus, leptin activates neural circuits involving an array of neuropeptides to control food intake and energy expenditure. In response to fasting, leptin levels decrease rapidly before and out of proportion to any changes in fat mass, and reproductive hormone levels decrease. Women with anorexia nervosa and exercise-induced HA are chronically energy-deprived and these conditions are associated with low circulating levels of leptin. Leptin treatment in replacement doses in women with HA improves gonadal function and restores ovulatory menstruation [130,173]. Numerous neurotransmitters and neuromodulators modulate GnRH pulsatile secretion. The recognition of the important roles of NKB and kisspeptin in the control of pulsatile GnRH secretion have led to a focus on these neuropeptides as targets and mediators of the effects of stress, inflammation, and negative energy homeostasis, including leptin, on GnRH secretion in the pathogenesis of HA [174]. Although abnormalities of GnRH secretion and menstrual function are well documented in female athletes, similar reproductive abnormalities have not been widely reported in male athletes or men with nutritional deficits. Although it is possible that the signs and symptoms of androgen deficiency in men may be subtle and, therefore, remain undetected, clinically important hypogonadism does not appear to be common in male endurance athletes. Serum testosterone and LH concentrations are usually normal or lownormal in male endurance athletes. Nonetheless, it

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would not be unexpected for similar perturbations in GnRH secretion to occur in men under conditions of stress, exercise, and malnutrition, as has been documented in acute illness, and it is likely that they are underdiagnosed.

Chronic Renal Failure and Gonadal Dysfunction Hypogonadism is very common in patients with end-stage renal disease. Abnormalities at multiple levels of the hypothalamic pituitary gonadal axis contribute to hypogonadism. Sperm concentrations and semen quality are usually depressed; steroidogenesis is also suppressed to varying degrees. Depression of libido and potency is common in uremic men, and menstrual irregularities and infertility are common in premenopausal women. Reproductive dysfunction in uremic patients is usually multifactorial in nature; atherosclerotic disease, neuropathy, malnutrition, chronic illness, hypertension, diabetes mellitus, and drugs all contribute. Hypogonadism usually does not improve after initiation of dialysis. However, restoration of normal kidney function after transplantation will often, although not always, lead to improvement.

Hemochromatosis Hemochromatosis is an iron-storage disorder in which parenchymal iron deposition results in damage to a number of tissues, including liver, pancreas, heart, pituitary, and testes [175]. Hypogonadism and testicular atrophy are common in men with hemochromatosis. Both the pituitary and the testis can be involved by excessive iron deposition. However, the pituitary defect is the predominant lesion in a majority of patients with hemochromatosis and hypogonadism. Thus, HH is by far the more common defect. Diagnosis of hemochromatosis is suggested by the association of diabetes mellitus, hepatic enlargement, heart disease, characteristic skin pigmentation, arthritis, and hypogonadism. Excessive parenchymal iron stores can be demonstrated by determination of high transferrin saturation, very high serum ferritin concentrations, high chelatable iron stores using the agent desferrioxamine, and liver biopsy, as well as by genetic testing [176].

Hyperprolactinemia and Hypogonadotropism Elevated levels of prolactin are often associated with suppression of LH and FSH secretion, resulting in menstrual cycle dysfunction and amenorrhea in women and reduced testosterone levels in men [177]. In fact, the clinical presentations of a small

prolactinoma may be primarily related to the associated HH. Gonadotrophin deficiency in hyperprolactinemic disorders may result from one or more mechanisms. First, prolactin can inhibit hypothalamic GnRH secretion, with current evidence suggesting that this occurs through suppression of kisspeptin [178]. Secondly, a prolactin-secreting tumor may destroy the surrounding gonadotrophs in the pituitary gland by direct invasion, compression, or interference with vascular supply. Third, a pituitary tumor may cause compression of the pituitary infundibulum resulting both in hyperprolactinemia through interference with hypothalamic dopamine delivery to lactotrophs and in hypogonadotropism through interference with GnRH delivery to gonadotrophs. The function of the reproductive axis is typically restored to normal after normalization of serum prolactin levels.

Space-Occupying Lesions Neoplastic and nonneoplastic lesions in the region of the hypothalamus and pituitary can directly or indirectly affect gonadotroph function. Lesions involving the hypothalamus or the hypothalamic pituitary connection may arise primarily in the hypothalamus, in the suprasellar structures, or within the sella itself and extend upwards. Pituitary tumors are discussed elsewhere in this book. In the adult human, pituitary adenomas constitute the largest single category of space-occupying lesions affecting gonadotroph function.

Hypothalamic Syndromes Tumors or lesions in the suprasellar region that affect hypothalamic function to cause HH can often be distinguished from those localized within the sella by the presence or absence of several unique clinical features: 1. Diabetes insipidus is distinctly unusual with lesions contained within the sella turcica. Its presence usually indicates a suprasellar lesion affecting the hypothalamic arginine vasopressin-secreting nuclei in the preoptic and paraventricular region, or compressing the hypothalamic pituitary stalk connection. The association of diabetes insipidus in patients with pituitary adenomas is usually indicative of suprasellar extension. 2. Visual field deficits indicate a suprasellar location. Pituitary adenomas can extend suprasellarly and compress the optic chiasm from below, resulting initially in superior temporal field defects. However, depending upon the origin and location of the suprasellar mass, a variety of visual field defects can result.

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3. The presence of neurologic and/or neuropsychiatric syndromes should alert the physician to the possibility of hypothalamic lesions. 4. Thermoregulatory and sleep defects favor hypothalamic lesions. 5. Autonomic dysregulation, characterized by extreme perspiration, sinus tachycardia, and low blood pressure, is seen only in hypothalamic lesions. 6. Disturbances of appetite and energy homeostasis favor hypothalamic involvement.

TABLE 7.5 Disorders Characterized by Premature or Excessive Gonadotrophin Secretion

Lesions in different regions of the hypothalamus may also present some unique clinical features that can assist in anatomic and functional localization. For examples, lesions in or around the median eminence and ventromedial area, or those resulting in stalk compression, often lead to panhypopituitarism, hyperprolactinemia, and diabetes insipidus. Lesions of the lateral hypothalamus may present with anorexia and weight loss, perhaps related to destruction of the feeding center. Lesions of the ventromedial area lead to hyperphagia and obesity. Lesions of the caudal hypothalamus may cause sexual precocity. The neoplastic lesions may also compress or erode a number of contiguous structures so that the clinical picture is often more complex.

Central nervous system tumors

HYPERGONADOTROPHIC DISORDERS: EXCESSIVE OR NONPHYSIOLOGIC SECRETION OF GONADOTROPHINS Physiologically inappropriate secretion of gonadotrophins may occur in several clinical disorders (Table 7.5). Autonomous secretion of LH, FSH, and/or free α-subunit may characterize pituitary gonadotroph adenomas (see chapter: Nonfunctioning and Gonadotrophin-Secreting Adenomas). Ectopic LH and FSH secretion is uncommon but does occur [179]. Premature reactivation of the hypothalamic pituitary gonadal axis because of premature GnRH secretion can present with the clinical syndrome of precocious puberty.

Ectopic Gonadotrophin Secretion Ectopic production of hCG has been described from a number of neoplasms of trophoblastic and nontrophoblastic origin [180]. These neoplasms include malignant melanoma, adrenocortical carcinoma, breast cancer, renal carcinoma, lung cancer, pancreas, stomach and colon cancer, and a variety of teratocarcinomas. Ectopic LH- and FSH-secreting tumors are exceedingly rare, likely reflecting the need for highly tissue-specific factors for the transcription of the

Idiopathic central precocious puberty Central precocious puberty due to genetic causes Mutations in MKRN3 Mutations in KISS1 Mutations in KISS1R Hypothalamic hamartoma

Other central nervous system lesions such as hydrocephalus, granulomas, cysts, head trauma Gonadotrophin-secreting adenomas Ectopic hCG secretion Ectopic LH or FSH secretion Polycystic ovarian syndrome Sexual precocity in children with congenital adrenal hyperplasia or androgen-secreting tumors McCune Albright syndrome Miscellaneous

subunit genes, together with the need for subunit glycosylation and heterodimerization for functional activity. A patient with a neuroendocrine thoracic carcinoid secreting functional FSH, presenting with symptoms of relapsing ovarian hyperstimulation syndrome (OHSS), was recently reported [179]. A few cases of ectopic LH secretion in association with pancreatic tumors, adrenocortical tumors, or urogenital tumors have been described, presenting with hyperandrogenism and elevated serum androgen and LH levels leading to hyperthecosis and luteinized granulosa-thecal cell tumors of the ovaries.

Central or Gonadotrophin-Dependent Precocious Puberty Puberty results when pulsatile secretion of GnRH is re-initiated and the hypothalamo pituitary gonadal axis is activated. The onset of puberty is marked by breast development in girls and testicular enlargement in boys [32]. Puberty onset is considered precocious if a boy develops secondary sex characteristics before the age of 9 years, or a girl before the age of 8 years. True or central sexual precocity is gonadotrophindependent and results from premature activation of GnRH secretion. The prevalence of CPP is about 10 times higher in girls than in boy. Genetic factors play a fundamental role in the timing of pubertal onset, as illustrated by the similar age

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at menarche among members of an ethnic group and in mother daughter, monozygotic twin, and sibling pairs. A 27.5% prevalence of familial CPP has been reported; indeed, as noted earlier, loss-of-function mutations in MKRN3 were identified as the most common genetic cause of CPP to date, in about a third of patients with familial CPP, suggesting that MKRN3 acts as an inhibitor or “brake” for GnRH secretion during the period of childhood quiescence of the hypothalamic pituitary gonadal axis [98]. In addition, isolated cases with mutations in KISS1 or KISS1R have been identified [32]. Several tumors, mostly intracranial, can cause precocious sexual development [181]. CNS tumors, both benign and malignant, are found more often in boys than in girls. Although hypothalamic hamartomas have been shown to express GnRH, the expression of GnRH did not differ between hamartomas associated or not associated with CPP. The majority of these tumors are hypothesized to cause sexual precocity by removing inhibitory influences on GnRH secretion. Astrocytomas, ependymomas, and gliomas of the optic nerve or hypothalamus have all been reported in association with central sexual precocity. The availability of computed tomography and MRI scanning has made it easier to diagnose CNS tumors. Some germinomas secrete hCG and may result in premature androgen production; others are associated with premature gonadotrophin secretion and true sexual precocity. Besides intracranial tumors, a variety of other intracranial lesions, including granulomas, suprasellar cysts, hydrocephalus, and head trauma, can cause sexual precocity. Patients with McCune Albright syndrome (cafe-aulait spots, fibrous dysplasia of bones, and sexual precocity) often have autonomously functioning follicular cysts in the ovary, which may produce estrogens. However, some children with this syndrome have true gonadotrophin-dependent sexual precocity.

Activating Mutations of the LH Receptor Activating or gain-of-function mutations of the LHR are associated with gonadotrophin-independent sexual precocity in boys, but do not produce a discernible phenotype in females. This is referred to as familial male-limited precocious puberty, also called testotoxicosis, and is an autosomal dominant disease caused by constitutive activating mutations in the human LHR (Fig. 7.8A). The disease generally presents at around 2 4 years of age with signs of pubertal onset, accelerated virilization, and excessive growth velocity ultimately leading to short stature in adulthood due to precocious closure of the epiphyses. Testosterone levels

are high despite low levels of basal gonadotrophins and a prepubertal response to the GnRH stimulation test. Treatment consists of drugs that block adrenal and testicular synthesis of androgens (e.g., ketoconazole) and/or androgenic receptor blockage (cyproterone acetate), ER blockers, and aromatase inhibitors [182]. Most activating mutations are situated in the cytoplasmic halves of the transmembrane segments or in the third intracellular loop and may involve increased or activating interactions with the Gs protein [24]. Activating LH receptor mutations appear to have no phenotype in the female, which may be explained by the absence of LHR expression in prepubertal girls [24].

Activating Mutations of the FSH Receptor The first case of an activating mutation of the FSHR was reported in a male patient who was hypophysectomized because of a pituitary tumor and continued to have normal spermatogenesis despite undetectable serum levels of gonadotrophins [24,183]. The mutation was able to cause ligand-independent constitutive activation of FSHR in vitro. In transgenic mice expressing this mutant FSHR, the constitutive activation of the receptor could completely compensate for the absence of FSH, with normal spermatogenesis and normal fertility (Fig. 7.8B). Subsequent changes in the FSHR were identified in women in association with OHSS [184 186]. OHSS is a common complication of in vitro fertilization, appearing after administration of exogenous FSH followed by hCG administration in the presence of high estradiol levels. Spontaneous OHSS, appearing in the absence of exogenous hormonal stimulation of follicular growth, is rare, but can occur due to the presence of elevated circulating endogenous hCG or TSH levels (mimicking FSH action on granulosa cells)—e.g., in the case of a hydatidiform mole, spontaneous multiple pregnancy, or hypothyroidism. Isolated cases of spontaneous OHSS have been described in women with FSHR variants having an increased sensitivity to normal TSH and/or hCG levels. These mutations confer the ability to respond to hCG or TSH to the receptor because of a conformational change that leads to a loss of specificity. As a result, these mutations increase the risk for OHSS during in vitro fertilization therapy and predispose to spontaneous OHSS in women during pregnancy or in women with hypothyroidism [184 186] (Fig. 7.20).

Polycystic Ovarian Syndrome Polycystic ovarian syndrome (PCOS) can be considered a disorder of gonadotrophin excess resulting from a state of increased GnRH secretion with an increased

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LH hCG

LH receptor

Embryo

LH or hCG activates cellular signals that induce luteinization

hCG Placenta

Theca cells

Granulosa cells

FSH, but not hCG, activates cellular signals that induce follicular proliferation

hCG from the placenta causes hyperstimulation of the ovary with multiple follicle formation

FSH receptor hCG

FSH

β

γ

Cellular signals by hCG that induce follicular proliferation

Cyclic AMP

α

FSH receptor

AC

AC

No cellular signals by hCG that induce follicular proliferation

β

α

γ

α

Mutations in FSH receptor

FSH receptor

Signaling by hCG

FSH

hCG Normal FSH receptor

hCG

FSH

Mutations in FSH receptor

FIGURE 7.20 Pathogenesis of familial gestational spontaneous ovarian hyperstimulation syndrome. hCG synthesized by the syncytiotrophoblast cells of the developing placenta in pregnancy circulates to act at the level of the ovary. In a normal pregnancy, its activity is limited to LH receptors. Stimulation of LH receptors in the corpus luteum results in continued progesterone production to allow the maintenance of pregnancy. Either of two mutations (depicted in red and orange) in the FSH receptor allows activation of downstream signaling events by hCG (modeled as a change in the conformation of the mutant FSH receptor, so that the low-affinity binding of hCG to the receptor ectodomain leads to activation of the serpentine domain). This conformational change results in the stimulation of FSH receptors by hCG in the granulosa cells of developing follicles, leading in turn to excessive follicular recruitment and enlargement. AC, adenylyl cyclase; α, β, and γ, G protein subunits. Source: From Kaiser UB. The pathogenesis of the ovarian hyperstimulation syndrome. N Engl J Med 2003;349(8):729 32.

GnRH pulse frequency. Patients with PCOS have elevated serum concentrations of LH with normal or low FSH. The high LH/FSH ratio may contribute to defective ovulation by causing a cycle initiation defect, since a relative deficiency of FSH during the follicular phase

is associated with impaired follicle maturation and inadequate luteinization. The high mean LH concentrations are due to an increase in LH pulse frequency and/or pulse amplitude, reflecting the increase in pulsatile secretion of hypothalamic GnRH [187].

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PCOS is one of the most common endocrine disorders in reproductive age women, affecting 7 10% of premenopausal women. PCOS is characterized by hyperandrogenism and oligo- or amenorrhea, classically with a peripubertal onset. The ovaries typically appear polycystic on ultrasound [188]. The syndrome is often associated with obesity, insulin resistance, and metabolic syndrome. Women with PCOS are at increased risk for obesity, insulin resistance, type II diabetes, and cardiovascular disease. The pathophysiologic basis of PCOS is unknown. Increased LH pulsatility and reduced FSH levels lead to increased androgen production and anovulation. Hyperandrogenemia and impaired luteinization, in turn, impair hypothalamic sensitivity to progesterone and prevent the slowing of GnRH pulses [189]. LH regulates androgen synthesis by theca cells, whereas FSH regulates aromatase activity in granulosa cells. When the concentration of LH increases relative to FSH, the ovaries preferentially synthesize androgen [187]. Furthermore, insulin stimulates ovarian synthesis of androgens. Insulin acts synergistically with LH in stimulating ovarian androgen production, and also upregulates LH receptors, thus increasing ovarian responsiveness to circulating LH. Insulin also inhibits hepatic synthesis of SHBG, the key circulating protein that binds to testosterone, thereby increasing the proportion of testosterone that circulates in the unbound, biologically available state (Fig. 7.21) [187]. Insulin can also bind to and activate the type 1 IGF-1 receptor, albeit with lower affinity than to its own receptor. IGF-1 receptor is expressed in granulosa cells and also in oocytes in dominant antral follicles, implicating this as another potential pathway in the pathogenesis of PCOS. Furthermore, insulin inhibits IGF binding protein-1 production, thereby augmenting IGF receptor signaling by IGF-1 (derived from the circulation) and IGF-2 (derived from local ovarian synthesis) [190]. Both PCOS and the accompanying insulin resistance appear to have major genetic components. There is evidence for familial aggregation of PCOS that is consistent with a genetic contribution to the disease. It is likely that PCOS is a complex multigenic disorder, as suggested by genome-wide association studies that have identified multiple loci associated with PCOS [191]. These loci include DENND1A, INSR, YAP1, C9orf3, RAB5B, HMGA2, TOX3, SUMO1P1/ZNF217, THADA, FSHR, and LHCGR. The FSHR/LHCGR, DENND1A, RAB5B, and THADA loci were most consistently identified in multiple cohorts. While the receptors for FSH, LH, and insulin are not unanticipated given their known pathophysiologic roles in PCOS, the identification of a link with the DENND1A gene has provided an opportunity for new insights into the pathophysiology of PCOS. DENND1A

functions as a guanine nucleotide exchange factor that interacts with members of the Rab family of small GTPases and is thought to be involved in clathrinmediated endocytosis, facilitating internalization of proteins and lipids, receptor recycling, and membrane trafficking. DENND1A is highly expressed in ovarian theca cells. Alternative splicing yields two variants of DENND1A, one of which (DENND1A. V2) is increased in PCOS theca cells and is associated with increased androgen biosynthesis. These discoveries have identified a potential diagnostic marker and potential therapeutic target for PCOS [191].

TREATMENT OF HYPOGONADOTROPHIC DISORDERS When a neoplasm or other mass lesion is responsible for HH, therapy should be directed at the underlying cause when possible, to evoke a cure. In many instances of pituitary macroadenomas, cure by resection is not possible and adjunct treatment directed at shrinking the tumor and lowering hormone secretion is desirable. If gonadotrophins are normalized, the resultant hypogonadism is usually corrected. The goals of therapy are to induce and maintain normal reproductive function, gonadal sex steroid hormone production, and stimulation of gametogenesis in those desiring fertility. If fertility is not an immediate objective, sex steroid hormone replacement is usually sufficient. For induction of spermatogenesis, therapy with gonadotrophins or GnRH is usually required.

Gonadotrophin Treatment of HH hCG and human menopausal gonadotrophin (hMG) preparations have been commercially available for four decades. hCG is purified from the urine of pregnant women, being secreted primarily by the human placenta during pregnancy. hMG is derived from the urine of postmenopausal women. While hCG primarily interacts with LHRs, hMG contains LH and FSH activities in almost equal proportions. Since their introduction into clinical practice in 1961, gonadotrophins extracted from the urine of postmenopausal women have played a central role in ovulation induction therapy. This initially crude preparation was refined to make available purified urinary FSH. Since 1996, recombinant human FSH (rhFSH), expressed in Chinese hamster ovary cell lines and purified to homogeneity, has been available, and recombinant hCG is also available [192,193]. For ovulation induction, rhFSH is administered subcutaneously with incremental dose increases until

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FIGURE 7.21 Hypothalamic pituitary ovarian axis and the role of insulin. Increased ovarian androgen biosynthesis in polycystic ovarian syndrome results from abnormalities at all levels of the hypothalamic pituitary ovarian axis. The increased frequency of GnRH pulses leads to increased frequency of LH pulses, favoring the production of LH over FSH. The relative increase in LH leads to an increase in ovarian theca cell androgen production. Insulin acts synergistically with LH to enhance androgen production. Scc, side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein; 3β-HSD, 3β-hydroxysteroid dehydrogenase. Solid arrows denote a higher degree of stimulation than dashed arrows. Source: From Ehrmann DA. Polycystic ovary syndrome. N Engl J Med 2005;352(12):1223 36.

serum estradiol concentrations begin to increase, reflecting induction of follicle maturation. The dose is then maintained or decreased and the ovarian response to gonadotrophin therapy is monitored by measuring serum estradiol levels and using transvaginal ultrasonography to measure follicular diameter. Criteria for follicle maturity include a follicle diameter of 18 mm and/or a serum estradiol concentration of 200 pg/mL per dominant follicle. Complications of ovulation induction therapy with gonadotrophins include multiple pregnancies, OHSS, and increased spontaneous miscarriage rates. Multiple pregnancy and OHSS rates can be kept to a minimum by using lower doses of rhFSH, which is particularly relevant in

patients with PCOS. If more than two follicles larger than 15 mm are present, stimulation should be stopped to prevent multiple pregnancies and OHSS. Once a dominant follicle is identified, follicle rupture is induced by hCG administration [192,193]. For induction of spermatogenesis in men with HH, the traditional approach is the administration of hCG to induce full steroidogenesis from Leydig cells and hMG or rhFSH to induce spermatogenesis [194]. A variety of treatment regimens have been used and there is no consensus on what constitutes the optimum dose and schedule of gonadotrophin administration. Doses are adjusted based on serum testosterone levels with the goal to achieve serum testosterone levels in the

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midnormal range. Higher testosterone concentrations tend to be associated with more elevated serum estrogen concentrations and an increased incidence of gynecomastia. Sperm counts should be monitored on a monthly basis. It may take several months for spermatogenesis to be restored. If spermatogenesis is not restored after 6 months of therapy with hCG alone with serum testosterone levels in the midnormal range, then it is recommended to add FSH. Men with initial testes of less than 5 mL almost always require the addition of FSH, whereas spermatogenesis often can be induced with hCG alone in men with larger testicular volumes. FSH doses are guided by testicular size and seminal fluid analysis. It may occasionally take 18 24 months or longer for spermatogenesis to be restored.

Pulsatile GnRH Therapy Pioneering studies by Knobil’s group had predicted that pulsatile administration of GnRH would be required to maintain normal LH and FSH output from the pituitary. As discussed earlier in this chapter, continuous infusion of GnRH in monkeys, made hypogonadotrophic by radiofrequency lesions of the hypothalamic GnRH-secreting nuclei, downregulates LH and FSH secretion [80]. Pulsatile administration of exogenous GnRH using an infusion pump is an effective therapy for the stimulation of endogenous LH and FSH secretion, follicle development, and ovulation in women with GnRH deficiency [195]. The resulting serum FSH and LH concentrations remain within the normal range and the chances of multiple pregnancies and OHSS are therefore low. However, success of GnRH therapy assumes normal pituitary and gonadal function. A pulse interval is 60 90 minutes and a dose of 2.5 10 μg per pulse is typically used, using the lowest dose required to induce ovulation to minimize the likelihood of multiple pregnancies. The agonist analogues of GnRH are not useful for restoring gonadotrophin secretion, because after an initial short-lived stimulatory phase, GnRH agonists downregulate pituitary LH and FSH output. More recently, the use of kisspeptin administration infertility induction protocols has been proposed, especially in patients with PCOS, to further reduce the risks of multiple pregnancies and OHSS [92]. Successful induction of puberty by pulsatile administration of low doses of GnRH has been achieved in boys with Kallmann syndrome or IHH [64,194,196]. Therapy is usually started with an initial dose of 25 ng/kg per pulse administered subcutaneously every 2 hours by a portable infusion pump. Serum testosterone, LH, and FSH levels are monitored, and the dose of GnRH is progressively increased until serum testosterone levels in

the midnormal range are reached. There is considerable variability in GnRH dose requirements and doses ranging from 25 to 200 ng/kg may be required to induce virilization. Once pubertal changes have been initiated, the dose of GnRH can often be reduced without adverse effects on serum testosterone, LH, and FSH levels. Development of anti-GnRH antibodies is an uncommon occurrence, but can be a cause of treatment failure. Treatment failure can also occur in patients with pituitary disease as their etiology of the HH. Pulsatile GnRH therapy is an effective but currently unapproved treatment for GnRH-deficient men seeking fertility and is used primarily in research studies. While induction of virilization by pulsatile GnRH administration in patients with HH has provided important insights into the mechanisms of puberty and regulation of gonadotrophin secretion by GnRH, this approach has no particular advantage over the traditional gonadotrophin therapy. In fact, wearing a portable infusion device can be quite cumbersome and follow-up of these patients often requires considerable physician supervision and laboratory monitoring. GnRH is currently not commercially available.

GnRH Analogues A large number of GnRH analogues are currently available for therapeutic use. The half-life of GnRH is very short (2 4 minutes) as it is degraded rapidly by peptidases in the hypothalamus and pituitary. These peptidases cleave bonds between amino acids 5 and 6, 6 and 7, and 9 and 10. Analogues have been synthesized by changing the amino acids at these positions. Over 2000 synthetic analogues have been synthesized and tested, which can be broadly divided into two classes: GnRH agonist analogues and GnRH antagonist analogues. Agonist analogues bind to the GnRH receptors and initiate the same series of postreceptor events that underlie LH release by native GnRH. However, chronic administration of GnRH agonists leads to a paradoxical decrease in LH and FSH secretion and inhibition of gonadal function, a phenomenon referred to as desensitization or downregulation. The antagonist analogues of GnRH, on the other hand, bind to GnRH receptors and block the action of GnRH. The antagonist analogues thus have no intrinsic ability to trigger the postreceptor events usually attributable to GnRH.

GnRH Agonists Comparison of the amino acid sequences of GnRH across species reveals conservation in the N-terminus (pGlu-His-Trp-Ser) and C-terminus (Pro-Gly NH2), supporting an important role for these residues in

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REFERENCES

receptor binding and activation. The Arg at position 8 is critical for binding, and substitutions at this position result in loss of binding affinity. Several key aspects of the structure activity relationships are important in the design of GnRH agonists [197,198]. Replacement of the Gly residue at position 6 with D-alanine increases the potency and also increases the peptide stability. Replacement of the C-terminal glycinamide residue by an ethylamine group is a key modification that has formed the basis of many subsequent structural modifications, increasing receptor binding affinity and also prolonging the duration of action. When these two modifications are combined, the biologic potency of the resulting compound is further amplified. GnRH agonists can be administered subcutaneously, intranasally, or intramuscularly. These compounds have an initial agonistic response with an increase in circulating levels of LH and FSH, followed by desensitization and downregulation to produce HH, due to desensitization and uncoupling of the receptor from its signaling pathways and downregulation of receptors on the plasma membrane, as well as postreceptor events [197]. The wide clinical applicability of GnRH agonists has spurred development and testing of a large number of agonist analogues. GnRH agonists have been found to be effective therapeutic agents in many sex steroid (androgen or estrogen)-dependent clinical disorders [199]. Their lack of systemic toxicity has been striking. Most side effects have been related to the decrease in androgens or estrogens due to the desired downregulation of gonadotrophin secretion. The main pharmacologic difference among the currently approved GnRH agonists is the method of administration. Leuprolide is the most commonly used GnRH agonist and can be administered as a daily subcutaneous injection or as a monthly depot injection. Osmotic pump implants are also available that deliver leuprolide acetate at a controlled rate for up to 12 months. Long-acting agonists are utilized therapeutically to suppress gonadotrophins in several conditions, including endometriosis, uterine fibroids, central gonadotrophin-dependent precocious puberty [181], and androgen-dependent prostate cancer. Their main drawback is that gonadotrophin suppression does not occur immediately; instead, there is a transient (several days) increase (“flare”) in sex hormone levels, followed by a lasting suppression of hormone synthesis and secretion.

GnRH Antagonists More recently, several antagonist analogues of GnRH have become available; these have several advantages over the agonist analogues. Antagonists are far more potent in inhibiting gonadotrophin secretion in men

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and experimental animals than are agonist analogues. In addition, the antagonist analogues have the potential advantage of rapid onset of inhibitory action and do not have the initial stimulatory effects that characterize GnRH agonists. Changes in the conserved N-terminal residues of GnRH result in analogues with antagonistic properties. This modification, together with the substitution of the Gly at position 6 with a D-amino acid, forms the basis of antagonists [197,200]. The GnRH antagonists cetrorelix and ganirelix are sometimes used in assisted reproduction; in the early to mid-follicular phase of the menstrual cycle, they suppress an early surge in LH, resulting in improved rates of implantation and pregnancy. GnRH antagonists also have applications for palliation of metastatic prostate cancer. In this situation, a direct GnRH antagonist has the advantage of avoiding the initial surge in testosterone seen with GnRH agonists [200].

Acknowledgments The author would like to acknowledge Drs. Shalendar Bhasin, Charles Fisher, and Ronald Swerdloff, the authors of the corresponding chapter in an earlier edition of this textbook, for the use of some of the material in this chapter.

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C H A P T E R

8 The Posterior Pituitary Daniel G. Bichet

INTRODUCTION Disorders of water balance are a common feature of clinical practice. An understanding of the physiology and pathophysiology of vasopressin, perception of thirst, vasopressin receptors, and aquaporin water channels is key to the diagnosis and management of these disorders. Mammals are osmoregulators: they have evolved mechanisms that maintain extracellular fluid osmolality near a stable value, and osmoregulatory neurons express a truncated delta-N variant of the Transient Receptor Potential Vanilloid (TRPV1) channel involved in hypertonicity and thermic perception, while systemic hypotonicity might be perceived by TRPV4 channels. Mutations in the vasopressin gene arginine vasopressin (AVP) are responsible for familial neurohypophyseal diabetes insipidus, a conformational disease, where misfolded fibrillar aggregates within the endoplasmic reticulum cause magnocellular toxicity and death. The antidiuretic action of vasopressin is explained by the interaction of vasopressin with a renal G (guanine nucleotide-binding)-protein-coupled V2 receptor coupled to adenylate cyclase. The activation of V2 receptors on principal cells of renal collecting tubules promotes the cyclic adenosine monophosphatemediated incorporation of water channels in the luminal membrane of these cells. Hereditary nephrogenic diabetes insipidus (NDI) is secondary to mutations in the gene coding for the vasopressin V2 receptor (AVPR2, X-linked NDI) or to mutations in the gene coding for the aquaporin-2 water channel (AQP2, autosomal dominant or recessive NDI). Some rare gain-offunction mutations of the vasopressin V2 receptor are responsible for the nephrogenic syndrome of inappropriate antidiuresis. Nonpeptide vasopressin V2 receptor antagonists are aquaretic compounds useful for the treatment of hyponatremic patients.

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00008-8

STRUCTURE OF THE NEUROHYPOPHYSIS: ANATOMY AND ELECTROPHYSIOLOGY OF VASOPRESSINPRODUCING CELLS The hypothalamus, a small, one cubic centimeter structure in humans, is located at the anterior and ventral part of the thalamus and embodies a group of nuclei that form the floor and ventrolateral walls of the triangular-shaped third ventricle [1]. The neurohypophysis consists of: (1) a set of hypothalamic nuclei, namely the supraoptic nuclei (SONs) and paraventricular nuclei (PVNs), which house the perikarya of the magnocellular neurons; (2) axonal processes of the magnocellular neurons form the supraoptical hypophyseal tract; and (3) the neurosecretory material of these neurons which is transported to the posterior pituitary gland (see Fig. 8.1). Large neurosecretory magnocellular cells of 20 40 μm and smaller parvocellular cells of 10 15 μm are neuroendocrine, autonomic, and circadian controllers and regulators. Tonicity is perceived specifically by neuronal groups on the anterior wall of the third cerebral ventricule (Fig. 8.1). Cells respond to dehydration or to hyperhydration by changing volume, but cells of the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and median preoptic nucleus (MnPO) are “perfect” osmoreceptors, that is, their changes in volume are maintained as long as the osmotic stimulus persists [4]. Cell shrinking during dehydration is mechanically coupled to the activation of delta-N TRPV1 channels [5], a molecular codetector of body temperature and osmotic stress, through densely interweaved microtubule networks present only in osmosensitive cells [4], including excitatory thirst neurons from the SFO [6] (Fig. 8.2). These excitatory SFO neurons project to magnocellular cells of the SONs and PVNs producing vasopressin

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FIGURE 8.1 Schematic representation of the osmoregulatory pathway of the hypothalamus (sagittal section of midline of ventral brain around the third ventricle in mice). Neurons (lightly filled circles) in the lamina terminalis (OVLT), median preoptic nucleus (MnPO), and subfornical organ (SFO) that are responsive to plasma hypertonicity send efferent axonal projections (black lines) to magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON). The axons of these magnocellular neurons form the hypothalamo-neurohypophyseal pathway that courses in the median eminence to reach the posterior pituitary, where neurosecretion of vasopressin and oxytocin occurs. Dendritic vasopressin release during dehydration stimulates sympathetic preautonomic cells in the PVN and directly increased renal nerve stimulation [2], a central integrated response to restore tonicity and volume. Source: Modified from Wilson Y, Nag N, Davern P, et al. Visualization of functionally activated circuitry in the brain. Proc Natl Acad Sci U S A 2002;99(5):3252 7, [3] with permission.

FIGURE 8.2 Cell autonomous osmoreception in vasopressin neurons. Changes in osmolality cause inversely proportional changes in soma volume. Shrinkage activates delta-N transient receptor vanilloid-type (TRPV1) channels and the ensuing depolarization increases action potential firing rate and vasopressin (VP) release from axon terminals in the neurohypophysis. Increased VP levels in blood enhance water reabsorption by the kidney (antidiuresis) to restore extracellular fluid osmolality toward the set-point. Hypotonic stimuli inhibit TRPV1. The resulting hyperpolarization and inhibition of firing reduces VP release and promotes diuresis. Source: Modified from Prager-Khoutorsky M, Bourque CW. Mechanical basis of osmosensory transduction in magnocellular neurosecretory neurones of the rat supraoptic nucleus. J Neuroendocrinol 2015;27(6):507 15.

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STRUCTURE OF THE NEUROHYPOPHYSIS: ANATOMY AND ELECTROPHYSIOLOGY OF VASOPRESSIN-PRODUCING CELLS

and, as a consequence, these neurosecretory cells are depolarized and vasopressin is released both from axonal and dendritic terminals. Dendritic vasopressin release during dehydration stimulates sympathetic

FIGURE 8.3 Cell shrinking of hypothalamic osmoreceptor cells during dehydration is mechanically coupled to activation of delta-N TRPV1 channels. As a result of cell shrinking, the plasma membrane shifts inward (right), increasing the proportion of microtubules that push onto (and activate) delta-N Trpv1 channels [4].

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preautonomic cells in the PVN and directly increases renal nerve stimulation, a central integrated response to restore tonicity and volume [2] (Fig. 8.1). Vasopressin-producing cells in SONs and PVNs also bear delta-N TRPV1 channels, which depolarize during dehydration and hyperpolarize during overhydration. Vasopressin release is the net result of depolarization (Fig. 8.3). Thirst cells of the anterior wall of the third ventricle also project to two conscious areas, the anterior cingulate cortex and the insula, delivering a conscious assessment of the dehydration state and, probably, of the necessary water volume to quench thirst (Fig. 8.4). This is a unique situation where tonicity is consciously perceived, analogous to hunger perception. Also, thirst-promoting neurons transmit negative valence teaching signals that are actively avoided in experimental animals [8].

FIGURE 8.4 Osmoregulatory circuits in the mammalian brain and the periphery. Neurons and pathways are color-coded to distinguish osmosensory, integrative, and effector areas. Afferent pathways from the OVLT to ACC are responsible for thirst perception. Central preautonomic neurons in the PVN are responsible for the increased renal sympathetic activity mediated by perception of dehydration by magnocellular cells in close proximity (see Fig. 8.1). ACC, anterior cingulate cortex; AP, area postrema; DRG, dorsal root ganglion; IML, intermediolateral nucleus; INS, insula; MnPO, median preoptic nucleus; NTS, nucleus tractus solitarius; OVLT, organum vasculosum laminae terminalis; PAG, periaqueductal gray; PBN, parabrachial nucleus; PP, posterior pituitary; PVN, paraventricular nucleus; SFO, subfornical organ; SN, sympathetic nerve; SON, supraoptic nucleus; SpN, splanchnic nerve; THAL, thalamus; VLM, ventrolateral medulla. Source: Reproduced from Bourque CW. Central mechanisms of osmosensation and systemic osmoregulation. Nat Rev Neurosci 2008;9(7):519 31, [7] with permission. I. HYPOTHALAMIC PITUITARY FUNCTION

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The OVLT, SFO, MnPO, and the pituitary gland do not have a blood brain barrier, that is, their capillary endothelium is fenestrated and allows for full exposure to plasma osmotic and hormonal variations including angiotensin II. Excitatory thirst neurons of the SFO specifically expressed AT1 angiotensin receptors [6], most probably explaining the osmoregulatory gain observed with increased circulating plasma levels of angiotensin [9]. This osmoregulatory gain is clinically important since, for the same osmotic stimulus, more vasopressin will be released when plasma angiotensin II is elevated, a common situation seen with hypotension, with decreased effective blood volume of heart failure, and with decompensated cirrhosis, where hyponatremia with high vasopressin levels is often observed. The supraoptic nucleus (SON) lies just dorsal to the optic chiasm and approximately 2 mm from the third ventricle. The paraventricular nucleus (PVN) lies closer to the thalamus in the suprachiasmatic portion of the hypothalamus, but it borders on the third ventricular space. These well-defined nuclei contain the majority of the large neuroendocrine cell bodies, known as the 100,000 magnocellular or neurosecretory cells, that manufacture arginine vasopressin (AVP) and oxytocin [3,10]. Axons and axonal collaterals project to the posterior pituitary as well as to the forebrain and limbic areas [11]. Dendritic release of vasopressin, of considerable importance during dehydration to induce a central preautonomic stimulation [2], leads to extremely high local brain concentrations of vasopressin [10,12]. Supraoptic magnocellular cells predominantly produce vasopressin and oxytocin; in contrast, PVN, constitute an important neuroendocrine center since they contain a diverse population of vasopressin, oxytocin, corticotrophin-releasing hormone (CRH), thyrotrophin-releasing hormone (TRH), and somatostatin (SS), and preautonomic cells [13] (Fig. 8.5). SONs only contain magnocellular cells, however PVNs contain magnocellular and parvocellular neurons. Parvocellular neurosecretory neurons send their axons to the median eminence, from where they release hypophysiotrophic hormones that control function of the anterior pituitary and the hypothalamo-pituitary axes. Parvocellular preautonomic neurons send long descending projections to sympathetic and parasympathetic centers in the brainstem and spinal cord, modulating sympathetic and parasympathetic outflows to a variety of target organs, including the heart, the peripheral vasculature, and the kidneys. In addition to neurosecretory and autonomic targets, PVNs also include neurons that project to hierarchically higher centers in the brain, including the central amygdala, projections recently shown to modulate fear-conditioned

FIGURE 8.5 Schematic model of the paraventricular nucleus (PVN) in the mouse. The anterior (rostral) two-thirds of the PVN contains the vast majority of magnocellular neuroendocrine neurons that project to the posterior pituitary (PP) and parvicellular neuroendocrine neurons that project to the median eminence, then to the anterior pituitary (AP) for thyrotrophin-releasing hormone (TRH), corticotrophin-releasing hormone (CRH), and somatostatin (SS) secretions. The posterior (caudal) one-third of the PVH contains most of the descending preautonomic neurons to the dorsal motor nucleus of the vagus nerve (DMX), the central gray spinal cord (CGS), and the intermediolateral column of the spinal cord (IML). Source: Modified from Biag J, Huang Y, Gou L, et al. Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing. J Comp Neurol 2012;520(1):6 33.

responses [12]. PVNs receive afferents from the nucleus solitarius and other spinal cardiovascular regulatory afferents, therefore PVN coordinate sympathetic, parasympathetic, and somatomotor responses to the endocrine activity. Oxytocin and AVP (Fig. 8.6) are synthesized in different populations of magnocellular neurons from the supraoptic, paraventricular, and accessory nuclei [11]. During evolution, secretion of these peptides in the cerebrospinal fluid (CSF) has been replaced by a vascular secretion. In advanced vertebrates, the axonal projections of these neurons are toward the posterior pituitary and also to forebrain, telencephalon, and diencephalon regions, probably explaining the behavioral effects of oxytocin and vasopressin [11]. Immunohistochemical studies have revealed a second vasopressin neurosecretory pathway that transports high concentrations of the hormone to the anterior pituitary gland from parvocellular neurons to the hypophyseal portal system. In the portal system, the high concentration of AVP acts synergistically with CRH to stimulate adrenocorticotrophin (ACTH) release from the anterior pituitary. More than half of the parvocellular neurons coexpress both CRH and AVP. In addition, while passing through the median eminence and the hypophyseal stalk, magnocellular axons can also release AVP into the long portal system. Furthermore, a number of neuroanatomical studies have shown the existence of short portal vessels that allow communication between the posterior and

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FIGURE 8.6

Contrasting structure of arginine vasopressin (AVP) and oxytocin (OT). The peptides differ only by two amino acids F3-I3 and R8-L8 in AVP and OT, respectively. The conformation of AVP was obtained from [14] and the conformation of OT was obtained from the Protein Data Bank (PDB Id 1XY1). Note: for both hormones, the formation of a disulfide bond between Cys residues at the 1 and 6 positions results in a peptide constituted of a six-amino-acid cyclic part and a three-amino-acid C-terminal part.

anterior pituitary. Thus, in addition to parvocellular vasopressin, magnocellular vasopressin is able to influence ACTH secretion [15,16].

THE VASOPRESSIN AND OXYTOCIN GENES Homologues of vasopressin and oxytocin have evolved over 700 million years and have been identified in insects to vertebrates [17,18]. The cis and trans components important for vasopressin and oxytocin expression in magnocellular neurons have been conserved over 450 million years in pufferfish isotocin and rat oxytocin genes [19,20]. Among these distant taxa (hydra, worms, insects, and vertebrates), oxytocinand vasopressin-related peptides also play a general role in the modulation of social and reproductive behavior [17]. In contrast to this apparent conservation of function, specific behaviors affected by these neuropeptides are notably species-specific [17]. Parents know the transformative nature of having and caring for a child, and effects of oxytocin in the biology of mammalian parenting and its effect on offspring social development is well demonstrated in mice and rats [21]. Virgin females and males of many species generally avoid infants, finding infant stimuli aversive. By contrast, parturient mothers typically find infants irresistible and display a suite of maternal nurturing behaviors to ensure survival of their offspring. For example, virgin female rats avoid or attack pups, but postpartum dams will press a lever more than 100 times per hour to have a pup delivered into their nest box with each press. The power of humoral factors to induce maternal behavior was first illustrated by showing that blood transfusions from a pregnant rat to

a virgin female elicited simultaneous onset of maternal responsiveness in both. Oxytocin helps make social interaction rewarding: in the mouse nucleus accumbens core, oxytocin is required both for social reinforcement and a form of presynaptic long-term depression of excitatory transmission onto medium spiny neurons [22]. Oxytocin and oxytocin receptor knockout mice exhibit related behavioral deficits (such as memory impairment, anxiety, stress, aggressiveness) and rewarding properties of social interaction in mice require the coordinated activity of oxytocin and 5hydroxytryptamine in the nucleus accumbens. Projections of PVN oxytocin neurons to the left auditory cortex facilitate the behavioral responses to cries of pups [23].

Gene Structure AVP and its corresponding carrier protein, neurophysin II, are synthesized as a composite precursor by the magnocellular and parvocellular neurons described previously. The precursor is packaged into neurosecretory granules and transported axonally in the stalk of the posterior pituitary (Fig. 8.7) [25]. On route to the neurohypophysis, the precursor is processed into the active hormone (Fig. 8.7). Preprovasopressin has 164 amino acids and is encoded by the 2.5-kb AVP gene located in chromosome region 20p13 [26]. The AVP gene (coding for AVP and neurophysin II) and the OXT gene (coding for oxytocin and neurophysin I) are located in the same chromosome region, at a very short distance from each other (12 kb in humans) in a head-to-head orientation. Data from transgenic mouse studies indicate that the intergenic region between the OXT and AVP genes contains the critical enhancer sites for

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FIGURE 8.7

Schematic representation of a magnocellular vasopressin-producing cell and general mechanism for vasopressin-containing vesicles to fuse to the plasma membrane during vasopressin release. A dendrite with release of dense core vesicles containing vasopressin is represented in the left upper part of the figure. In the central part, a microtubule is tracting a dense core vesicle along the very long axon. In the lower right part, osmotic stimuli depolarize magnocellular cells producing vasopressin and generate a depolarization signal towards the axon. When the action potential invades the axon or dendrite terminals, voltage-gated channels open, allowing calcium to flow into neurosecretory cell endings and this triggers the fusion of the vesicle membranes with the presynaptic membrane and results in vasopressin release. Similar molecular mechanisms are used for neurosecretion and neurotransmission and deciphering of fusion mechanisms has been recognized by the Nobel Prize in Medicine 2013 awarded to Thomas Su¨dhof, James Rothman, and Randy Sheckman. Two membrane proteins from neurosecretory vesicles, namely, synaptotagmin and synaptobrevin and two axonal membrane proteins synthaxin and SNAP-25 (synaptosomal associated protein, 25 kDa) are required. The structure of synaptotagmin includes a single transmembrane region and two calcium-binding domains, the calcium sensor of neurosecretion [24].

cell-specific expression in the magnocellular neurons [25]. It is phylogenetically interesting to note that cis and trans components of this specific cellular expression have been conserved between the Fugu isotocin (the homologue of mammalian oxytocin) and rat oxytocin genes [19]. Exon 1 of the AVP gene encodes the signal peptide, AVP, and the NH2-terminal region of NPII. Exon 2 encodes the central region of NPII, and exon 3 encodes the COOH-terminal region of NPII and the glycopeptide (Fig. 8.8). Provasopressin is generated by the removal of the signal peptide from preprovasopressin and the addition of a carbohydrate chain to the glycopeptide. Additional posttranslation processing occurs within neurosecretory vesicles during transport of the precursor protein to axon terminals in the

posterior pituitary, yielding AVP, NPII, and glycopeptide. The AVP NPII complex forms tetramers that can self-associate to form higher oligomers [28]. In the posterior pituitary, AVP is stored in vesicles. Exocytotic release is stimulated by minute increases in serum osmolality (hypernatremia, osmotic regulation) and by more pronounced decreases in extracellular fluid (ECF) (hypovolemia, nonosmotic regulation). Oxytocin and neurophysin I are released from the posterior pituitary by the suckling response in lactating females. The neuropeptides oxytocin and vasopressin are involved in new fascinating studies of the neurobiology of attachment [22,29] and central vasopressin and oxytocin receptors may regulate the autonomic expression of fear [30].

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THE VASOPRESSIN AND OXYTOCIN GENES

257 FIGURE 8.8 Cascade of vasopressin biosynthesis. SP, signal peptide; AVP, arginine vasopressin; NP, neurophysin; GP, glycoprotein. Source: From Richter D, Schmale H. Molecular aspects of the expression of the vasopressin gene. In: Czernichow P, Robinson AG, editors. Frontiers of hormone research. Basel: S. Karger; 1985 [27].

Expression of the Vasopressin Gene in Diabetes Insipidus Rats (Brattleboro Rats) The animal model of diabetes insipidus that has been most extensively studied is the Brattleboro rat. Discovered in 1961, the rat lacks vasopressin and its neurophysin, whereas the synthesis of the structurally related hormone oxytocin is not affected by the mutation [31]. Its inability to synthesize vasopressin is inherited as an autosomal semirecessive trait. Schmale and Richter [32] isolated and sequenced the vasopressin gene from homozygous Brattleboro rats and found that the defect is due to a single-nucleotide deletion of a G residue within the second exon encoding the carrier protein neurophysin. The shift in the reading frame caused by this deletion predicts a precursor with an entirely different C-terminus (Fig. 8.9). The messenger RNA (mRNA) produced by the mutated gene encodes a normal AVP but an abnormal NPII moiety [32], which impairs transport and processing of the AVP NPII precursor and its retention in the endoplasmic reticulum of the magnocellular neurons where it is produced [33,34]. Homozygous Brattleboro rats may still demonstrate some V2 (see below) antidiuretic effects since the administration of a selective nonpeptide V2 antagonist (SR121463A, 10 mg/kg i.p.) induced a further increase in urine flow rate (200 354 6 42 mL/24 h) and a decline in urinary osmolality (170 to 92 6 8 mmol/kg) [35]. This decline in urine osmolality following the administration of a nonpeptide V2R antagonist could also be secondary to the “inverse agonist” properties of SR121463A: the intrinsic activity, or “tone,” of the V2R would be

FIGURE 8.9 Neurophysin II genomic and amino acid sequence showing the 1 bp (G) deleted in the Brattleboro rat. The human sequence (GenBank entry M11166) is also shown. It is almost identical to the rat prepro sequence. In the Brattleboro rat, G1880 is deleted with a resultant frameshift after 63 amino acids (amino acid 1 is the first amino acid of neurophysin II).

deactivated by the SR121463A compound (for the inverse agonist properties of SR121463A see [36]). There is also an alternative explanation to this relatively high urine osmolality of 170 since, in Brattleboro rats, low levels of hormonally active AVP are produced from alternate forms of AVP preprohormone. Due to a process called molecular misreading, one transcript contains a 2-bp deletion downstream from the single-nucleotide deletion that restores the reading frame and produces a variant AVP preprohormone that is smaller in length by one amino acid and differs from the normal product by only 13 amino acids in the neurophysin II moiety [37]. Oxytocin, which is present at enhanced plasma concentrations in Brattleboro rats, may be responsible for the antidiuretic activity

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observed [38,39]. Oxytocin is not stimulated by increased plasma osmolality in humans.

Expression of the Vasopressin Gene in Autosomal Dominant and Autosomal Recessive Diabetes Insipidus in Humans Repaske et al. reported in 1990 [40] that the genetic locus for autosomal dominant central diabetes insipidus was within or near the gene encoding for AVP and suggested that a defective AVP gene might be the basis for this disease. Neurogenic diabetes insipidus (OMIM 125700) [41] is a now well-characterized entity, secondary to mutations in the AVP gene (OMIM 192340; GenBank: M11166). This disorder is also referred to as central, cranial, pituitary, or more commonly, familial neurohypophyseal diabetes insipidus (FNDI) [42]. Patients with autosomal dominant FNDI retain some limited capacity to secrete AVP, and the polyuropolydipsic symptoms usually appear after the first year of life, but there is considerable variation in age of onset. Over 50 AVP mutations segregating with autosomal neurogenic diabetes insipidus have been described [43,44]. Most are dominant, but rare recessive mutations have also been reported [43,45,46] as well as a single family with X-linked inheritance and no identified gene so far [42]. The majority of mutations are located in the neurophysin (NP) II domain (codons 32 124), a region important for protein folding and sorting (see Fig. 8.8) [47]. Transfection studies in mouse neuroblastoma Neuro2A cells support that the mechanism(s) by which a dominant mutant allele causes neurogenic diabetes insipidus is a result of the accumulation of AVP fibrillar aggregates within the endoplasmic reticulum, a so-called toxic gain of function [44,46,48 51]. This process is mechanistically similar to that seen in other neurodegenerative diseases such as Huntington and Parkinson diseases [51]. Although the handling of misfolded AVP mutants could account for the delayed onset and progressive nature of dominant FNDI, the precise mechanism of magnocellular toxicity is still unknown [44]. An extremely rare form of FNDI is recessively inherited where the symptoms appear in infancy. Here, one mutation in the AVP gene has been reported to date in five families [45,52 54]. Comparative expression studies in Neuro2A cells show that dominant forms accumulate in the cytoplasm, whereas recessive forms localize to the secretory granules at the tips of the cellular projections [48]. Moreover, in contrast to the dominant AVP mutants, it appears that recessive mutants do not exert progressive neurocytotoxicity since polyuria is not observed in parents with only one mutated allele.

Diabetes insipidus of nephrogenic origin nephrogenic diabetes insipidus (NDI), while rare, occurs more frequently than FNDI. Here, the disease results from the kidney’s inability to use available AVP and is associated with mutations in AVPR2 or in aquaporin-2 (AQP2) [55]. In contrast to FNDI, the polyuropolydipsic symptoms are present during the first week of life. Although errors in protein folding also represent the underlying basis for AVPR2 and AQP2 mutants responsible for NDI, the pathogenic mechanism is clearly different from FNDI. AVPR2 missense mutations lead to the rapid degradation of the affected polypeptide but not to the accumulation of toxic aggregates, since the other important functions of the principal cells of the collecting ducts (where AVPR2 is expressed) are unaffected.

CHEMISTRY, PROCESSING, AND METABOLISM OF AVP AVP is a nonapeptide with a molecular weight of 1084 Da. The chemical structure of AVP and related peptides are given in Table 8.1 and Fig. 8.6. It is a strongly basic molecule (isoelectric point pH 10.9) due to the amidation of three carboxyl groups. Lysine vasopressin, the antidiuretic hormone of the pig family, has the less basic amino acid lysine at position 8, resulting in a lower isoelectric point (pH 10.0). Biological activity of these hormones is destroyed by oxidation or reduction of the disulfide bond [57,58]. Members of the vasopressin hormone family have been detected throughout the animal kingdom [59], comprising more than half a dozen variants including peptides such as vasotocin of nonmammalian vertebrates, the diuretic hormone of insects, and the conopressins of mollusks. Invertebrates, their endocrine hormonal activity—controlling mainly water retention—is well documented, whereas in invertebrates, they may function primarily as neurotransmitters, although a hormonal diuretic activity has been demonstrated in the locust [60]. Acher and Chauvet [59] postulated the existence of a single ancestral peptide that developed along two evolutionary lines: one vasotocin vasopressin and the other isotocin mesotocin oxytocin. However, recent evidence suggests that multiple genes, which code for numerous vasopressin-like hormones, are present in Australian macropods. Neurophysins were first thought to be carrierproteins for vasopressin and oxytocin. It is now recognized that NPI (for oxytocin) and NPII (for vasopressin) belong to the precursor of the respective hormone (see above section on the vasopressin and the oxytocin gene). After synthesis in the hypothalamic neurons, the vasopressin precursor migrates along the neuronal

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TABLE 8.1 Amino Acid Sequence of Arginine Vasopressin and Related Neurohypophyseal Nonapeptides Arginine vasopressin

12345 6789 Cys-Tyr-Phe-Glu(NH2)-Asp(NH2)-Cys-Pro-Arg-Gly(NH2)

Distribution Most mammals

Lysine vasopressin

Phe Glu(NH2)

Lys

Pig family

Arginine vasotocin

lle Glu(NH2)

Arg

Nonmammalian vertebrates

Oxytocin

lle Glu(NH2)

Leu

Mammals, birds

Mesotocin

lle Glu(NH2)

lle

Reptiles

Isotocin

lle Ser

lle

Fish

Glumitocin

lle Ser

Glu(NH2)

Fish

Valitocin

lle Glu(NH2)

Val

Fish

Aspartocin

lle Asp(NH2)

Leu

Fish

From Baylis P. Vasopressin and its neurophysin. In: Degroot LG, Besser J, Cahill GFJ, Marshall JC, Nelson DH, Odell WD, editors. Endocrinology. 2nd ed. Philadelphia, PA: WB Saunders; 1989:213 29 [56], with permission.

axons, many of which terminate in the posterior pituitary. The time from synthesis to release of the hormone into the systemic circulation is about 1.5 hours [61]. Pulse chase experiments indicate that cleavage occurs continuously during axonal transport [62], but both cleaved and uncleaved precursors [63] are present in neurosecretory granules of the posterior pituitary. Only a small percentage of synthetic peptide is released; some vasopressin-containing neurosecretory granules move away from nerve endings and are unavailable for release. Once secreted into the circulation, vasopressin is accompanied, but not bound, by its specific neurophysin. Neurophysins themselves do not appear to have biological activity, but since they are synthesized and released with vasopressin and oxytocin, their concentrations in the plasma reflect changes in release of the active hormones (see below). The plasma half-life of vasopressin is short, being about 5 15 minutes. Clearance is independent of plasma vasopressin concentration as it involves a liverand kidney-dependent process. Vasopressin is not protein-bound, but large quantities of vasopressin are associated with platelets in humans [64] and dogs [65]. Platelet-rich plasma AVP concentrations are approximately five- to sixfold higher than those of platelet-depleted plasma. Furthermore, irreversible platelet aggregation may bring about intraplatelet AVP release [66]. However, osmotic stimulation of AVP release does not influence platelet-associated AVP concentrations [64].

CONTROL OF AVP SECRETION Osmotic Stimulation Central mechanisms of osmosensation and systemic osmoregulation have been reviewed by Bourque [7].

Vasopressin release can be regulated by changes in either osmolality or CSF Na concentration. Mammals are osmoregulators; they have evolved mechanisms that maintain ECF osmolality near a stable value. Yet, although mammals strive to maintain a constant ECF osmolality, values measured in an individual can fluctuate around the set-point owing to intermittent changes in the rates of water intake and water loss (through evaporation or diuresis), and to variations in the rates of Na intake and excretion (natriuresis). In humans, e.g., 40 minutes of strenuous exercise in the heat [67,68], or 24 h of water deprivation [69], causes plasma osmolality to rise by more than 10 mosmol/kg. In a dehydrated individual, drinking the equivalent of two large glasses of water (B850 mL) lowers osmolality by approximately 6 mosmol/kg within 30 minutes [70]. Similarly, ingestion of 13 g of salt increases plasma osmolality by approximately 5 mosmol/kg within 30 minutes [71]. Although osmotic perturbations larger than these can be deleterious to health, changes in the 1 3% range play an integral part in the control of body-fluid homeostasis. Differences between the ECF osmolality and the desired set-point induce proportional homeostatic responses according to the principle of negative feedback [7]. ECF hyperosmolality stimulates the sensation of thirst to promote water intake and the release of vasopressin to enhance water reabsorption in the kidney. By contrast, ECF hypoosmolality suppresses basal VP secretion in rats and humans [72].

Osmoreceptors in the Brain and the Periphery As summarized elegantly by Bourque [7], early studies provided clear evidence that “cellular dehydration” (i.e., cell shrinking) was required for thirst and VP release to be stimulated during ECF

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hyperosmolality; these responses could be induced by infusions of concentrated solutions containing membrane-impermeable solutes, which extract water from cells, but not by infusions of solutes that readily equilibrate across the cell membrane (such as urea). Verney coined the term “osmoreceptor” to designate the specialized sensory elements. He further showed that these were present in the brain and postulated that they might comprise “tiny osmometers” and “stretch receptors” that would allow osmotic stimuli to be “transmuted into electrical” signals [73]. Osmoreceptors are therefore defined functionally as neurons that are endowed with an intrinsic ability to detect changes in ECF osmolality, and it is now known that both cerebral and peripheral osmoreceptors contribute to the body-fluid balance. The identity of deltaN TRPV1, a molecular codetector of body temperature and osmotic stress, is now well demonstrated in osmosensory central organs [5]. Hepatic sensory neurons also function as osmoreceptors: they express TRPV4 channels and signal hypoosmotic stimuli from portal blood via the thoracic dorsal root ganglia with connections to vasopressinproducing cells. This explains why liver transplant patient osmolality is increased, since, in these liverdenervated transplant patients, there is no inhibition of central vasopressin release by portal hypoosmolality [74]. Portal osmoreceptors can signal changes in blood osmolality well before water intake impacts systemic blood osmolality. Osmotic Threshold: Sensitivity or Gain of the Osmoreceptor/AVP-Releasing Unit Levels of plasma osmolality at which hydrated subjects first respond to an intravenous infusion of 5% saline with a statistically significant fall in free water clearance (without a fall in osmolal clearance or creatinine excretion) were termed the osmotic threshold for vasopressin release [75]. This osmotic threshold, determined to be 288.5 mosmol/kg [76], was raised by administration of hydrocortisone [75] and plasma volume expansion [77] and lowered by plasma volume contraction [78]. With the development of sensitive radioimmunoassays, it was later demonstrated that, in healthy adults, the infusion of concentrated saline (850 mmol/L) caused a progressive rise in plasma osmolality and in plasma AVP concentrations [79 81]. A direct correlation between the two variables was established, defined by the function: pAVP 5 0.30 (Posm 280) (Fig. 8.10). The abscissal intercept, 280 mmol/kg, is the osmotic threshold. Because this intercept falls below the limit of detection of the assay methods, this “set” of the osmoreceptor mechanism should be referred to as the theoretical threshold for vasopressin release.

FIGURE 8.10 The relationship between plasma AVP and plasma osmolality during the infusion of hypertonic saline solution (A). Patients with primary polydipsia and nephrogenic diabetes insipidus have values within the normal range (open area) in contrast to patients with neurogenic diabetes insipidus, who show subnormal plasma ADH responses (light gray area). Relationship between urine osmolality and plasma ADH during dehydration and water loading (B). Patients with neurogenic diabetes insipidus and primary polydipsia have values within the normal range (open area) in contrast to patients with nephrogenic diabetes insipidus, who have hypotonic urine despite high plasma ADH (dark gray area). Source: Modified from Zerbe RL, Robertson GL. Disorders of ADH. Med North Am 1984;13:1570, [82].

Whether AVP secretion can be completely suppressed or whether a linear versus an exponential model should be used remains unclear [81,83]. A close relationship has also been demonstrated between urine osmolality and AVP concentrations, except in patients with NDI (Fig. 8.10). The exquisite sensitivity and gain of the osmoreceptor AVP renal reflex is given by the following example (Fig. 8.11). A normally hydrated man may have a plasma osmolality of 287 mmol/kg, a plasma vasopressin concentration of 2 pg/mL and a urinary osmolality of 500 mmol/kg. With an increase of 1% in total body water, plasma osmolality will fall by 1% (2.8 mmol/kg), plasma AVP will decrease to 1 pg/mL and urinary osmolality will diminish to 250 mmol/kg. Similarly, it is only necessary to increase total body water by 2% to suppress the plasma AVP maximally (,0.25 pg/mL) and to maximally dilute the urine (,100 mmol/kg). In the opposite direction, a 2% decrease in total body water will increase plasma osmolality by 2% (5.6 mmol/kg), plasma AVP will rise from 2 to 4 pg/mL and urine will be maximally concentrated ( . 1000 mmol/kg). Thus, in the context of these sensitivity changes, a 1 mmol rise in plasma osmolality would be expected to increase plasma AVP by 0.38 pg/mL and urinary osmolality by 100 mmol/ kg. Such a small change in plasma osmolality (measured by freezing point depression) or plasma AVP

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FIGURE 8.11 Schematic representation of the effect of small alterations in the basal plasma osmolality on (left) plasma vasopressin and (right) urinary osmolality in healthy adults. Source: Modified from Robertson GL, Shelton RL, Athar S. The osmoregulation of vasopressin. Kidney Int 1976;10(1):25 37.

(by radioimmunoassay) may be undetectable yet of extreme physiological importance. For example, a patient with a 24-hour urinary solute load of 600 mmol must excrete 6 L of urine with an osmolality of 100 mmol/kg to eliminate the solute; however, if the urine osmolality increases from 100 to 200 mmol/kg (due to an undetectable rise of 1 mmol in plasma osmolality and 0.38 pg/mL in plasma AVP), the obligatory 24-hour urine volume to excrete the 600 mmol solute load decreases substantially from 6 to 3 L. Examination of Fig. 8.11 demonstrates that a maximal antidiuresis is obtained when the plasma AVP concentration reaches 5 pg/mL. Greater hyperosmolality, although releasing more AVP, fails to conserve any more renal water, thus exposing the body to the potential of severe dehydration. This can be avoided by the stimulation of the thirst osmoreceptor at a plasma osmolality of 298 mmol/kg. However, recent studies, using a visual analogue scale, have demonstrated that the onset of thirst occurs at a considerably lower plasma osmolality than was previously recognized; the values were similar to those of the threshold for vasopressin release [84,85]. It has been shown in both animals [86] and humans [87 89] that the act of drinking ameliorates thirst and inhibits the secretion of vasopressin before changes occur in the ECF volume or osmolality. In humans, it has been shown that AVP secretion is inhibited independent of osmotic or gastric factors by the activation of the cold-sensitive oropharyngeal receptors [89]. The presence of such cold-sensitive oropharyngeal receptors may explain the desire of severely dehydrated patients, i.e., patients with diabetes insipidus (neurogenic or nephrogenic), for cold liquids. There are considerable variations between individuals in osmoreceptor sensitivity and in the threshold for vasopressin release; however, these individual values remain

constant for a relatively short period of time [90]. To determine whether these interindividual differences are genetically influenced, Zerbe [90] compared vasopressin osmolality relationships within monozygotic and dizygotic twin pairs. The threshold and sensitivity values correlated significantly within monozygotes but not within dizygotes, suggesting a genetic determinant for the set of the osmoregulatory system. Pregnancy causes a lowering of the threshold for vasopressin secretion without altering the gain of the osmoreceptors in both rats [91] and humans [92], thus accounting for the hypoosmolality of pregnancy. A role for human chorionic gonadotrophin in lowering this osmotic threshold has been postulated [93].

Baroregulation It is now well established that afferent neural impulses arising from stretch receptors in the left atrium, carotid sinus, and aortic arch inhibit the secretion of vasopressin. Conversely, when the discharge rate of these receptors is reduced, vasopressin secretion is enhanced (for review see Ref. [94]). Moreover, the relative potency of the cardiac and sino-aortic reflexes in the release of vasopressin appears to vary among species. For example, the increase in plasma vasopressin that occurs during moderate hemorrhage in the dog is attributable primarily to reflex effects from cardiac receptors; sino-aortic receptors appear to exert only minor influences on vasopressin release in this situation. In contrast, sino-aortic receptors appear to play the dominant role in eliciting vasopressin secretion during blood loss in nonhuman primates and humans [94]. In humans, blood pressure reductions of as little as 5%, induced by the ganglion-blocking agent trimetaphan, significantly altered plasma AVP

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severe, plasma AVP concentrations attain extremely high values and baroregulation overrides the osmoregulatory system. An enhanced osmoreceptor sensitivity, but blunted baroregulation, has been described in elderly subjects [99].

Hormonal Influences on the Secretion of Vasopressin

FIGURE 8.12

Increase in plasma arginine vasopressin (AVP) during hypotension (vertical lines). Note that a large diminution in blood pressure in normal humans induces large increments in AVP. Source: From Zerbe RL, Henry DP, Robertson GL. Vasopressin response to orthostatic hypotension. Etiologic and clinical implications. Am J Med 1983;74(2):265 71 [96], with permission.

FIGURE 8.13 Schematic representation of the relationship between plasma vasopressin and plasma osmolality in the presence of differing states of blood volume and/or pressure. The line labeled N represents normovolemic normotensive conditions. Minus numbers to the left indicate percent fall, and positive numbers to the right, percent rise in blood volume or pressure. Source: Data from Vokes TP, Robertson, GL. Physiology of secretion of vasopressin. In: Czernichow AGRP, editor. Frontiers in hormone research: diabetes insipidus in man, vol. 13. Basel: S. Karger; 1985. p. 127 55, [97].

concentration [95]. Furthermore, an exponential relationship between plasma vasopressin and the percentage decline in mean arterial blood pressure has been observed with large decreases in blood pressure (Fig. 8.12). Since an interdependence exists between osmoregulated and baroregulated AVP secretion (Fig. 8.13) [98], under conditions of moderate hypovolemia, renal water excretion can be maintained around a lower set-point of plasma osmolality, thus preserving osmoregulation. As hypovolemia becomes more

Studies on the direct effects of various peptides and other biological substances on the release of vasopressin may be confounded by the hemodynamic effects of these substances, which indirectly modulate vasopressin release via the cardiovascular reflexes. For example, the infusion of pressor doses of norepinephrine increases both arterial blood pressure and left atrial pressure. Each of these changes is capable of eliciting a reflex inhibition of vasopressin release which should reduce plasma vasopressin. However, the inhibitory effects of the sino-aortic and cardiac reflexes on vasopressin release seem to be offset by the direct stimulatory effect of circulating norepinephrine. A similar situation may exist with the possible stimulation of vasopressin release by angiotensin. The direct stimulatory effect of angiotensin may be offset by inhibitory influences elicited from the cardiovascular reflexes. Angiotensin is a well-known dipsogen and has been shown to cause drinking in all the species tested [100]. Angiotensin II receptors have been described in the SFO and OVLT (for review see Ref. [101]). Brooks et al. [102] found that the infusion of exogenous angiotensin II increased vasopressin secretion and altered the baroreflex function in conscious dogs. Philips et al. [103] found that thirst and vasopressin secretion were stimulated in four of 10 healthy subjects infused with angiotensin II. Furthermore, the AVP concentrations were higher in the responders than in the nonresponders. These effects occurred at plasma angiotensin concentrations that were well above those measured under physiological conditions associated with thirst and vasopressin secretion, such as water deprivation. To further assess the potential importance of angiotensin II in the regulation of vasopressin secretion in man, Morton et al. [104] submitted six normal subjects to a 3-day diet containing 10 mmol of sodium and 60 mmol of potassium per day. The mean cumulative sodium loss ( 6 SD) for the six subjects was 208 6 94 mmol. Sodium restriction had no effect on serum sodium concentrations. Sodium depletion increased the circulating concentrations of angiotensin II more than fivefold (p , 0.001), but had no effect on plasma AVP concentrations. In short, physiologic concentrations of angiotensin II do not increase plasma vasopressin concentration in normal subjects.

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VASOPRESSIN RECEPTORS AND ANTAGONISTS

However, the complex interaction between the direct stimulatory and cardiovascular inhibitory influences has not been studied. Of interest, knockout of angiotensinogen [105] or AT1A receptor [106,107] did not alter thirst or water balance. Disruption of the AT2 receptor only induced mild abnormalities of thirst postdehydration [108]. The presence of endogenous opioid peptides and opioid receptors [109] in the neural lobe has led to the suggestion that opioid peptides play a role in the release of neurohypophyseal hormones. Vasopressin coexists with dinorphin in large dense core vesicles of magnocellular cells [12]. It is now recognized that opioid drugs exert their pharmacologic effects through an interaction with specific receptors. These receptors are classified into several types: μ, δ, σ, and κ. μ Agonists such as morphine and methadone are responsible for the classical opiate effects of analgesia, respiratory depression, and physical dependence. They typically cause an antidiuresis in hydrated animals and humans [110]. In contrast, κ agonists have analgesic properties, but do not cause respiratory depression or physical dependence at the dose required for analgesia. They have been shown to cause a water diuresis in experimental animals and in humans, probably by the inhibition of vasopressin secretion [111]. κ Opioid agonists could have potential therapeutic benefits in the treatment of hyponatremia secondary to increased AVP secretion. A very rapid and robust release of AVP is seen in humans after cholecystokinin (CCK) injection [112]. Nitric oxide is an inhibitory modulator of the hypothalamo neurohypophyseal system in response to osmotic stimuli [113]. Vasopressin secretion is under the influence of glucocorticoid-negative feedback [114] and the vasopressin responses to a variety of stimuli (hemorrhage, hypoxia, hypertonic saline) in normal humans and animals appear to be attenuated or eliminated by pretreatment with glucocorticoids. Finally, nausea and emesis are potent stimuli of AVP release in humans and seem to involve dopaminergic neurotransmission [115]. The osmotic stimulation of AVP release by dehydration or hypertonic saline infusion, or both, is regularly used to test the AVP secretory capacity of the posterior pituitary. This secretory capacity can be assessed directly by comparing the plasma AVP concentration measured sequentially during a dehydration procedure with the normal values and then correlating the plasma AVP with the urinary osmolality measurements obtained simultaneously [116]. The AVP release can also be assessed indirectly by measuring plasma and urine osmolalities at regular intervals during the dehydration test [117]. The maximum urinary osmolality obtained during dehydration

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is compared with the maximum urinary osmolality obtained after the administration of vasopressin or desmopressin (1-desamino-8-D-arginine vasopressin) (dDAVP) (Pitressin: 5 U SQ in adults; 1 U SQ in children) or dDAVP (1 4 μg intravenously over 5 10 minutes). The nonosmotic stimulation of AVP release can be used to assess the vasopressin secretory capacity of the posterior pituitary in a rare group of patients with essential hyponatremia and hypodipsia syndrome. Although some of these patients may have partial central diabetes insipidus, they respond normally to nonosmolar AVP release signals such as hypotension, emesis, and hypoglycemia. In all other cases of suspected central diabetes insipidus, these nonosmotic stimulation tests will not give additional clinical information.

VASOPRESSIN RECEPTORS AND ANTAGONISTS The four different receptor subtypes, respectively V1a, V1b, V2, and oxytocin, have been cloned in mammals, lower vertebrates, and invertebrates. These are four of 701 members of the rhodopsin family within the superfamily of guanine nucleotide (G)-protein-coupled receptors (see also the perspective by Perez [118] and comments on X-ray structure breakthroughs in the transmembrane-spanning region [119]). The V1a, V1b, V2, and OT receptors are strikingly similar in both size and amino acid sequence. However, the V1a, V1b, and OT receptors are selectively coupled to G-proteins of the Gq/11 family which mediate the activation of distinct isoforms of phospholipase Cβ resulting in the breakdown of phosphoinositide lipids. The V2 receptor, on the other hand, preferentially activates the G-protein, Gs, resulting in the activation of adenylyl cyclase. The classical vascular smooth muscle contraction, platelet aggregation, and hepatic glycogenolysis actions of AVP are mediated by the V1a receptor that increases cytosolic calcium. In situ hybridization histochemistry using [39] S-labeled cRNA probes specific for the V1a receptor mRNA showed high levels of V1a receptor transcripts in the liver among hepatocytes surrounding central veins and in the renal medulla among the vascular bundles, the arcuate, and interlobular arteries [120]. V1a receptor mRNA was found to be extensively distributed throughout the brain where AVP may act as a neurotransmitter or a neuromodulator in addition to its classical role on vascular tone [29]. Brain AVP receptors have been proposed to mediate the effect of AVP on memory and learning, antipyresis, brain development, selective aggression, and partner preference in rodents, cardiovascular

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responsivity, blood flow to the choroid plexus and CSF production, regulation of smooth muscle tone in superficial brain vasculature, and analgesia. It is, however, not known whether V1a brain receptors respond to AVP released within the brain proper or whether the receptors also respond to AVP from the peripheral circulation [121]. V1b receptors are expressed in the anterior pituitary [122] and kidney [123] as originally reported and also in brain, uterus, thymus, heart, breast, and lung. The physiologic role of these extrapituitary V1b receptors remains unknown, but some functions of AVP attributed in the past to V1a receptors or OT receptors may be due to the activation of V1b receptors [124]. In the rat adrenal medulla, AVP may regulate adrenal functions by paracrine/autocrine mechanisms involving distinct AVP receptor subtypes: V1a in the adrenal cortex and V1b in the adrenal medulla [125]. V2 transcripts are abundantly expressed in cells of the renal collecting ducts (in humans and rodents) and in cells of the thick ascending limbs of the loops of Henle (in rodents only) [126]. Species specificity and partial agonist activity have frustrated the search to discover antidiuretic hormone receptor antagonists that are effective aquaretic agents in vivo. The compound SKF101926 (desGlyd(CH2)5DTyr(Et)VAVP) was shown to be a potent V2 receptor antagonist having aquaretic activity in several animal species, including a primate species. However, SKF101926 lacked aquaretic activity and was a vasopressin agonist in humans [127]. The orally effective nonpeptide V2 antagonists are aquaretic drugs potentially useful to treat various clinical syndromes with abnormal water retention [128].

CELLULAR ACTIONS OF VASOPRESSIN The neurohypophyseal hormone AVP has multiple actions, including the inhibition of diuresis, contraction of smooth muscle, aggregation of platelets, stimulation of liver glycogenolysis, modulation of adrenocorticotrophic hormone release from the pituitary, and central regulation of somatic functions (thermoregulation and blood pressure) and modulation of social and reproductive behavior. These multiple actions of AVP can be explained by the interaction of AVP with at least three types of G-protein-coupled receptors: the V1a (vascular, hepatic, and brain) and V1b (anterior pituitary) receptors act through phosphatidylinositol hydrolysis to mobilize calcium, and the V2 (kidney) receptor is coupled to adenylate cyclase [129]. The transfer of water across the principal cells of the collecting ducts is now known at such a detailed level that billions of molecules of water traversing the

membrane can be represented; see useful teaching tools at http://www.mpibpc.gwdg.de/abteilungen/ 073/gallery.html and http://www.ks.uiuc.edu/ research/aquaporins. The 2003 Nobel Prize in chemistry was awarded to Peter Agre and Roderick MacKinnon, who solved two complementary problems presented by the cell membrane: how does a cell let one type of ion through the lipid membrane to the exclusion of other ions? And how does it permeate water without ions? This contributed to a momentum and renewed interest in basic discoveries related to the transport of water and indirectly to diabetes insipidus. The first step in the action of AVP on water excretion is its binding to AVP type 2 receptors (hereafter referred to as V2 receptors) on the basolateral membrane of the collecting duct cells (Fig. 8.14). The human AVPR2 gene that codes for the V2 receptor is located in chromosome region Xq28 and has three exons and two small introns [132,133]. The sequence of the cDNA predicts a polypeptide of 371 amino acids with seven transmembrane, four extracellular, and four cytoplasmic domains (Fig. 8.15). The activation of the V2 receptor on renal collecting tubules stimulates adenylyl cyclase via the stimulatory G-protein (Gs) and promotes the cyclic adenosine monophosphate (cAMP)mediated incorporation of water channels into the luminal surface of these cells. There are two ubiquitously expressed intracellular cAMP receptors: (1) the classical protein kinase A (PKA) that is a cAMPdependent protein kinase, and (2) the recently discovered exchange protein directly activated by cAMP that is a cAMP-regulated guanine nucleotide exchange factor [134]. Like PKA, Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular cAMP levels to control diverse biological functions. PKA and Epac may act independently on long-term regulation of AQP2 abundance [135]. The increase in cyclic AMP activates PKA, which could phosphorylate AQP2 channels at five cytoplasmic carboxyterminal tail residues, Thr 244, Ser256, Ser261, Ser264, and Ser269 (Thr269 in human AQP2) [136]. Ser256, Ser264, and Ser269 phosphorylation are increased in abundance upon administration of dDAVP. Phosphorylation at Ser261 is thought to stabilize AQP2 ubiquitination [137]. ˚ resolution The X-ray structure of AQP2 at 2.75 A [138] shows the structure of carboxy- and aminotermini with striking differences between AQP2 and other mammalian AQP structures due to the highly variable position of the short C-terminal helix with high flexibility likely arising from two consecutive prolines (Pro225 and Pro226) that form a hinge region. Only one proline residue is present in the corresponding position in other mammalian AQP structures [138]. The crystal structure of AQP2 provided novel insights

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FIGURE 8.14 Schematic representation of the effect of arginine vasopressin (AVP) to increase water permeability in the principal cells of the collecting duct. AVP is bound to the V2 receptor (a G-protein-linked receptor) on the basolateral membrane. The basic process of Gprotein-coupled receptor signaling consists of three steps: a hepta-helical receptor that detects a ligand (in this case, AVP) in the extracellular milieu, a G-protein that dissociates into α-subunit bound to GTP and β- and γ-subunits after interaction with the ligand-bound receptor, and an effector (in this case, adenylyl cyclase) that interacts with dissociated G-protein subunits to generate small-molecule second messengers. AVP activates adenylyl cyclase, increasing the intracellular concentration of cyclic adenosine monophosphate (cAMP). The topology of adenylyl cyclase is characterized by two tandem repeats of six hydrophobic transmembrane domains separated by a large cytoplasmic loop and terminates in a large intracellular tail. Generation of cAMP follows receptor-linked activation of the heteromeric G-protein (Gs) and interaction of the free Gas-chain with the adenylyl cyclase catalyst. Protein kinase A (PKA) and possibly the exchange factor directly activated by cAMP (EPAC) are the target of the generated cAMP. In the long term, vasopressin also increases AQP2 expression via phosphorylation of the cAMP responsive element binding protein (CREB), which stimulates transcription from the AQP2 promoter. Cytoplasmic vesicles carrying the water channel proteins (represented as homotetrameric complexes) are fused to the luminal membrane in response to AVP, thereby increasing the water permeability of this membrane. Microtubules and actin filaments are necessary for vesicle movement toward the membrane. The mechanisms underlying docking and fusion of aquaporin-2 (AQP2)-bearing vesicles are not known. The detection of the small GTP binding protein Rab3a, synaptobrevin 2, and syntaxin 4 in principal cells suggests that these proteins are involved in AQP2 trafficking [130]. When AVP is not available, water channels are retrieved by an endocytic process, and water permeability returns to its original low rate. Internalized AQP2 can either be targeted to recycling pathways or to degradation via lysosomes. AQP3 and AQP4 water channels are expressed on the basolateral membrane. The importance of an endoplasmic reticulum calcium sensor for vasopressin/aquaporin signaling is also represented with coupling between STIM1 and Orai1 during calcium depletion possibly induced by aquaporin-2 signaling [131].

to understand NDI, AQP2 mutations affecting folding, the selectivity filter region, and the cadmium21/ calcium21- binding sites [138]. The importance of an endoplasmic reticulum calcium sensor for vasopressin/aquaporin signaling has been demonstrated with an autosomal recessive Stim1 mutation responsible for partial NDI in SHR-A3 rats [131] (Fig. 8.14). The NDI phenotype observed in SHR-A3 rats is mild with basal urine osmolalities around 500 mmol/kg with corresponding plasma osmolalities of 303 mmol/kg and a urine osmolality of 2300 mmol/kg after 24 hours of dehydration. It is unlikely that loss of function of this gene results in a severe phenotype in humans.

AVP also increases renal water reabsorptive capacity by regulating the urea transporter UT-A1 present in the inner medullary collecting duct [139]. AVP also increases permeability of principal collecting duct cells to sodium [140]. In summary, in the absence of AVP stimulation, collecting duct epithelia exhibit very low permeabilities to sodium urea and water. These specialized permeability properties permit the excretion of large volumes of hypotonic urine formed during intervals of water diuresis. By contrast, AVP stimulation of the principal cells of the collecting ducts leads to selective increases in the permeability of the apical membrane to water, urea, and sodium.

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diuretics. Osmotic diuresis should be considered when solute excretion exceeds 60 mmol/h.

CLINICAL CHARACTERISTICS OF DIABETES INSIPIDUS DISORDERS Neurogenic Diabetes Insipidus Common Forms

FIGURE 8.15 Schematic representation of the V2 receptor and identification of 193 putative disease-causing AVPR2 mutations. Predicted amino acids are shown as the one-letter amino acid code. A solid symbol indicates a codon with a missense or nonsense mutation; a number indicates more than one mutation in the same codon; other types of mutations are not indicated in the figure. There are 95 missense, 18 nonsense, 46 frameshift deletion or insertion, seven inframe deletion or insertion, four splice-site and 22 large deletion mutations, and one complex mutation.

These actions of vasopressin in the distal nephron are possibly modulated by prostaglandin E2, nitric oxide [141], and by luminal calcium concentration. High levels of E-prostanoid-3 receptors are expressed in the kidney [142]. However, mice lacking E-prostanoid-3 receptors for prostaglandin E2 were found to have quasi-normal regulation of urine volume and osmolality in response to physiological stimuli [142]. An apical calcium/polycation receptor protein expressed in the terminal portion of the inner medullary collecting duct of the rat reduces AVPelicited osmotic water permeability when luminal calcium concentration rises [143]. This possible link between calcium and water metabolism may play a role in the pathogenesis of renal stone formation [143].

QUANTITATING RENAL WATER EXCRETION Diabetes insipidus is characterized by the excretion of abnormally large volumes of hypoosmotic urine (,250 mmol/kg). This definition excludes osmotic diuresis, which occurs when excess solute is being excreted, as with glucose in the polyuria of diabetes mellitus. Other agents that produce osmotic diuresis are mannitol, urea, glycerol, contrast media, and loop

Failure to synthesize or secrete vasopressin normally limits maximal urinary concentration and, depending on the severity of the disease, causes varying degrees of polyuria and polydipsia. Experimental destruction of the vasopressin-synthesizing areas of the hypothalamus (supraoptic and PVN) causes a permanent form of the disease. Similar results are obtained by sectioning the hypophyseal hypothalamic tract above the median eminence. Sections below the median eminence, however, produce only transient diabetes insipidus. Lesions to the hypothalamic pituitary tract are frequently associated with a three-stage response both in experimental animals and in humans [144]: (1) an initial diuretic phase lasting from a few hours to 5 6 days; (2) a period of antidiuresis unresponsive to fluid administration. This antidiuresis is probably due to vasopressin release from injured axons and may last from a few hours to several days (because urinary dilution is impaired during this phase, continued water administration can cause severe hyponatremia); and (3) a final period of diabetes insipidus. The extent of the injury determines the completeness of the diabetes insipidus and, as already discussed, the site of the lesion determines whether the disease will be permanent. Twenty-five percent of patients studied after transsphenoidal surgery developed spontaneous isolated hyponatremia, 20% developed diabetes insipidus, and 46% remained normonatremic. Normonatremia, hyponatremia, and diabetes insipidus were associated with increasing degrees of surgical manipulation of the posterior lobe and pituitary stalk during surgery [145]. Central diabetes insipidus observed after transsphenoidal surgery is often transient and only 2% of patients need long-term treatment with dDAVP [146]. Central diabetes insipidus is observed both before but mainly after surgery for craniopharyngiomas [147]. The etiologies of central diabetes insipidus in adults and children are listed in Table 8.2 [148 151]. Rare causes of central diabetes insipidus include leukemia, thrombotic thrombocytopenic purpura, pituitary apoplexy, sarcoidosis [152] and Wegener’s granulomatosis, xanthoma disseminatum [153], septo-optico dysplasia and agenesis of the corpus callosum [154], metabolic (anorexia nervosa), lymphocytic hypophysitis

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TABLE 8.2 Etiology of Hypothalamic Diabetes Insipidus in Children and Adults Children (%)

Children and young adults (%)

Adults (%)

Primary brain tumora

49.5

22

30

Before surgery

33.5

13

After surgery

16

17

Idiopathic (isolated or familial)

29

58

Histiocytosis

16

12

b

Metastatic cancer Traumac

2.2

2.0

Postinfectious disease

2.2

6.0

25

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dominant neurogenic diabetes insipidus [161]. Neurogenic diabetes insipidus (OMIM 125700) [41] is a well-characterized entity, secondary to mutations in AVP (OMIM 192340). Patients with autosomal dominant neurogenic diabetes insipidus retain limited capacity to secrete AVP during severe dehydration, and the polyuropolydipsic symptoms usually appear after the first year of life [162] when the infant’s demand for water is more likely to be understood by adults. Expression of the vasopressin gene in autosomal dominant and autosomal recessive diabetes insipidus in humans is described above.

8

Wolfram Syndrome

17

Wolfram syndrome, also known as DIDMOAD, is an autosomal recessive neurodegenerative disorder accompanied by insulin-dependent diabetes mellitus and progressive optic atrophy with an estimated prevalence in Japan of 1/710,000 [163]. The acronym DIDMOAD describes the following clinical features of the syndrome: diabetes insipidus, diabetes mellitus, optic atrophy, and sensorineural deafness. An unusual incidence of psychiatric symptoms has also been described in patients with this syndrome. These include paranoid delusions, auditory or visual hallucinations, psychotic behavior, violent behavior, organic brain syndrome typically in the late or preterminal stages of their illness, progressive dementia, and severe learning disabilities or mental retardation, or both. Wolfram syndrome patients develop diabetes mellitus and bilateral optical atrophy mainly in the first decade of life, the diabetes insipidus is usually partial and of gradual onset, and the polyuria can be wrongly attributed to poor glycemic control. Furthermore, a severe hyperosmolar state can occur if untreated diabetes mellitus is associated with an unrecognized posterior pituitary deficiency. The dilatation of the urinary tract observed in DIDMOAD syndrome may be secondary to chronic high urine flow rates and, perhaps, to some degenerative aspects of the innervation of the urinary tract. The gene responsible for Wolfram syndrome, located in chromosome region 4p16.1, encodes a putative 890-aminoacid transmembrane protein referred to as wolframin. Wolframin is an endoglycosidase H-sensitive glycoprotein, which localizes primarily in the endoplasmic reticulum of a variety of neurons including neurons in the SON and neurons in the lateral magnocellular division of the PVN [164,165]. Disruption of the Wfs1 gene in mice causes progressive β-cell loss and impaired stimulus-secretion coupling in insulin secretion, but central diabetes insipidus is not observed in Wfs2/2 mice [166]. Miner1, another endoplasmic reticulum protein coded by ZCD2 [167], is causative in Wolfram syndrome 2 [168].

a

Primary malignancy: craniopharyngioma, dysgerminoma, meningioma, adenoma, glioma, astrocytoma. Secondary: metastatic from lung or breast, lymphoma, leukemia, dysplastic pancytopenia. c Trauma could be severe or mild. Data from Czernichow P, Pomarede R, Brauner R, Neurogenic diabetes insipidus in children. In: Czernichow P, Robinson AG, editors. Frontiers in hormone research: diabetes insipidus in man no. 13. Basel: S. Karger; 1985. Greger NG, Kirkland RT, Clayton GW, Kirkland JL. Central diabetes insipidus. 22 years’ experience. Am J Dis Child 1986;140(6):551 4. Moses AM, Blumenthal SA, Streeten DH, editors. Acidbase and electrolyte disorders associated with endocrine disease: pituitary and thyroid. Arieff AI, de Fronzo RA, editors. Fluid, electrolyte and acid-base disorders. New York, NY: Churchill Livingstone; 1985. and Maghnie M, Cosi G, Genovese E, et al. Central diabetes insipidus in children and young adults. N Engl J Med 2000;343 (14):998 1007. b

[155], necrotizing infundibulo-hypophysitis [156], IgG4 hypophysitis, and administration of ipilimumab [157,158]. Maghnie et al. [151] studied 79 patients with central diabetes insipidus. Additional deficits in anterior pituitary hormones were documented in 61% of patients, a median of 0.6 years (range: 0.1 18.0) after the onset of diabetes insipidus. The most frequent abnormality was growth hormone deficiency (59%), followed by hypothyroidism (28%), hypogonadism (24%), and adrenal insufficiency (22%). Seventy-five percent of the patients with Langerhans cell histiocytosis had an anterior pituitary hormone deficiency that was first detected a median of 3.5 years after the onset of diabetes insipidus [151]. None of the patients with central diabetes insipidus secondary to AVP mutations developed anterior pituitary hormone deficiencies.

Rare Forms Autosomal Dominant and Recessive Neurogenic Diabetes Insipidus Lacombe [159] and Weil [160] described a familial non-X-linked form of diabetes insipidus without associated mental retardation. Descendants of the family described by Weil were later found to have autosomal

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Syndrome of Hypernatremia and Hypodipsia Some patients with the hypernatremia and hypodipsia syndrome may have partial central diabetes insipidus. These patients also have persistent hypernatremia that is not due to any apparent extracellular volume loss, absence or attenuation of thirst, and a normal renal response to AVP. In almost all the patients studied to date, the hypodipsia has been associated with cerebral lesions in the vicinity of the hypothalamus. It has been proposed that in these patients there is a “resetting” of the osmoreceptor because their urine tends to become concentrated or diluted at inappropriately high levels of plasma osmolality. However, using the regression analysis of plasma AVP concentration versus plasma osmolality, it has been shown that in some of these patients the tendency to concentrate and dilute urine at inappropriately high levels of plasma osmolality is due solely to a marked reduction in sensitivity or a gain in the osmoregulatory mechanism [169]. This finding is compatible with the diagnosis of partial central diabetes insipidus. In other patients, however, plasma AVP concentrations fluctuate in a random manner, bearing no apparent relationship to changes in plasma osmolality. Such patients frequently display large swings in serum sodium concentration and frequently exhibit hypodipsia. It appears that most patients with “essential hypernatremia” fit one of these two patterns (Fig. 8.16). Both of these groups of patients consistently respond normally to nonosmolar AVP release signals, such as hypotension, emesis, or hypoglycemia, or all three. These observations suggest that: (1) the osmoreceptor may be anatomically as well as functionally separate from the nonosmotic efferent pathways and neurosecretory neurons for vasopressin and a hypothalamic lesion may impair the osmotic release of AVP while the nonosmotic release of AVP remains intact; and (2) the osmoreceptor neurons that regulate vasopressin secretion are not totally synonymous with those that regulate thirst, although they appear to be anatomically close if not overlapping. These two suggestions have been confirmed by the deconstruction of thirst and vasopressin secretion [4]. Nephrogenic Diabetes Insipidus In NDI, the kidney is unable to concentrate urine despite normal or elevated concentrations of the antidiuretic hormone AVP [55]. In congenital NDI, the obvious clinical manifestations of the disease, that is polyuria and polydipsia, are present at birth and need to be immediately recognized to avoid severe episodes of dehydration. It is clinically useful to distinguish two types of hereditary NDI: a “pure” type characterized by loss of water only and a complex type characterized

FIGURE 8.16 Plasma vasopressin as a function of “effective” plasma osmolality in two patients with adipsic hypernatremia. Unfilled circles indicate values obtained on admission; filled squares indicate those obtained during forced hydration; filled triangles indicate those obtained after 1 2 weeks of ad libitum water intake. Shaded areas indicate range of normal values. Source: From Robertson G. The pathophysiology of ADH secretion. In: Tolis G, Labrie F, Martin JB, editors. Clinical neuroendocrinology: a pathophysiological approach. New York, NY: Raven Press; 1979 [170], with permission, Wolters Kluwer.

by loss of water and ions. Patients who have congenital NDI and bear mutations in the AVPR2 or AQP2 genes have a “pure” NDI phenotype with loss of water but normal conservation of sodium, potassium, chloride, and calcium. Patients who bear inactivating mutations in genes (SLC12A1, KCNJ1, CLCNKB, CLCNKA, and CLCNKB in combination, or BSND) that encode the membrane proteins of the thick ascending limb of the loop of Henle have a complex polyuropolydipsic syndrome with loss of water, sodium, chloride, calcium, magnesium, and potassium. Most ( . 90%) of “pure” congenital NDI patients have mutations in the AVPR2 gene, the Xq28 gene coding for the vasopressin V2 (antidiuretic) receptor. In less than 10% of the families studied, congenital NDI has an autosomal recessive inheritance and mutations have been identified in the AQP2 gene located in chromosome region 12q13, i.e., the vasopressin-sensitive water channel. When studied in vitro, most AVPR2 mutations lead to receptors that are trapped intracellularly and are unable to reach the plasma membrane. A minority of the mutant receptors reach the cell surface but are unable to bind AVP or to trigger an intracellular cAMP signal. Similarly, AQP2 mutant proteins are trapped intracellularly and cannot be expressed at the luminal membrane. AVPR2 and AQP2 trafficking defects are correctable by chemical chaperones.

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Loss-of-Function Mutations of the AVPR2 X-linked NDI (OMIM 304800) is secondary to AVPR2 mutations, which result in a loss-of-function or dysregulation of the V2 receptor [171]. Rareness and Diversity of AVPR2 Mutations X-linked NDI is generally a rare disease in which the affected male patients do not concentrate their urine after administration of AVP [172]. Because this form is a rare, recessive X-linked disease, females are unlikely to be affected, but heterozygous females can exhibit variable degrees of polyuria and polydipsia because of skewed X chromosome inactivation. In Quebec, the incidence of this disease among males was estimated to be approximately 8.8 in 1,000,000 male live births [173]. A founder effect of two particular AVPR2 mutations [174], one in Ulster Scot immigrants (the Hopewell mutation, W71X) and one in a large Utah kindred (the Cannon pedigree), results in an elevated prevalence of X-linked NDI in their descendants in certain communities in Nova Scotia, Canada and in Utah, USA [174]. These founder mutations have now spread all over the North American continent. To date, we have identified the W71X mutation in 42 affected males who reside predominantly in the Maritime Provinces of Nova Scotia and New Brunswick, and the L312X mutation has been identified in eight affected males who reside in central USA. We know of 98 living affected males of the Hopewell kindred and 38 living affected males of the Cannon pedigree. We also determined that the historical case report by Perry et al. [175] was related to the Hopewell pedigree and had the W71X mutation. To date, .250 putative disease-causing AVPR2 mutations have been published in over 300 NDI families [176]. Approximately half of the mutations are missense mutations. Frameshift mutations owing to nucleotide deletions or insertions (24%), nonsense mutations (9%), large deletions (11%), in-frame deletions or insertions (4%), splice-site mutations (2%), and one complex mutation account for the remainder of the mutations [176]. Mutations have been identified in every domain, but on a per nucleotide basis about twice as many mutations occur in transmembrane domains compared with extracellular or intracellular domains. We previously identified private mutations, recurrent mutations, and mechanisms of mutagenesis [173]. Ten recurrent mutations (D85N, V88M, R113W, Y128S, R137H, S167L, R181C, R202C, A294P, and S315R) were found in 35 ancestrally independent families. The occurrence of the same mutation on different haplotypes was considered evidence for recurrent mutation. In addition, the most frequent mutations (D85N, V88N, R113W, R137H, S167L, R181C, and R202C) occurred at potential

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mutational hotspots (a C-to-T or G-to-A nucleotide substitution at a CpG dinucleotide). Most Mutant V2 Receptors are not Transported to the Cell Membrane and are Retained in the Intracellular Compartments Classification of the defects of naturally occurring mutant human V2 receptors can be based on a similar scheme to that used for the low-density lipoprotein receptor. Mutations have been grouped according to the function and subcellular localization of the mutant protein whose cDNA has been transiently transfected in a heterologous expression system [177]. Using this classification, type 1 mutant V2 receptors reach the cell surface but display impaired ligand binding and are consequently unable to induce normal cAMP production. The dose-dependent AVP-stimulated cAMP production could be compared with the cAMP production obtained with the wild-type receptor using a proteinbased bioluminescence resonance energy transfer cAMP biosensor that allows for the measurement of cAMP in living cells [178]. The presence of mutant V2 receptors on the surface of transfected cells can be determined pharmacologically. By carrying out saturation binding experiments using tritiated AVP, the number of cell surface mutant V2 receptors and their apparent binding affinity can be compared with that of the wild-type receptor. In addition, the presence of cell surface receptors can be assessed directly by using immunodetection strategies to visualize epitope-tagged receptors in whole-cell immunofluorescence assays. Type 2 mutant receptors have defective intracellular transport. This phenotype is confirmed by carrying out, in parallel, immunofluorescence experiments on cells that are intact (to demonstrate the absence of cell surface receptors) or permeabilized (to confirm the presence of intracellular receptor pools). In addition, protein expression is confirmed by western blot analysis of membrane preparations from transfected cells. It is likely that these mutant type 2 receptors accumulate in a pre-Golgi compartment because they are initially glycosylated but fail to undergo glycosyltrimming maturation. Type 3 mutant receptors are ineffectively transcribed and lead to unstable mRNAs which are rapidly degraded. This subgroup seems to be rare since northern blot analysis of cells expressing mutant AVPR2 receptors showed mRNAs of normal quantity and molecular size. Most of the AVPR2 mutants that we and other investigators have tested are type 2 mutant receptors. They did not reach the cell membrane and were trapped in the interior of the cell [179]. Other mutant G-protein-coupled receptors [180] and gene products

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causing genetic disorders are also characterized by protein misfolding. Mutations that affect folding of secretory proteins, integral plasma membrane proteins, or enzymes destined to the endoplasmic reticulum, Golgi complex, and lysosomes result in loss-offunction phenotypes irrespective of their direct impact on protein function because these mutant proteins are prevented from reaching their final destination [181]. Folding in the endoplasmic reticulum is the limiting step: mutant proteins that fail to fold correctly are initially retained in the endoplasmic reticulum and subsequently often degrade. Key proteins involved in the urine countercurrent mechanisms are good examples of this basic mechanism of misfolding. AQP2 mutations responsible for autosomal recessive NDI are characterized by misrouting of the misfolded mutant proteins and are trapped in the endoplasmic reticulum [182]. Other mutant renal membrane proteins responsible for Gitelman syndrome [183], Bartter syndrome [184,185], and cystinuria [186] are also retained in the endoplasmic reticulum. The AVPR2 missense mutations are likely to impair folding and to lead to rapid degradation of the misfolded polypeptide and not to the accumulation of toxic aggregates (as is the case for AVP mutants that cause neurohypophyseal diabetes insipidus), because the other important functions of the principal cells of the collecting duct (where AVPR2 is expressed) are entirely normal. These cells express the epithelial sodium channel (ENaC). Decreased function of this channel results in a sodium-losing state [187]. This has not been observed in patients with AVPR2 mutations. However, dDAVP did not stimulate sodium reabsorption in male patients with NDI bearing AVPR2 mutations [140], but this is a V2R-specific effect [188]. By contrast, another type of conformational disease is characterized by toxic retention of the misfolded protein. The relatively common Z mutation in α1-antitrypsin deficiency not only causes retention of the mutant protein in the endoplasmic reticulum, but also affects the secondary structure by insertion of the reactive center loop of one molecule into a destabilized β-sheet of a second molecule [189]. These polymers clog up the endoplasmic reticulum of hepatocytes and lead to cell death and juvenile hepatitis, cirrhosis, and hepatocarcinomas in these patients [190]. Nonpeptide Vasopressin Receptor Antagonists Act as Pharmacological Chaperones to Functionally Rescue Misfolded Mutant V2 Receptors Responsible for X-linked NDI If the misfolded protein/traffic problem responsible for so many human genetic diseases can be overcome and the mutant protein can be transported out of the endoplasmic reticulum to its final destination, these mutant proteins might be sufficiently functional [191].

Therefore, using pharmacological chaperones to promote escape from the endoplasmic reticulum is a possible therapeutic approach. We used selective nonpeptide V2 and V1 receptor antagonists to rescue the cell surface expression and function of naturally occurring misfolded human V2 receptors [179]. Since the beneficial effect of nonpeptide V2 antagonists could be secondary to prevention or interference with endocytosis, we studied the R137H mutant previously reported to lead to constitutive endocytosis [192]. We found that the antagonist did not prevent the constitutive β-arrestin-mediated endocytosis. These results indicate that as for other AVPR2 mutants, the beneficial effects of the treatment result from the action of the pharmacological chaperones. In clinical studies, we administered a nonpeptide vasopressin antagonist SR49059 to five adult NDI patients bearing the del62 64, R137H, or W164S mutation. SR49059 significantly decreased urine volume and water intake and increased urine osmolality, while sodium, potassium, and creatinine excretions and plasma sodium were constant throughout the study [193]. This new therapeutic approach could be applied to the treatment of several hereditary diseases resulting from errors in protein folding and kinesis [191]. Since most human gene therapy experiments using viruses to deliver and integrate DNA into host cells are potentially dangerous [194], other treatments are being actively pursued. Torsten Scho¨neberg and colleagues [195] used aminoglycoside antibiotics because of their ability to suppress premature termination codons. They demonstrated that geneticin, a potent aminoglycoside antibiotic, increased AVP-stimulated cAMP in cultured collecting duct cells prepared from E242X mutant mice. The urine-concentrating ability of heterozygous mutant mice was also improved. Gain-of-Function of the Vasopressin V2 Receptor: Nephrogenic Syndrome of Inappropriate Antidiuresis The clinical phenotype here is the opposite to NDI. Rare cases of infants or adults with hyponatremia, concentrated urine, and suppressed AVP plasma concentrations have been described bearing the mutations R137C, R137L, or F229V in their AVPR2 gene [196,197]. In contrast to R137C/L mutant receptors, F229V receptors do not undergo spontaneous desensitization, which results in sustained high basal activity. Also, the V2R selective inverse agonists tolvaptan and satavaptan completely silenced the constitutive signaling activity of the F229V mutant receptor, indicating that this substitution does not lock the receptor in an irreversible active state [197]. It is interesting to note that another mutation in the same codon (R137H) is a relatively frequent mutation causing classical NDI, albeit the phenotype may be milder in

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some patients [198]. With cell-based assays, both R137C and R137L were found to have elevated basal signaling through the cAMP pathway and to interact with β-arrestins in an agonist-independent manner. It is my opinion that AVPR2 mutations with gain-offunction are extremely rare. We have sequenced the AVPR2 gene in many patients with hyponatremia and never found a mutation. By contrast, we continue to identify new and recurrent loss-of-function AVPR2 mutations in patients with classical NDI.

Loss-of-Function Mutations of AQP2 On the basis of 1-desamino-8-D-arginine vasopressin (dDAVP) infusion studies and measurements of plasma cAMP levels following pharmacological intravenous doses of dDAVP, a vasopressin V2 synthetic analogue, we first suggested that X-linked NDI was a precyclic AMP defect [199,200]. Male patients with Xlinked NDI did not stimulate coagulation factor release or plasma cyclic AMP level after a pharmacological infusion of dDAVP, a suggestion of a loss of function of both renal and extrarenal vasopressin V2 receptors. Using dDAVP infusion studies and other families with severe polyuric characteristics in both male and female individuals, a non-X-linked form of NDI with a postreceptor (post-cAMP) defect was suggested [201,202]. A patient who presented shortly after birth with typical features of NDI but who exhibited normal coagulation and normal fibrinolytic and vasodilatory responses to dDAVP was shown to be a compound heterozygote for two missense mutations (R187C and S217P) in the AQP2 gene [203]. Expression of each of these two mutations in Xenopus oocytes revealed nonfunctional water channels. We used sequencing data provided by Deen et al. to solve the molecular identification of NDI in two inbred Pakistani girls with nonX-linked NDI originally reported by Langley et al. [202]. They were found to be homozygous for the AQP2 V71M mutation, a recurrent mutation in Pakistani kindred since two other children from two other Pakistani families living in the UK, said to be unrelated, were found to bear the same mutation on the same AQP2 haplotype [204]. To date, more than 51 putative disease-causing AQP2 mutations have been identified in 59 NDI families (Fig. 8.17). The oocytes of the African clawed toad (Xenopus laevis) have provided a most useful experimental system for studying the function of many channel proteins. This convenient expression system was key to the discovery of AQP1 by Agre [205] because toad oocytes have very low permeability and survive even in freshwater ponds. Control oocytes are injected with water alone; test oocytes are injected with various quantities of synthetic transcripts from AQP1 or AQP2 DNA (cRNA).

FIGURE 8.17 A representation of the AQP2 protein and identification of 46 putative disease-causing AQP2 mutations. A monomer is represented with six transmembrane helices. The extracellular, transmembrane, and cytoplasmic domains are defined according to Deen et al. [203]. Solid symbols indicate the location of the mutations: M1I; L22V; V24A; L28P; G29S; A47V; Q57P; G64R, N68S, A70D; V71M; R85X; G100X; G100V; G100R; I107D; 369delC; T125M; T126M; A147T; D150E; V168M; G175R; G180S; C181W; P185A; R187C; R187H; A190T; G196D; W202C; G215C; S216P; S216F; K228E; R254Q; R254L; E258K; and P262L. GenBank accession numbers— AQP2: AF147092, exon 1; AF147093, exons 2 4. NPA motifs, the Nglycosylation site, the ubiquitination site (Ub), and phosphorylation sites (P) in the C-terminus are also indicated.

When subjected to a 20-mOsm osmotic shock, control oocytes have exceedingly low water permeability but test oocytes become highly permeable to water. These osmotic water permeability assays demonstrated an absence or very low water transport for all of the cRNA with AQP2 mutations. Immunofluorescence and immunoblot studies demonstrated that these recessive mutants were retained in the endoplasmic reticulum. AQP2 mutations in autosomal recessive NDI, which are located throughout the gene, result in misfolded proteins that are retained in the endoplasmic reticulum. In contrast, the dominant mutations reported to date are located in the region that codes for the carboxyl terminus of AQP2. Dominant AQP2 mutants form heterotetramers with wt-AQP2 and are misrouted.

Complex Polyuropolydipsic Syndrome In contrast to a “pure” NDI phenotype, with loss of water but normal conservation of sodium, potassium, chloride, and calcium, in Bartter syndrome, patients’ renal wasting starts prenatally and polyhydramnios often leads to prematurity. Bartter syndrome (OMIM 601678, 241200, 607364, and 602522) refers to a group

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of autosomal recessive disorders caused by inactivating mutations in genes (SLC12A1, KCNJ1, CLCNKB, CLCNKA, and CLCNKB in combination, or BSND) that encode membrane proteins of the thick ascending limb of the loop of Henle (for review see Ref. [206]). Although Bartter syndrome and Bartter’s mutations are commonly used as a diagnosis, it is likely, as explained by Jeck et al. [207], that the two patients with a mild phenotype originally described by Dr. Bartter had Gitelman syndrome, a thiazide-like saltlosing tubulopathy with a defect in the distal convoluted tubule [207]. As a consequence, salt-losing tubulopathy of the furosemide type is a more physiologically appropriate definition. Thirty percent of the filtered sodium chloride is reabsorbed in the thick ascending limb of the loop of Henle through the apically expressed sodium potassium chloride cotransporter NKCC2 (encoded by the SLC12A1 gene), which uses the sodium gradient across the membrane to transport chloride and potassium into the cell. The potassium ions must be recycled through the apical membrane by the potassium-channel ROMK (encoded by the KCNJ1 gene). In the large experience of Seyberth and colleagues [208], who studied 85 patients with a hypokalemic salt-losing tubulopathy, all 20 patients with KCNJ1 mutations (except one) and all 12 patients with SLC12A1 mutations were born as preterm infants after severe polyhydramnios. Of note, polyhydramnios was never seen during the pregnancies that led to infants bearing AVPR2 or AQP2 mutations. The most common causes of increased amniotic fluid include maternal diabetes mellitus, fetal malformations and chromosomal aberrations, twin-to-twin transfusion syndrome, rhesus incompatibility, and congenital infections [209]. Postnatally, polyuria was the leading symptom in 19 of the 32 patients. Renal ultrasound revealed nephrocalcinosis in 31 of these patients. These patients with complex polyuropolydipsic disorders are often poorly recognized and may be confused with “pure” NDI. As a consequence, congenital polyuria does not suggest automatically AVPR2 or AQP2 mutations, and polyhydramnios, salt wasting, hypokalemia, and nephrocalcinosis are important clinical and laboratory characteristics that should be assessed. In patients with Bartter syndrome (salt-losing tubulopathy/furosemide type), the dDAVP test will only indicate a partial type of NDI. The algorithm proposed by Peters et al. [208] is useful since most mutations in SLC12A1 and KCNJ1 are found in the carboxyl terminus or in the last exon and, as a consequence, are amenable to rapid DNA sequencing. Nephropathic cystinosis, nephronophthisis, and apparent mineralocorticoid excess are also characterized by polyuria, polydipsia, and poor urinary osmolality response to AVP [210].

Acquired NDI (Table 8.3) Acquired NDI is much more common than congenital NDI but it is rarely as severe. The ability to produce hypertonic urine is usually preserved even though there is inadequate concentrating ability of the

TABLE 8.3 Acquired Causes of Nephrogenic Diabetes Insipidus Chronic renal disease

Polycystic disease Medullary cystic disease Pyelonephritis Ureteral obstruction Far-advanced renal failure

Electrolyte disorders

Hypokalemia Hypercalcemia

Drugs

Alcohol Phenytoin Lithium Demeclocycline Acetohexamide Tolazamide Glyburide Propoxyphene Amphotericin Methoxyflurane Norepinephrine Vinblastine Colchicine Gentamicin Methicillin Isophosphamide Angiographic dyes Osmotic diuretics Furosemide and ethacrynic acid

Sickle cell disease Dietary abnormalities

Excessive water intake Decreased sodium chloride intake Decreased protein intake

Miscellaneous

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Multiple myeloma Amyloidosis Sjo¨gren disease Sarcoidosis

CLINICAL CHARACTERISTICS OF DIABETES INSIPIDUS DISORDERS

nephron. Polyuria and polydipsia are therefore moderate (3 4 L/day). Among the more common causes of acquired NDI, lithium administration has become the most frequent cause; 54% of 1105 unselected patients on chronic lithium therapy developed NDI [211]. Nineteen percent of these patients had polyuria, as defined by a 24-hour urine output exceeding 3 L. The dysregulation of AQP2 expression is the result of cytotoxic accumulation of lithium which enters via the ENaC on the apical membrane and leads to the inhibition of signaling pathways that involve glycogen synthase kinase type 3 beta [212]. The urinary concentration of lithium in patients on well-controlled lithium therapy (i.e., 10 40 mOsmol/L) is sufficient to exert this effect. For patients on long-term lithium therapy, amiloride prevents uptake of lithium in the collecting ducts, thus preventing inhibitory effects of intracellular lithium on water transport [213]. Other new experimental treatments have been tested in animal models [214]. Primary Polydipsia Primary polydipsia is a state of hypotonic polyuria secondary to excessive fluid intake. Primary polydipsia was extensively studied by Barlow and de Wardener in 1959 [215]; however, the understanding of the pathophysiology of this disease has made little progress. Barlow and de Wardener [215] described seven women and two men who were compulsive water drinkers; their ages ranged from 48 to 59 years except for one patient who was 24 years old. Eight of these patients had histories of previous psychological disorders, which ranged from delusions, depression, and agitation, to frank hysterical behavior. The other patient appeared normal. The consumption of water fluctuated irregularly from hour to hour or from day to day; in some patients, there were remissions and relapses lasting several months or longer. In eight of the patients, the mean plasma osmolality was significantly lower than normal. Vasopressin tannate in oil made most of these patients feel ill; in one, it caused overhydration. In four patients, the fluid intake returned to normal after electroconvulsive therapy or a period of continuous narcosis; the improvement in three was transient, but in the fourth it lasted 2 years. Polyuric female subjects might be heterozygous for de novo or previously unrecognized AVPR2 mutations, may bear AQP2 mutations, and may be classified as compulsive water drinkers [216]. Therefore, the diagnosis of compulsive water drinking must be made with care and may represent our ignorance of yet undescribed pathophysiological mechanisms. Robertson [216] has described under the name “dipsogenic diabetes insipidus” a selective defect in the osmoregulation of thirst. Three studied

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patients had, under basal conditions of ad libitum water intake, thirst, polydipsia, polyuria, and highnormal plasma osmolality. They had a normal secretion of AVP, but osmotic threshold for thirst was abnormally low. These dipsogenic diabetes insipidus cases might represent up to 10% of all patients with diabetes insipidus [216]. Diabetes Insipidus and Pregnancy PREGNANCY IN A PATIENT KNOWN TO HAVE DIABETES INSIPIDUS

An isolated deficiency of vasopressin without a concomitant loss of hormones in the anterior pituitary does not result in altered fertility, and with the exception of polyuria and polydipsia, gestation, delivery, and lactation are uncomplicated [217]. Patients may require increasing dosages of dDAVP. The increased thirst may be due to a resetting of the thirst osmostat [93]. Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, stimulates thirst and vasopressin secretion and could explain the water/electrolyte changes observed during pregnancy [218]. Increased polyuria also occurs during pregnancy in patients with partial NDI [219]. These patients may be obligatory carriers of the NDI gene [220] or may be homozygotes, compound heterozygotes, or may have dominant AQP2 mutations. SYNDROMES OF DIABETES INSIPIDUS THAT BEGIN DURING GESTATION AND REMIT AFTER DELIVERY

Barron et al. [221] described three pregnant women in whom transient diabetes insipidus developed late in gestation and subsequently remitted postpartum. In one of these patients, dilute urine was present despite high plasma concentrations of AVP. Hyposthenuria in all three patients was resistant to administered aqueous vasopressin. Because excessive vasopressinase activity was not excluded as a cause of this disorder, Barron et al. labeled the disease vasopressin-resistant rather than NDI. A well-documented case of enhanced activity of vasopressinase has been described in a woman in the third trimester of a previously uncomplicated pregnancy [222]. She had massive polyuria and markedly elevated plasma vasopressinase activity. The polyuria did not respond to large intravenous doses of AVP but responded promptly to dDAVP, a vasopressinaseresistant analogue of AVP. The polyuria disappeared with the disappearance of the vasopressinase. It is suggested that pregnancy may be associated with several different forms of diabetes insipidus, including central, nephrogenic, and vasopressinasemediated [219,223].

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INVESTIGATION OF A PATIENT WITH POLYURIA Plasma sodium and osmolality are maintained within normal limits (136 143 mOsmol/L for plasma sodium; 275 290 mOsmol/kg for plasma osmolality) by a thirst AVP renal axis. Thirst and AVP release, both stimulated by increased osmolality, is a “doublenegative” feedback system [224]. Even when the AVP component of this “double-negative” regulatory feedback system is lost, the thirst mechanism still preserves the plasma sodium and osmolality within the normal range, but at the expense of pronounced polydipsia and polyuria. Thus, the plasma sodium concentration or osmolality of an untreated patient with diabetes insipidus may be slightly greater than the mean normal value, but these small increases have no diagnostic significance. Theoretically, it should be relatively easy to differentiate between neurogenic diabetes insipidus, NDI, and primary polydipsia by comparing the osmolality of urine obtained during dehydration with that of urine obtained after the administration of dAVP. Patients with neurogenic diabetes insipidus should reveal a rapid increase in urinary osmolality, whereas it should increase normally in response to moderate dehydration in patients with primary polydipsia. However, for several reasons, these distinctions may not be as clear as one might expect [225]. First, chronic polyuria resulting from any cause interferes with the maintenance of the medullary concentration gradient and this “washout” effect diminishes the maximum concentrating ability of the nephron. The extent of the blunting varies in direct proportion to the severity of the polyuria. Hence, for any given basal urine output, the maximum urine osmolality achieved in the presence of saturating concentrations of AVP is depressed to the same extent in patients with primary polydipsia, neurogenic diabetes insipidus, or NDI (Fig. 8.18). Second, most patients with neurogenic diabetes insipidus maintain a small, but detectable, capacity to secrete AVP during severe dehydration, and urinary osmolality may then increase to greater than the plasma osmolality. Third, patients referred to as having partial diabetes insipidus (either neurogenic or nephrogenic) and patients with acquired NDI have an incomplete response to AVP and are able to concentrate their urine to varying degrees in a dehydration test. Finally, all polyuric states (whether neurogenic, nephrogenic, or psychogenic) can induce large dilatations of the urinary tract and bladder [226,227]. As a consequence, the urinary bladder of these patients has an increased residual capacity, and changes in urinary osmolality induced by diagnostic maneuvers might be difficult to demonstrate.

FIGURE 8.18 The relationship between urine osmolality and plasma vasopressin in patients with polyuria of diverse etiology and severity. Note that for each of the three categories of polyuria—neurogenic diabetes insipidus, nephrogenic diabetes insipidus, and primary polydipsia, the relationship is described by a family of sigmoid curves that differ in height. These differences in height reflect differences in maximal concentrating capacity owing to “washout” of the medullary concentration gradient. They are proportional to the severity of the underlying polyuria (indicated in liters per day at the right end of each plateau) and are largely independent of the etiology. Thus, the three categories of diabetes insipidus differ principally in the submaximal or ascending portion of the dose response curve. In patients with partial neurogenic diabetes insipidus, this part of the curve lies to the left of normal, reflecting increased sensitivity to the antidiuretic effects of very low concentrations of plasma arginine vasopressin (AVP). In contrast, in patients with partial nephrogenic diabetes insipidus, this part of the curve lies to the right of normal, reflecting decreased sensitivity to the antidiuretic effects of normal concentrations of plasma AVP. In primary polydipsia, this relationship is relatively normal. Source: From Robertson GL. Diagnosis of diabetes insipidus. In: Czernichow AGRP, editor. Frontiers of hormone research, vol. 13. Basel: S. Karger; 1985: p. 176 [225], with permission, Karger AG Basel.

Indirect Tests for Diabetes Insipidus The measurement of urinary osmolality after dehydration and dDAVP administration is usually referred to as “indirect testing” because AVP secretion is indirectly assessed through changes in urinary osmolalities [117]. The patient is maintained on a complete fluidrestriction regimen until urinary osmolality reaches a plateau, as indicated by an hourly increase of less than 30 mOsmol/kg for at least three successive hours. After measuring the plasma osmolality, 2 μg of dDAVP is administered subcutaneously. Urinary osmolality is measured 30 and 60 min later. The last urinary osmolality value obtained before the dDAVP injection and the highest value obtained after the

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INVESTIGATION OF A PATIENT WITH POLYURIA

injection are compared. In patients with severe neurogenic diabetes insipidus, urinary osmolality after dehydration is usually low (,200 mOsmol/kg) and increases more than 50% after dDAVP administration. In patients with severe NDI, urinary osmolality after dehydration is also low (,200 mOsmol/kg) but does not increase after dDAVP administration (,20%). Urinary osmolality increases to variable degrees (10 50%) after dDAVP administration to patients with partial neurogenic or partial NDI. In patients with primary polydipsia, maximum urinary osmolality will be obtained after dehydration ( . 295 mOsmol/kg) and does not increase after dDAVP administration (,10%). Alternatively, plasma sodium and plasma and urinary osmolalities can be measured at the beginning of the dehydration procedure and at regular intervals (usually hourly) thereafter depending on the severity of the polyuria. For example, an 8-year-old patient (body weight 31 kg) with a clinical diagnosis of congenital NDI (later found to bear an AVPR2 mutation) continued to excrete large volumes of urine (300 mL/h) during a short 4-hour dehydration test. During this time, the patient suffered from severe thirst, his plasma sodium was 155 mOsmol/L, plasma osmolality was 310 mOsmol/kg, and urinary osmolality was 85 mOsmol/kg. The patient received 1 μg of dDAVP intravenously and was allowed to drink water. Repeated urinary osmolality measurements demonstrated a complete urinary resistance to dDAVP. It would have been dangerous and unnecessary to prolong the dehydration further in this young patient. Thus, the usual prescription of overnight dehydration should not be used in patients, and especially children, with severe polyuria and polydipsia (more than 30 mL/kg body weight per day). Great care should be taken to avoid any severe hypertonic state, arbitrarily defined as plasma sodium greater than 155 mOsmol/L.

Direct Tests of Diabetes Insipidus The two approaches of Zerbe and Robertson are used [116], although they are expensive, timeconsuming, and difficult to carry out on young patients. In the first approach, during the dehydration test, plasma is collected hourly and assayed for AVP. The results are plotted on a nomogram depicting the normal relationship between plasma sodium or osmolality and plasma AVP in normal individuals (Fig. 8.10). If the relationship goes below the normal range, the disorder is diagnosed as neurogenic diabetes insipidus. In the second approach, NDI can be differentiated from primary polydipsia by analyzing the relationship between plasma AVP and urinary osmolality at the

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end of the dehydration period (Fig. 8.10). However, definitive differentiation might be impossible because a normal or even supranormal AVP response to increased plasma osmolality occurs in polydipsic patients. None of the patients with psychogenic or other forms of severe polydipsia studied by Robertson showed any evidence of pituitary suppression [225]. In a comparison of diagnoses based on indirect versus direct tests of AVP function in 54 patients with polyuria of diverse cause, Robertson [225] found that the indirect test was reliable only for patients with severe defects. Three severe NDI patients and 16 of 17 patients with severe neurogenic diabetes insipidus were accurately diagnosed. However, the error rate of the indirect test was about 50% in diagnosing partial neurogenic diabetes insipidus, partial NDI, or primary polydipsia in patients who were able to concentrate their urine to varying degrees when water-deprived. The benefits of combined direct and indirect testing of AVP function have been discussed by Stern and Valtin [228]. The diagnosis of primary polydipsia remains one of exclusion and the cause could be psychogenic [215] or inappropriate thirst [216,229]. Psychiatric patients with polydipsia and hyponatremia have unexplained defects in urinary dilution, the osmoregulation of water intake or the secretion of vasopressin [230]. Therapeutic Trial of dDAVP In selected patients with an uncertain diagnosis, a closely monitored therapeutic trial of dDAVP (10 μg intranasally twice a day for 2 3 days) may be used to distinguish partial NDI from partial neurogenic diabetes insipidus or primary polydipsia. If dDAVP at this dosage causes a significant antidiuretic effect, NDI is effectively excluded. If polydipsia as well as polyuria is abolished and plasma sodium does not go below the normal range, the patient probably has neurogenic diabetes insipidus. Conversely, if dDAVP causes a reduction in urine output without reduction in water intake and hyponatremia appears, the patient probably has primary polydipsia. Since fatal water intoxication is a remote possibility, the dDAVP trial should be closely monitored. The methods of differential diagnosis of diabetes insipidus are described in Tables 8.4 and 8.5. Carrier Detection, Perinatal Testing, and Early Treatment The identification of mutations in the genes that cause hereditary diabetes insipidus allows the early diagnosis and management of at-risk members of families with identified mutations. We encourage physicians who follow families with autosomal neurogenic, X-linked and autosomal NDI to recommend mutation

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TABLE 8.4 Urinary Responses to Fluid Deprivation and Exogenous Vasopressin in Recognition of Partial Defects in Antidiuretic Hormone Secretion Number of cases

Maximum Uosma

Uosm after vasopressin

Percent change (Uosm)

Uosm increase after vasopressin

Normal subjects

9

1068 6 69

979 6 79

29 6 3

,9%

Complete central diabetes insipidus

8

168 6 13

445 6 52

183 6 41

.50%

Partial central diabetes insipidus

11

438 6 34

549 6 28

28 6 5

.9% , 50%

Nephrogenic diabetes insipidus

2

123.5

174.5

42

,50%

Compulsive water drinking

7

738 6 73

780 6 73

5.0 6 2.2

,9%

a

Urinary osmolality (Uosm) in mmol/kg. Data from Miller M, Dalakos T, Moses AM, Fellerman H, Streeten DH. Recognition of partial defects in antidiuretic hormone secretion. Ann Intern Med 1970;73(5):721 9.

TABLE 8.5 Differential Diagnosis of Diabetes Insipidus 1. Measure plasma osmolality and/or sodium concentration under conditions of ad libitum fluid intake. If they are above 295 mmol/kg and 143 mmol/L, the diagnosis of primary polydipsia is excluded and the work-up should proceed directly to step 5 and/or 6 to distinguish between neurogenic and nephrogenic diabetes insipidus. Otherwise: 2. Perform a dehydration test. If urinary concentration does not occur before plasma osmolality and/or sodium reach 295 mmol/kg or 143 mmol/L, the diagnosis of primary polydipsia is again excluded and the work-up should proceed to step 5 and/ or 6. Otherwise: 3. Determine the ratio of urine to plasma osmolality at the end of the dehydration test. If it is less than 1.5, the diagnosis of primary polydipsia is again excluded and the work-up should proceed to step 5 and/or 6. Otherwise: 4. Perform a hypertonic saline infusion with measurements of plasma vasopressin and osmolality at intervals during the procedure. If the relationship between these two variables is subnormal, the diagnosis of diabetes insipidus is established. Otherwise: 5. Perform a vasopressin infusion test. If urine osmolality rises by more than 150 mosmol/kg above the value obtained at the end of the dehydration test, nephrogenic diabetes insipidus is excluded. Alternately: 6. Measure urine osmolality and plasma vasopressin at the end of the dehydration test. If the relationship is normal, the diagnosis of nephrogenic diabetes insipidus is excluded. Data from Robertson GL. Diseases of the posterior pituitary. In: Felig D, Baxter JD, Broadus AE, editors. Endocrinology and metabolism. New York, NY: McGraw-Hill; 1981: p. 251 [231].

analysis before the birth of an infant because early diagnosis and treatment can avert the physical and mental retardation associated with episodes of dehydration. Diagnosis of X-linked NDI was accomplished by mutation testing of cultured amniotic cells (n 5 7), chorionic villus samples (n 5 10), or cord blood obtained at birth (n 5 57) in our patients. Three infants who had mutation testing done on amniotic cells or chorionic villous samples also had the diagnosis confirmed by cord blood testing. Of 74 offspring tested, 35 were found to be affected males, 22 were unaffected

males, and nine were noncarriers (M.-F. Arthus, M. Lonergan, and D.G. Bichet, unpublished data). Affected patients were immediately given abundant water intake, a low-sodium diet, and hydrochlorothiazide. They never experienced episodes of dehydration, and their physical and mental development is normal. Gene analysis is also important for the identification of nonobligatory female carriers in families with X-linked NDI. Most females heterozygous for a mutation in the V2 receptor do not have clinical symptoms; few are severely affected [173]. Mutation detection in families with inherited neurogenic diabetes insipidus provides a powerful clinical tool for early diagnosis and management of subsequent cases, especially in early childhood, when diagnosis is difficult and the clinical risks are the greatest [232]. Neurogenic diabetes insipidus (central or Wolfram) is easily treated with dDAVP [233]. All complications of congenital NDI are prevented by an adequate water intake. Thus, patients should be provided with unrestricted amounts of water from birth to ensure normal development. In addition to a low-sodium diet, the use of diuretics (thiazides) or indometacin may reduce urinary output. This advantageous effect has to be weighed against the side effects of these drugs (thiazides: electrolyte disturbances; indometacin: reduction of the glomerular filtration rate and gastrointestinal symptoms).

RADIOIMMUNOASSAY OF AVP, COPEPTIN, AND OTHER LABORATORY DETERMINATIONS Radioimmunoassay of AVP Three developments were basic to the elaboration of a clinically useful radioimmunoassay for plasma AVP [234]: (1) the extraction of AVP from plasma with petrol ether and acetone and the subsequent elimination of nonspecific immunoreactivity; (2) the use of

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RADIOIMMUNOASSAY OF AVP, COPEPTIN, AND OTHER LABORATORY DETERMINATIONS

highly specific and sensitive rabbit antiserum; and (3) the use of a tracer (125I-AVP) with high specific activity. These same extraction procedures are still widely used [79,92], and commercial tracers (125I-AVP) and antibodies are available. AVP can also be extracted from plasma by using Sep-Pak C18 cartridges [235]. Blood samples collected in chilled 7 mL lavenderstoppered tubes containing EDTA are centrifuged at 4 C, 1000 g (3000 rpm in a usual laboratory centrifuge), for 20 minutes. This 20-minute centrifugation is mandatory for obtaining platelet-poor plasma samples because a large fraction of the circulating vasopressin is associated with the platelets in humans [79,236]. The tubes may be kept for 2 hours on slushed ice prior to centrifugation. Plasma is then separated, frozen at 20 C, and extracted within 6 weeks of sampling. Details for sample preparation and assay procedure can be found in writings by Bichet and colleagues [79]. An AVP radioimmunoassay should be validated by demonstrating: (1) a good correlation between plasma sodium or osmolality and plasma AVP during dehydration and infusion of hypertonic saline solution (Fig. 8.10); and (2) the inability to obtain detectable values of AVP in patients with severe central diabetes insipidus. Plasma AVPimmunoreactivity may be elevated in patients with diabetes insipidus following hypothalamic surgery [237]. In pregnant patients, the blood contains high concentrations of cystine aminopeptidase which can (in vitro) inactivate enormous quantities (ng/mL per min) of AVP. However, phenanthrolene effectively inhibits these cystine aminopeptidases. Measurements of copeptin by ELISA could, in the future, replace the difficult radioimmunological measure of AVP [238]. Copeptin, the C-terminal glycoprotein of the AVP prohormone, is an easily measureable and stable surrogate of endogenous plasma AVP. It is more used in Europe and of interest for evaluation of polyuric disorders [239 241].

AQP2 Measurements Urinary AQP2 excretion could be measured by radioimmunoassay or quantitative western analysis and could provide an additional indication of the responsiveness of the collecting duct to AVP [242]

Plasma Sodium, Plasma, and Urine Osmolality Measurements of plasma sodium, plasma, and urinary osmolality should be immediately available at various intervals during dehydration procedures. Plasma sodium is easily measured by flame photometry or with a sodium-specific electrode [243]. Plasma and urinary osmolalities are also reliably measured by

277

freezing point depression instruments with a coefficient of variation at 290 mmol/kg of less than 1%. At variance with published data [79,116], we have found that plasma and serum osmolalities are equivalent (i.e., similar values are obtained). Blood taken in heparinized tubes is easier to handle because the plasma can be more readily removed after centrifugation. The tube used (green-stoppered tube) contains a minuscule concentration of lithium and sodium, which does not interfere with plasma sodium or osmolality measurements. Frozen plasma or urinary samples can be kept for further analysis of their osmolalities because the results obtained are similar to those obtained immediately after blood sampling, except in patients with severe renal failure. In the latter patients, plasma osmolality measurements are increased after freezing and thawing but the plasma sodium values remain unchanged. Plasma osmolality measurements can be used to demonstrate the absence of unusual osmotically active substances (e.g., glucose and urea in high concentrations, mannitol, ethanol). With this information, plasma or serum sodium measurements are sufficient to assess the degree of dehydration and its relationship to plasma AVP. Nomograms describing the normal plasma sodium/plasma AVP relationship (Fig. 8.10) are equally as valuable as classic nomograms describing the relationship between plasma osmolality and effective osmolality (i.e., plasma osmolality minus the contribution of “ineffective” solutes: glucose and urea).

Magnetic Resonance Imaging in Patients With Diabetes Insipidus Magnetic resonance imaging (MRI) permits visualization of the anterior and posterior pituitary glands and the pituitary stalk. The pituitary stalk is permeated by numerous capillary loops of the hypophyseal portal blood system. This vascular structure also provides the principal blood supply to the anterior pituitary lobe, because there is no direct arterial supply to this organ. In contrast, the posterior pituitary lobe has a direct vascular supply. Therefore, the posterior lobe can be more rapidly visualized in a dynamic mode after administration of a gadolinium (gadopentate dimeglumine) as contrast material during MRI. The posterior pituitary lobe is easily distinguished by a round, high-intensity signal (the posterior pituitary “bright spot”) in the posterior part of the sella turcica on T1-weighted images. Loss of the pituitary hyperintense spot or bright spot on a T1-weighted MRI image reflects loss of functional integrity of the neurohypophysis and is a nonspecific indicator of neurohypophyseal diabetes insipidus regardless of the underlying cause [151] since it is also observed in

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X-linked severe NDI [244]. It is now considered that the bright spot represents normal AVP storage in the posterior lobe of the pituitary that the intensity is correlated with the amount of AVP and that after 60 years of age the signal is often less intense with irregularities in the normally smooth convex edge [245,246]. MRI is reported to be the best technique with which to evaluate the pituitary stalk and infundibulum in patients with idiopathic polyuria. A thickening or enlargement of the pituitary stalk may suggest an infiltrative process destroying the neurohypophyseal tract [247].

Treatment In most patients with complete hypothalamic diabetes insipidus, the thirst mechanism remains intact. Thus, hypernatremia does not develop in these patients and they suffer only from the inconvenience associated with marked polyuria and polydipsia. If hypodipsia develops or access to water is limited, then severe hypernatremia can supervene. The treatment of choice for patients with severe hypothalamic diabetes insipidus is dDAVP, a synthetic, long-acting vasopressin analogue, with minimal vasopressor activity but a large antidiuretic potency [233]. The usual intranasal daily dose is between 5 and 20 μg. To avoid the potential complication of dilutional hyponatremia, which is exceptional in these patients as a result of an intact thirst mechanism, dDAVP can be withdrawn at regular intervals to allow the patients to become polyuric. Aqueous vasopressin (Pitressin) or dDAVP (4.0 μg/mL ampule) can be used intravenously in acute situations such as after hypophysectomy or for the treatment of diabetes insipidus in the brain-dead organ donor. Pitressin tannate in oil and nonhormonal antidiuretic drugs are somewhat obsolete and now rarely used. For example, chlorpropamide (250 500 mg daily) appears to potentiate the antidiuretic action of circulating AVP, but troublesome side effects of hypoglycemia and hyponatremia do occur. In the treatment of congenital NDI, an abundant unrestricted water intake should always be provided, and affected patients should be carefully followed during their first years of life. Water should be offered every 2 hours, day and night, and temperature, appetite, and growth should be monitored. The parents of these children easily accept setting their alarm clock every 2 hours during the night. Hospital admission may be necessary to allow for continuous gastric feeding. A low-osmolar and low-sodium diet, hydrochlorothiazide (1 2 mg/kg per day) alone or with amiloride, and indometacin (0.75 1.5 mg/kg) substantially reduce water excretion and are helpful in the treatment of children. Many adult patients receive no treatment.

SYNDROME OF INAPPROPRIATE SECRETION OF THE ANTIDIURETIC HORMONE (SIADH) Hyponatremia (defined as a plasma sodium below 130 mmol/L) is the most common disorder of bodyfluid and electrolyte balance encountered in the clinical practice of medicine, with incidences ranging from 1% to 2% in both acutely and chronically hospitalized patients [248 250]. Because a defect in renal water excretion, as reflected by hypoosmolality, may occur in the presence of an excess or deficit of total body sodium or nearly normal total body sodium, it is useful to classify the hyponatremic states accordingly [251]. Moreover, since total body sodium is the primary determinant of the ECF volume, bedside evaluation of the ECF volume allows for a convenient means of classifying hyponatremic patients [251] (Fig. 8.19). Patients with hyponatremia who show no evidence of either hypovolemia or edema constitute a select group. Although many of these patients may have SIADH (this syndrome is so named because the secretion of AVP cannot be accounted for by recognized osmotic or nonosmotic stimuli), endocrine disorders such as hypothyroidism, hypopituitarism with glucocorticoid deficiency, various pharmacological agents, and emotional and physical stress may also cause euvolemic hyponatremia [128]. Psychotic patients with polydipsia and hyponatremia are also classified in this group of hyponatremic patients with normal total body sodium [230,252]. The diagnosis of SIADH is made primarily by excluding other causes of hyponatremia. It should be considered in the absence of hypovolemia, edematous disorders, endocrine dysfunction (including primary and secondary adrenal insufficiency and hypothyroidism), renal failure and drugs, all of which impair water excretion. Psychotic patients with polydipsia and hyponatremia have multiple disturbances in water regulation including alteration in osmoreceptor function, inappropriate thirst response, renal hypersensitivity to vasopressin, and vasopressinindependent perturbation of urinary dilution. Thus, we recommend that these patients should not be classified as presenting with SIADH. An animal model of antidiuretic-induced hyponatremia closely resembling clinical SIADH was developed by Verbalis and coworkers using a continuous subcutaneous infusion of dDAVP in combination with dextrose drinking or the self-ingestion of a concentrated, nutritionally balanced liquid diet. A chronic, severe hyponatremia accompanied by an antidiuretic effect was obtained. Multiple hemodynamic and hormonal adaptive responses were also observed [253].

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SYNDROME OF INAPPROPRIATE SECRETION OF THE ANTIDIURETIC HORMONE (SIADH)

10

Type I random

Type II reset

8

Plasma vasopressin (pg/mL)

6 Normal range

4

Normal range

2

279

FIGURE 8.19 Plasma vasopressin as a function of plasma osmolality during the infusion of hypertonic saline in patients with syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Source: From Robertson G. The pathophysiology of ADH secretion. In: Tolis G, Labrie F, Martin JB, editors. Clinical neuroendocrinology: a pathophysiological approach. New York, NY: Raven Press; 1979, with permission, Wolters Kluwer.

0

10

Type III leak

Type IV “Normal”

8 6 4

Normal range

Normal range

2 0 250 260 270 280 290 300 310 250 260 270 280 290 300 310 Plasma osmolality (moSmol/kg)

Since 1957, when Schwartz et al. [254] first described SIADH in two patients with bronchogenic carcinoma who were hyponatremic, clinically euvolemic with normal renal and adrenal function, and who had less than maximally dilute urine with appreciable urinary sodium concentrations (greater than 20 mmol/L), SIADH has been recognized in a variety of pathological processes. Various diseases, which may be accompanied by SIADH, are listed in Table 8.6. These diseases generally fall into three categories: (1) malignancies; (2) pulmonary disorders; and (3) central nervous system disorders. Human immunodeficiency virus infection forms a new category of patients with SIADH, with as many as 35% of hospitalized patients affected. In these patients, Pneumocystis carinii pneumonia, CNS infections, and malignancies play a role in the development of SIADH [255]. Tumors can synthesize and secrete AVP. Many tumors contain typical secretory granules and cultured tumor tissue has been shown to synthesize not only AVP but also the entire AVP precursor peptide, propressophysin [256,257]. Furthermore, tumor extracts have been found to contain AVP bioactivity and immunologically recognizable AVP [258]. Numerous reports have called attention to a rare tumor, olfactory neuroblastoma, which is frequently associated with chronic, occasionally symptomatic, hyponatremia [259]. In spite of hyponatremia, patients with SIADH have a concentrated urine in which the urinary sodium concentration closely parallels the sodium intake, i.e., it is

usually above 20 mmol/L. However, in the presence of sodium restriction or volume depletion, these patients can conserve sodium normally and decrease their urinary sodium concentration to less than 10 mmol/L [260]. Serum uric acid has been found to be reduced in SIADH patients, whereas patients with other causes of hyponatremia have normal concentrations of serum uric acid. Similarly, low serum blood urea nitrogen concentrations have been found in SIADH [261]. This is probably due to an increase in total body water, where urea is normally distributed, but a decrease in protein intake could also contribute. Plasma atrial natriuretic factor concentration has been found to be increased in patients with SIADH and to correlate with urinary sodium excretion [262]. Abnormal osmoregulation of vasopressin has been studied in 79 patients with SIADH [263]. These patients underwent either hypertonic saline or water loading or both and four patterns of responses were identified. The type I pattern, observed in 37% of the patients studied, consisted of large, erratic changes in plasma vasopressin concentrations with no relationship to the plasma osmolality. In type II (33% of the patients), the release of vasopressin was found, as in normal subjects, to correlate closely with the plasma osmolality; however, the osmotic threshold for vasopressin release was abnormally low. Theoretically, this group could correspond to the previously described patients with a “reset osmostat” which enabled the urine of these patients to become maximally dilute if they were sufficiently

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TABLE 8.6 Disorders Associated With Syndrome of Inappropriate Secretion of the Antidiuretic Hormone (SIADH) Carcinomas

Small-cell carcinoma of the lung Carcinoma of the duodenum Carcinoma of the pancreas Thymoma Carcinoma of the ureter Lymphoma Ewing’s sarcoma Mesothelioma Carcinoma of the bladder Prostatic carcinoma Olfactory neuroblastoma

Central nervous Encephalitis (viral or bacterial) system disorders Meningitis (viral, bacterial, tuberculous, fungal)

hyponatremic. Unfortunately, Zerbe et al. [263] did not publish any water load studies for these patients. Thus the ability of this group of SIADH patients to excrete a water load normally at a reduced osmotic threshold remains to be documented. In the type III patients, a constant, nonsuppressible basal “leak” of vasopressin with an otherwise normal osmotic release of vasopressin was observed. In type IV patients (14%), no detectable abnormalities in vasopressin secretion were observed. This suggested either a nonvasopressinmediated mechanism or an increased sensitivity to normal amounts of vasopressin. These four types of SIADH did not correlate with the underlying clinical problems (Table 8.7). For example, bronchogenic carcinoma, a disorder that might be expected to exhibit ectopic production of vasopressin, was associated with all four categories of SIADH. The clinical relevance of this categorization of SIADH therefore remains to be elucidated.

Head trauma Brain abscess

SIGNS, SYMPTOMS, AND TREATMENT OF HYPONATREMIA

Brain tumors Guillain Barre´ syndrome Acute intermittent porphyria Subarachnoid hemorrhage of subdural hematoma Cerebellar and cerebral atrophy Cavernous sinus thrombosis Neonatal hypoxia Hydrocephalus Shy Drager syndrome Rocky mountain spotted fever Delirium tremens Cerebrovascular accident (cerebral thrombosis or hemorrhage) Acute psychosis Peripheral neuropathy Multiple sclerosis Pulmonary disorders

Viral pneumonia Bacterial pneumonia Pulmonary abscess Tuberculosis Aspergillosis Positive-pressure breathing Asthma Pneumothorax Cystic fibrosis

The majority of the manifestations of hyponatremia are of a neuropsychiatric nature and include lethargy, psychosis, seizures, and coma [264]. Elderly and young children with hyponatremia are most likely to become symptomatic. The degree of the clinical impairment is not strictly related to the absolute value of the lowered serum sodium concentration, but, rather, it relates to both the rate and the extent of the fall of ECF osmolality [264]. The mortality rate from acute symptomatic hyponatremia is difficult to determine. Arieff quotes a mortality rate of approximately 50% [265]. On the other hand, none of the 10 acutely hyponatremic patients reported by Sterns had permanent neurologic sequelae [266]. As Berl [267] commented, the 50% mortality rate might be an exaggeration, but estimates suggesting that acute hyponatremia is a benign condition greatly underevaluate this potentially catastrophic electrolyte disturbance. Most patients who have seizures and coma have plasma sodium concentrations less than 120 mmol/L. The signs and symptoms are most likely related to the cellular swelling and cerebral edema that are associated with hyponatremia. Patients with SIADH whose plasma sodium concentrations are usually greater than 125 mmol/L rarely have significant symptoms related to hyponatremia itself and may not require specific treatment to raise their plasma sodium. The treatment of symptomatic hyponatremic patients has been the subject of a large-scale debate in the literature. This debate has been prompted by the description of both pontine (central pontine

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TABLE 8.7 Principal Clinical Diagnoses in Each of the Four Types of Syndrome of Inappropriate Secretion of the Antidiuretic Hormone (SIADH) Identified by Saline Infusion in 25 Patients Type

Number of patients

Diagnoses

I

2

Acute respiratory failure

1

Bronchogenic carcinoma

1

Pulmonary tuberculosis

1

Schizophrenia

1

Rheumatoid arthritis

4

Bronchogenic carcinoma

2

Cerebrovascular disease

1

Tuberculous meningitis

1

Acute respiratory disease

1

Carcinoma of pharynx

3

Central nervous system disease

2

Bronchogenic carcinoma

1

Pulmonary tuberculosis

1

Schizophrenia

1

Bronchogenic carcinoma

1

Diabetes mellitus, arteriosclerosis

II

III

IV

From Robertson G. The pathophysiology of ADH secretion. In: Tolis G, Labrie F, Martin JB, editors. Clinical neuroendocrinology: a pathophysiological approach. New York, NY: Raven Press; 1979, with permission, Wolters Kluwer.

myelinolysis) and extrapontine demyelinating lesions in patients whose hyponatremia has been treated. Blood brain barrier disruption is now recognized as probably pathogenetic in osmotic demyelination [268]. Virtually all investigators now agree that self-induced water intoxication, symptomatic hospital-acquired hyponatremia, and hyponatremia associated with intracranial pathology are true emergencies that demand prompt and definitive intervention with hypertonic saline. A 4 6 mmol/L increase in serum sodium concentration is adequate in the most seriously ill patients and this is best achieved with bolus infusions of 3% saline. Virtually all investigators now agree that overcorrection of chronic hyponatremia (which we define as more than 6 8 mmol/L in 24 hours, 12 mmol/L in 48 hours, and 18 mmol/L in 72 hours) risks iatrogenic brain damage. The following calculations and statements are important to consider [250]: • Loss or gain of approximately 3 mL of water per kilogram of body weight will change plasma sodium concentrations by approximately 1 mmol/L. • Maximally dilute urine, whether resulting from untreated diabetes insipidus, spontaneous recovery from hyponatremia, or administration of a vasopressin antagonist, will increase plasma sodium concentrations by about 2.5 mmol/L per hour.

• In the absence of urinary loss of water,1 mL of 3% saline per kilogram of body weight will increase plasma sodium concentrations by about 1 mmol/L. • In a woman with a body weight of 50 kg, the increase in plasma sodium levels caused by a maximum water diuresis is similar to the increase caused by infusion of approximately 125 mL of 3% saline per hour. Appropriate therapy should keep the patient safe from serious complications of hyponatremia while staying well clear of correction rates that risk iatrogenic injury. Accordingly, we suggest therapeutic goals of 4 6 mmol/L immediately, 10 mmol/L in 24 hours, and 18 mmol/L in 48 hours. We recommend to stay well below these limits in patients at high risk of osmotic demyelination, including patients with liver disease, cirrhosis, or malnutrition [250]. Inadvertent overcorrection owing to a water diuresis may complicate any form of therapy, including the newly available vasopressin antagonists. Frequent monitoring of the serum sodium concentration and urine output are mandatory. Administration of desmopressin to terminate an unwanted water diuresis, or to clamp plasma sodium in a patient with severe, double-digit, hyponatremia, is an effective strategy to avoid or reverse overcorrection [269].

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Acknowledgments This work was supported by the Canadian Institutes of Health Research, the Kidney Foundation of Canada, and the Fonds de la Recherche en Sante´ du Que´bec. The author thanks his coworkers, Marie-Franc¸oise Arthus, Miche`le Lonergan, Ellen Buschman, Mary Fujiwara, and Kenneth Morgan, and many colleagues who contributed to the work. Graphical expertise is from Danielle Binette.

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C H A P T E R

9 The Hypothalamus Andrea Giustina, Stefano Frara, Alfio Spina and Pietro Mortini

INTRODUCTION The hypothalamus plays a key role in regulation of endocrine (pituitary function), metabolic (food intake, energy balance, and water metabolism), and nonendocrine (body temperature, sleep/wake cycle) functions. Diseases involving the hypothalamus give rise to variable associations of endocrine, metabolic, neurologic, and other systemic signs and symptoms. Causes of hypothalamic dysfunction include genetic diseases such as Prader Willi and Bardet Biedl syndromes, neoplastic lesions (e.g., craniopharyngioma) or hematologic systemic disorders such as sarcoidosis and Langerhans’ cell histiocytosis, traumatic, and postirradiation brain disorders. Due to the pivotal role of the hypothalamus in regulation of food intake, obesity is a common finding in patients with hypothalamic disorders or in those undergoing hypothalamic pituitary surgery.

Corpus callosum

2 1 3

6 7

9

8 4

10

11

5 Infundibulum

12 Pituitary

ANATOMY

1

2

3

4

Frontal section planes

The hypothalamus is one of the major components of the diencephalon, and is situated at the base of the brain below the thalamus and above the pituitary (Fig. 9.1). The anterior margin of the optic chiasm forms the anterior boundary of the hypothalamus, while the posterior margins of the mammillary bodies delineate the posterior boundary. The lateral borders are constituted by the optic tracts, internal capsule, pes pedunculi, globus pallidus, and ansa lenticularis [2]. Between the chiasm and the mammillary bodies on the ventral surface is the tuber cinereum from which the pituitary stalk arises. The third ventricle lies at the center of the hypothalamus and is connected to the lateral ventricles through the foramen of Monro, and to the fourth ventricle by the aqueduct of Sylvius. The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00009-X

FIGURE 9.1 Schematic representation of lateral brain section demonstrating hypothalamic nuclei. Dashed lines represent the frontal (coronal) section planes. Key to numbers: 1, preoptic nucleus; 2, paraventricular nucleus; 3, anterior hypothalamic area; 4, supraoptic nucleus; 5, arcuate nucleus; 6, dorsal hypothalamic area; 7, dorsomedial nucleus; 8, ventromedial nucleus; 9, posterior hypothalamic area; 10, mammillary body; 11, optic chiasm; 12, optic nerve. Source: Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary. 3rd ed. Boston: Academic Press; 2002. p. 303 41, [1].

Although it is relatively a small structure of 4 cm3, the hypothalamus contains many groups of nerve cell bodies forming distinct nuclei (Table 9.1), with highly diverse structural, molecular, and functional organizations [1 3] (Table 9.2). Hypothalamic nuclei are connected through

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TABLE 9.1 Major Hypothalamic Nuclei Region

Zone periventricular

Medial

Lateral

Preoptic

Preoptic periventricular nucleus

Medial preoptic nucleus

Lateral preoptic nucleus

Suprachiasmatic nucleus

Anterior hypothalamic nucleus

Lateral portion of supraoptic nucleus

Paraventricular nucleus

Medial portion of supraoptic nucleus

Arcuate (infundibular) nucleus

Dorsomedial hypothalamic nucleus

Anterior periventricular nucleus Supraoptic

Tuberal

Lateral hypothalamic nucleus

Ventromedial hypothalamic nucleus Mammillary

Posterior hypothalamic nucleus

Premammillary nucleus

Lateral mammillary nucleus

Medial mammillary nucleus

Intercalatus nucleus

Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary. 3rd ed. Boston: Academic Press; 2002. p. 303 41.

TABLE 9.2 Hypothalamic Functions, the Nuclei or Regions Involved With the Specific Functions, and the Disorders Resulting From Stimulatory or Destructive Lesions in the Regions Function

Nuclei [n] or region [r] involved

Disorders

Water metabolism

Supraoptic [n]; paraventricular [n] Circumventricular organs [r]

Diabetes insipidus Essential hypernatremia SIADH

Temperature regulation

Preoptic anterior hypothalamic [r] Posterior hypothalamus [r]

Hyperthermia Hypothermia Poikilothermia

Appetite control

Ventromedial [n] (satiety center)

Hypothalamic obesity Cachexia

Lateral hypothalamic [r] (feeding center)

Anorexia nervosa Diencephalic syndrome Diencephalic glycosuria

Ventrolateral preoptic anterior hypothalamic [r] (sleep center)

Somnolence Reversal of sleep wake cycle

Posterior hypothalamic [r] including tuberomammillary [n] (arousal center)

Akinetic mutism

Suprachiasmatic [n]

Coma

Posterior medial [r] (sympathetic region)

Sympathetic activation

Preoptic anterior hypothalamus [r] (parasympathetic region)

Parasympathetic activation

Ventromedial [n] Medial and posterior hypothalamus [r]

Sham rage Fear or horror Apathy

Caudal hypothalamic [r]

Hypersexual behavior

Ventromedial [n]

Short-term memory loss

Sleep wake cycle and circadian rhythm

Visceral (autonomic) fraction

Emotional expression and behavior

Memory

Mammillary bodies Control of anterior pituitary

Arcuate [n]; preoptic [n]

Hyperfunction syndromes Hypofunction syndromes

Suprachiasmatic [n] Paraventricular [n] Neovascular zone (median eminence) Reprinted from Giustina A, Braunstein GD. Hypothalamic syndromes. In: Jameson JL, De Groot LJ, editor. Endocrinology: adult and pediatric. 7th ed. Philadelphia: Elsevier Saunders; 2016; p. 174 87. [4]. SIADH, syndrome of inappropriate secretion of antidiuretic hormone.

HYPOTHALAMIC PHYSIOLOGY

afferent and efferent nerve fibers to different parts of the brain and brain stem. The hypothalamus is divided into four regions: from anterior to posterior, the preoptic, supraoptic, tuberal, and mammillary regions; and three zones: laterally from the third ventricle, the periventricular, medial, and lateral zones [1,2] (Table 9.2, Fig. 9.1). In addition to the nuclei, numerous afferent and efferent fibers connect the hypothalamus to cerebral cortex and the brain stem. Hypothalamic actions can be subdivided into endocrine and nonendocrine. This chapter deals with hypothalamic physiology and pathophysiology as well as with clinical manifestations of diseases affecting the hypothalamus. The main hypothalamic endocrine action is the control of anterior pituitary function via a connection by the hypothalamo-pituitary stalk, and forms a distinct anatomic and functional structure [2]. The hypothalamus is also responsible for essential metabolic homeostatic mechanisms such as water and electrolyte metabolism and regulation of body weight by controlling food intake, energy expenditure, and energy storage [5,6]. To perform these latter tasks, the hypothalamus receives afferent messages from peripheral structures such as adipose tissue, the gastrointestinal tract, liver, and pancreatic β cells, and sends efferent messages to the same organs, as well as to the muscles via sympathetic and parasympathetic signals [3,7]. Other important nonendocrine hypothalamic functions include regulation of body temperature, sleep/wake cycle and circadian rhythms, and behavioral and cognitive aspects such as emotional expression and memory [4,5,8]. Table 9.2 lists the various functions of the hypothalamus, the hypothalamic nuclei or hypothalamic regions that have been identified as being responsible for these functions, and the disorders that result from lesions in or around the nuclei or region. Hypothalamic diseases may be congenital such as developmental malformations, genetic, often as part of complex syndromes (e.g., Prader Willi syndrome (PWS)), or acquired due to tumors, infections, vascular problems, trauma, nutritional and metabolic problems, degenerative processes, immunologic abnormalities, and infiltrative diseases. Extrahypothalamic manifestations generally coexist in systemic disorders. Hypothalamic lesions may also cause activation or loss of function and their clinical presentation may vary depending on the disorder, the site of hypothalamic involvement, the rate of progression of the disease process, whether the disease affects a single hypothalamic nucleus or is bilateral, and the age of onset of the disorder [1,5].

HYPOTHALAMIC PHYSIOLOGY A number of endocrine and nonendocrine functions have been attributed to the hypothalamus based upon animal studies, clinical observations of disease states and

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electrical stimulation or destruction of hypothalamic regions in humans. Because of the close association of hypothalamic nuclei to afferent and efferent tracts from other cortical and noncortical (thalamic, limbic, midbrain, and spinal) regions, it is not always possible to precisely ascribe every function to specific nuclei in the hypothalamus [1]. In fact, many nuclei appear to subserve multiple functions, and more than one pair of nuclei may be involved with the same function. For example, the ventromedial nucleus is involved in appetite control, emotional expression, and short-term memory retention [1,6,9].

Hypothalamic Endocrine and Metabolic Functions Control of Anterior Pituitary Function The hypothalamus synthesizes and secretes several hypophysiotrophic- releasing and -inhibitory hormones that regulate anterior pituitary function. Guillemin and Schally received the Nobel Prize in 1977 for the discovery of peptide hormone production of the brain, a discovery that opened a new field in endocrinology: neuroendocrinology [10]. Pituitary hormones in turn regulate target gland/organ function and five specific hypothalamo-pituitary peripheral gland/organ axes are identified. Several immunohistochemical studies have localized the sources of hormones produced in the hypothalamus. Many in vivo observations have elucidated the mode of function as well as the role of the hypothalamus in maintaining the homeostasis of the various axes, including feedback connections with peripheral and pituitary hormones [11]. Although nerve cell bodies in which the factors are synthesized are widely distributed throughout the hypothalamus, the axons converge at the median eminence (neurovascular zone) as part of the tuberoinfundibular system and terminate on or near the hypothalamohypophyseal portal vessels, within the hypothalamo-pituitary stalk, where they discharge hypophysiotrophic substances under appropriate stimulation. In fact, hypothalamic hormones are usually not detected in the systemic circulation. Measurable levels of these hormones in serum are likely due to extrahypothalamic sources of secretion, generally gastrointestinal, since most of these substances are gastroneuropeptides [12]. The highest concentrations of nerve cell bodies for gonadotrophin-releasing hormone (GnRH) are located in the medial basal hypothalamus and preoptic areas. Thyrotrophin-releasing hormone (TRH) neurons are found in the suprachiasmatic, preoptic medial, and paraventricular nuclei [13]; while corticotrophinreleasing hormone (CRH) has been localized to the paraventricular nucleus [14]. Growth-hormonereleasing hormone (GHRH)-containing neurons are found in the arcuate nucleus, as are neurons

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synthesizing somatostatin [15]. Dopaminergic neurons, which inhibit prolactin secretion through dopamine release into the hypothalamohypophyseal portal vessels, are found primarily in the arcuate nucleus, with smaller amounts found in the dorsomedial, ventromedial, periventricular, paraventricular nuclei, and median forebrain bundle [13]. In addition to peptides and amines with established physiologic pituitary regulatory functions, the hypothalamus is replete with a large number of biologically active substances, many of which are located in the same neurons that harbor hypophysiotrophic factors, that modulate their action (s) at the pituitary level [16]. Appetite Control The main hypothalamic areas involved in energy regulation are the ventromedial nucleus, the paraventricular nuclei, the arcuate nucleus, and the lateral hypothalamic area. The arcuate nucleus contains two sets of neurons: one generates orexigenic responses via agouti-related protein and neuropeptide Y, and the other generates anorexigenic responses via proopiomelanocortin (POMC) and cocaine- and amphetamine-related transcript. POMC is a precursor for α-melanocyte-stimulating hormone (α-MSH), whose main effect on weight regulation is through melanocortin 4 receptor (MC4R) [3,17]. The paraventricular nucleus expresses both MC4R and neuropeptide Y receptors, and secretes neuropeptides, including CRH and oxytocin, which have an anorexigenic effect [1,4,5]. Among the hormonal factors involved in appetite a relevant role is played by leptin, derived from adipocytes, which interacts with leptin receptors present in the two sets of neurons in the arcuate nucleus [18]. Additional hypothalamic factors that affect appetite experimentally include orexins, endocannabinoids, brain-derived neutrotrophic factor, and nesfatin-1. Other peripherally produced factors that affect appetite include cholecystokinin, peptide YY, ghrelin, obestatin, and glucagon-like peptide-1 (GLP-1) [19]. Water Metabolism Arginine vasopressin (AVP) is synthesized in nerve cell bodies of the magnocellular neurons of the supraoptic and paraventricular nuclei. The hormone is packaged in secretory granules with a specific neurophysin and transported through axoplasmic streaming down long axons that terminate in the pituitary stalk and posterior pituitary. AVP is released into the blood when serum osmolarity increases or vascular volume decreases. Blood volume status is sensed by stretch receptors present in the left atrium and large pulmonary veins, while serum osmolarity changes are detected by peripheral and hypothalamic osmoreceptors. Increased serum osmolarity is the dominant

stimulus for AVP release, mediated primarily through hypothalamic osmoreceptors located in the medial preoptic anterior hypothalamic region [1,5]. Osmoreceptors located in the lateral preoptic anterior hypothalamic region stimulate thirst in response to increased serum osmolarity. Hypovolemia and hypotension also stimulate thirst. AVP acts on the V2 receptors of the renal distal tubules and collecting ducts and increases water permeability through aquaporin-II water channels, allowing water to be reabsorbed from the urine into the hypertonic renal medullary interstitial region, from which it re-enters the bloodstream [20]. Reabsorbed water along with water ingested in response to activation of the thirst mechanism re-establishes volume and decreases osmolarity, thereby closing the feedback loop [21]. Other factors that stimulate release of AVP include hypotension, nausea, vomiting, nicotine, hypoglycemia, hypoxia, barbiturates, β-adrenergic drugs, morphine, tricyclic antidepressants, cholinergic drugs, and angiotensin II infusions. AVP release is inhibited by ethanol, atropine, α-adrenergic drugs, diphenylhydantoin, and chlorpromazine [21].

Hypothalamic Nonendocrine Functions Temperature Regulation The preoptic anterior hypothalamus harbors receptors for warmth (“warm receptors”), as well as “cold receptors” that respond to cold. Peripheral warm receptors are stimulated by a rise in ambient temperature, and the hypothalamic adrenergic warm receptors are activated by an increase in blood temperature. Efferent signals are transmitted to the lateral portion of the posterior hypothalamus via the median forebrain bundle, leading to activation of heat-dissipating responses of vasodilatation and sweating. In contrast, activation of peripheral cold receptors through decreased environmental temperature, or activation of serotonergic hypothalamic cold receptors, leads to medially placed neurons in the posterior hypothalamus activating heat production and conservation mechanisms of shivering and vasoconstriction [22]. Sleep Wake Cycle and Circadian Rhythm Control The most important area governing wakefulness is the reticular activating system of the brain stem. Lesions in this area result in coma, a state in which the individual cannot be aroused even with noxious stimuli. The anterior hypothalamus contains a “sleep center,” stimulation of which leads to inhibition of the reticular activating system and sleep, from which, in contrast to coma, the animal or individual can be aroused. Stimulation of the posterior hypothalamus

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(“wakefulness center”) leads to wakefulness and arousal. The normal sleep wake cycle is regulated in part by the suprachiasmatic nucleus, which integrates retinal stimuli during the day and pineal gland melatonin secretion at night [23]. Suprachiasmatic nuclei also control circadian rhythms of anterior pituitary hormone release, as well as other physiologic rhythms, many of which are entrained through the visual system via the retinohypothalamic tract [24]. Regulation of Visceral (Autonomic) Functions Integration of sympathetic and parasympathetic autonomic nervous system activity is an important function of the hypothalamus. Stimulation of the “sympathetic region” in the posteromedial hypothalamus results in activation of thoracolumbar autonomic responses and a “fight-or-flight” reaction with pupillary dilatation, a rise in blood pressure, tachycardia, increased cardiac output, tachypnea, piloerection, vasoconstriction of the α-adrenergic receptor visceral vascular beds, and vasodilatation of the β-adrenergic responsive blood vessels in skeletal muscle. Stimulation of the “parasympathetic region” in the preoptic anterior hypothalamus leads to increased vagal and sacral autonomic response with pupillary constriction, bradycardia, hypotension, increased blood flow in the visceral vascular bed, and decreased flow in the muscle blood vessels. Other types of autonomic function described in animal studies include stimulation of defecation with electrical stimulation of the medial tuberal region, increased motility activity of the gastrointestinal tract with stimulation of the preoptic anterior hypothalamus and posterior dorsolateral regions, and reduced bowel motility with ventromedial hypothalamic stimulation. Gastric juice volume, acidity, and pepsin content are increased with stimulation of the anteromedial hypothalamus, as well as the tegmentum of the brain stem [1]. Emotional and Cognitive Functions Through the use of electrode stimulation or production of lesions in animal hypothalamic regions, as well as clinical observations of humans, the ventromedial nucleus has been found to play an important role in integrating cortical input influencing behavior. Lesions in this area lead to rage with aggressive, often violent, behavior associated with activation of the sympathetic nervous system. This behavior is referred to as “sham rage,” distinguished from voluntary or cortical rage. The autonomic response is probably mediated through activation of the posterior hypothalamic sympathetic area. In humans, electrical stimulation of the medial or posterior hypothalamus results in sensations of fear or horror, while apathy and reduced activity are found with destructive lesions in these areas. Lesions in the

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limbic system in the region of the caudal hypothalamus have been associated with aggressive, hypersexual behavior [1]. A “pleasure center” located in the medial forebrain bundle in the lateral hypothalamus of rats has a “nourishing region” around the septal area, stimulation of which leads to lapping, licking, and chewing [1]. Memory is a complex process that requires an intact brain stem reticular formation, limbic system, and hypothalamus. Short-term or recent memory requires intact ventromedial nuclei and hippocampus [1].

PATHOPHYSIOLOGY OF HYPOTHALAMIC SYNDROMES Due to the large number of important physiological functions that depend upon the integrity of the hypothalamus, the close proximity of the nuclei and tracts, and the small overall size of the structure, diseases involving the hypothalamus give rise to a plethora of clinical syndromes, according to general pathophysiological principles [1,5,25,26]. 1. The spectrum of diseases that can affect the hypothalamus is large, and different lesions may produce identical signs and symptoms of hypothalamic damage and it is rare to find a lesion involving only one nucleus or a single tract (Table 9.3). Bauer in the 1950s reviewed 60 patients with hypothalamic involvement by a variety of diseases documented by autopsy [25,26]. Despite the diversity of pathological abnormalities, 78% had neuro-ophthalmologic abnormalities (in 13%, these were the first manifestations), 75% developed pyramidal tract or sensory nerve involvement, 65% had headaches, 62% showed extrapyramidal cerebellar signs, and 40% exhibited recurrent vomiting. Findings more specific to the hypothalamus included precocious puberty in 40% (undoubtedly reflecting a selection bias due to the type of cases in whom autopsies were performed), diabetes insipidus (DI) in 35%, hypogonadism in 32%, somnolence in 30%, dysthermia in 28%, and obesity or emaciation in 25%. On the other hand, some pathological processes result in rather specific symptoms. For instance, gliosis of the supraoptic and paraventricular nuclei has DI as its only hypothalamic manifestation. Similarly, hamartomas have precocious puberty and galastic seizures as their primary manifestations, due to their endocrine activity and/or their specific location in the tuber cinereum. Many of the pathological processes have characteristic appearances on magnetic resonance imaging (MRI) that are diagnostically helpful [27].

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TABLE 9.3 Causes of Hypothalamic Dysfunction

TABLE 9.3 (Continued)

Congenital

Nutritional/metabolic

Tumors

Acquired

Anorexia nervosa

Neuroblastoma

Drugs

Developmental malformations

Kernicterus

Pinealomas

Hayek Peake syndrome

Anencephaly

Wernicke Korsakoff syndrome

Pituitary tumors

Idiopathic SIADH

Porencephaly

Weight loss

Plasmacytoma

Kleine Levin syndrome

Sarcoma

Periodic syndrome of Wolff

Metastatic tumors

Psychosocial deprivation syndrome

Agenesis of the corpus callosum Septooptic dysplasia

Degenerative

Suprasellar arachnoid cyst

Glial scarring

Infiltrative

Colloid cyst of the third ventricle

Parkinson’s

Histiocytosis

Hamartoma

Infectious

Aqueductal stenosis

Bacterial meningitis

Trauma

Mycobacterial tuberculosis

Intraventricular hemorrhage

Spirochetal syphilis

Functional

Leukemia Sarcoidosis Other

Genetic (familial or sporadic cases)

Immunologic

Radiation

Idiopathic diabetes insipidus

Porphyria

Paraneoplastic syndrome

Toluene exposure

Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary, 3rd ed. Boston: Academic Press; 2002. p. 303 41.

Hypothalamic hypopituitarism

Viral

Familial diabetes insipidus

Cytomegalovirus

Prader Willi syndrome

Encephalitis

Bardet Biedl syndrome

Jakob Creutzfeldt

Wolfram’s syndrome

Kuru

Pallister Hall syndrome

Poliomyelitis Varicella

Tumors Primary intracranial tumors

Vascular

Angioma of the third ventricle

Aneurysm

Craniopharyngioma

Arteriovenous malformation

Ependymoma

Pituitary apoplexy

Ganglioneuroma

Subarachnoid hemorrhage

Germ cell tumors

Vasculitis

Glioblastoma multiforme Glioma

Trauma

Hamartoma

Birth injury

Hemangioma

Head injury

Lipoma

Postneurosurgical

Lymphoma Medulloblastoma

Functional

Meningioma

Diencephalic epilepsy (Continued)

2. As a general rule, patients with systemic illnesses such as sarcoidosis, histiocytosis, and infections that involve the hypothalamus usually, but not uniformly, have nonhypothalamic manifestations of the disease process, including ophthalmologic and extracranial disease in sarcoidosis and bony lesions in histiocytosis. 3. Clinical manifestations depend in part upon the rate of progression of the disease process. Patients with small, rapidly progressive lesions often develop symptoms early, while slowly progressive lesions may remain asymptomatic for long periods (bigger size). Acute insults, such as vascular accidents or trauma, tend to result in decreased consciousness, hyperthermia, and DI, which may be transient if the patient survives the initial injury. Chronic lesions tend to alter cognitive ability and endocrine function, and are not reversible. 4. Most lesions resulting in chronic hypothalamic syndromes are bilateral. In fact, most hypothalamic functions are controlled by one or more pairs of nuclei and destruction of a single nucleus is not generally sufficient to result in a clinical syndrome. Therefore, pathological processes that are multiple (i.e., metastatic tumors, granulomatous diseases), arise in or around the third ventricle (colloid cysts), cause enlargement of the third ventricle

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(pinealomas, germ cell tumors (GCTs), midbrain gliomas, aqueductal stenosis), or impinge upon or invade the floor of the hypothalamus (craniopharyngiomas, optic gliomas, pituitary adenomas) will more likely result in clinical signs and symptoms of hypothalamic disease. 5. Lesions involving hypothalamic nuclei may cause specific syndromes depending upon whether the lesion results in stimulation or destruction of the nuclei. 6. Clinical manifestations of hypothalamic disease depend upon the age of onset. As a rule, the neonatal hypothalamus is quite immature, and diseases afflicting the neonatal or infant hypothalamus present different symptoms than the same disease affecting the same region in an older child or an adult. For example, in the diencephalic syndrome of infancy due to a glioma involving the anterior hypothalamus affected infants lose weight despite eating seemingly adequate quantities of food. After the age of 2, the surviving infants gain weight. Moreover, gonadotrophin deficiency that occurs before puberty results in a lack of pubertal changes with maintenance of the sexually infantile state. Acquired hypothalamic hypogonadism that has its onset in an adult may lead to some regression of secondary sexual characteristics, but such individuals do not appear sexually infantile. Growth hormone (GH) deficiency due to hypothalamic disease in a prepubertal individual is associated with short stature, while a similar deficiency in adults causes mainly metabolic changes [1,4,5].

CLINICAL FEATURES OF HYPOTHALAMIC SYNDROMES Clinical features of hypothalamic syndromes and their possible etiologies (Table 9.3) are described while maintaining the schematic subdivision for endocrine and metabolic and nonendocrine manifestations.

Endocrine and Metabolic Anterior Pituitary Dysfunction A certain degree of hypothalamo-pituitary unit involvement, not necessarily clinically symptomatic, is almost invariably present in hypothalamic syndromes and is the main reason for referral of affected patients to endocrinologists. Hypothalamic lesions or disease may lead to an activation of hypothalamic neurons with consequent hyperproduction of a hypothalamic hormone or

TABLE 9.4 Causes of Central Precocious Puberty Congenital abnormalities Hypothalamic hamartoma

Neoplasms Optic nerve glioma

Arachnoid cyst

Hypothalamic glioma

Myelomeningocele

Neurofibroma

Aqueductal stenosis with hydrocephalus

Astrocytoma

Tuberous sclerosis

Ependymoma

Congenital optic nerve hypoplasia

Infundibuloma

Congenital adrenal hyperplasia

Medulloblastoma

McCune Albright syndrome

Meningoma

Subdural hematoma

Pinealoma

Primary hypothyroidism

Neuroblastoma

Idiopathic

Germinoma Craniopharyngioma

Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary, 3rd ed. Boston: Academic Press; 2002. p. 303 41.

to a loss of hypothalamic function with associated hypopituitarism and/or hyperprolactinemia [4]. Activating Lesions CENTRAL PRECOCIOUS PUBERTY

Pubertal development with early appearance of secondary sexual characteristics in girls younger than 8 years, or boys younger than 9 years, is generally due to premature activation of the hypothalamic pituitary gonadal axis. In most subjects, this form represents a functional abnormality and no organic cause is found (idiopathic central precocious puberty). There is a marked gender difference in the underlying etiologies accounting for central precocious puberty. While most of these girls have idiopathic early activation of the hypothalamic pituitary ovarian axis, idiopathic precocious puberty accounts for only 10% of cases in boys [28]. The spectrum of etiologies responsible for central precocious puberty is shown in Table 9.4. Precocious puberty may be associated not only with hypothalamic hamartomas [29] but also with other benign or malignant neoplasms such as craniopharyngiomas, gliomas, and astrocytomas or with infiltrative and inflammatory lesions. Some of these lesions may cause early activation of the hypothalamic pituitary gonadal axis through increased intracranial pressure or irritation of the basal hypothalamus. Hypothalamic hamartomas involving the tuber cinereum may prematurely activate normal hypothalamic GnRH secretory mechanisms or may

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directly secrete GnRH located immunohistochemically within hamartomatous neurons [29,30]. In addition to pressure effects, GCTs may result in precocious puberty through secretion of human chorionic gonadotrophin (hCG). Precocious puberty may be found in polyostotic fibrous dysplasia (McCune Albright) syndrome. In both sexes, accelerated growth velocity is observed initially with tall stature for age. This, however, is associated with increased velocity of bone maturation and premature growth cessation and final short stature, the severity of which is generally dependent on the age of onset of the disorder. Gonadotrophin and sex steroid serum levels are increased with respect to chronological age and gonadotrophin response to GnRH is typically pubertal with normal gonadotrophin pulsatility [4]. Precocious puberty in children with primary hypothyroidism and galactorrhea with elevated levels of thyroidstimulating hormone (TSH) and prolactin (Van Wyk Grumbach syndrome) is probably not due to premature activation of the hypothalamic pituitary gonadal axis, since treatment of such patients with thyroxine halts further progression of precocious puberty [31].

GHRH hypersecretion can also be managed medically with long-acting somatostatin analogs [32,33].

ACROMEGALY

HYPERPROLACTINEMIA

Acromegaly is generally (98% of patients) caused by a GH-secreting pituitary adenoma [32]. Rarely, it can be due to a eutopic or ectopic GHRH hypersecretion. Hypothalamic GHRH is secreted into the portal system, binds to specific surface receptors of the somatotroph cell, and elicits intracellular signals that modulate pituitary GH synthesis and/or secretion. GHRH-producing neurons have been well characterized in the hypothalamus by immunostaining techniques [12]. Hypothalamic tumors, including hamartomas, choristomas, gliomas, and gangliocytomas may produce excessive GHRH with subsequent somatotroph cell hyperstimulation and increased GH secretion. The structure of hypothalamic GHRH was in fact elucidated from material extracted from pancreatic GHRH-secreting tumors in two patients with acromegaly [12]. Immunoreactive GHRH (but not acromegaly) is present in several tumors, including carcinoid tumors, pancreatic cell tumors, small-cell lung cancers, adrenal adenomas, and pheochromocytomas. Measuring GHRH plasma levels provides a precise test for the diagnosis of ectopic acromegaly in the absence of pituitary adenoma. Peripheral GHRH levels are not elevated in patients with hypothalamic GHRHsecreting tumors, supporting the notion that excess eutopic hypothalamic GHRH secretion into the hypophyseal portal system does not appreciably enter the systemic circulation. An enlarged pituitary is often found on MRI of patients with GHRH-secreting tumors [12]. Surgical resection of the tumor secreting GHRH should reverse GH hypersecretion, and pituitary surgery should not be necessary in these patients. Eutopic

Prolactin (PRL) is secreted in a pulsatile manner and has a circadian fluctuation with high levels during nonrapid eye movement sleep [36]. The most important physiologic stimuli are suckling, stress, and increased levels of ovarian steroids, primarily estrogens [36]. In response, the hypothalamus elaborates a host of prolactin-releasing factors (PRFs) and prolactin-inhibiting factors (PIFs). The general view is that lactotrophs exhibit spontaneously high secretory activity. Therefore, pituitary PRL secretion is under a tonic and predominantly inhibitory control exerted by the hypothalamus. Dopamine, the most important PIF, is present in the portal blood in sufficient concentrations to inhibit PRL release. Dopamine suppresses PRL synthesis and secretion via the D2 subtype dopamine receptors. These actions constitute the physiological basis for the therapeutic effect of dopamine agonists in hyperprolactinemia [37]. Other neurotransmitters regulate PRL secretion at the hypothalamic level, and at the level of the tuberoinfundibular dopaminergic system (TIDA) in most cases. Inhibitory neurotransmitters, such as serotonin and norepinephrine, increase PRL secretion by means of a decrease in TIDA activity. Adrenergic modulation, mediated at the β-receptors by norepinephrine and epinephrine, plays an important role in stress-induced PRL secretion [4,36]. Among hypothalamic neurohormones, TRH, oxytocin, and vasoactive intestinal peptide are well-known PRFs. Neuropeptides, such as galanin [16], largely present in hypothalamic neurons, are also abundantly expressed in the anterior lobe of the pituitary,

CUSHING DISEASE

Cushing disease is the pituitary-dependent form of Cushing’s syndrome associated with bilateral adrenal cortical hyperplasia, excessive secretion of ACTH and cortisol, and the presence of a pituitary adenoma. This form should be differentiated from pituitarydependent ACTH hypersecretion secondary to excessive ectopic production of CRH, and pituitary-independent ectopic ACTH syndrome, adrenal neoplasms, and bilateral micronodular adrenal hyperplasia. Almost invariable MRI findings of pituitary (micro)adenomas as well as postsurgical characterization of adenoma clonal origin exclude a relevant role for CRH hypersecretion for most patients with Cushing disease [34]. Conversely, a very unusual cause of pituitary-dependent Cushing disease is secretion of CRH by intracranial neoplasms such as gangliocytoma [35]. Lesions with Hypothalamic Loss of Function

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determining an increase in PRL secretion. Because PRL secretion is predominantly under hypothalamic dopamine inhibitory control, it is not surprising that patients with a variety of hypothalamic disorders, such as germinomas, may exhibit hyperprolactinemia [38]. Most of these patients have PRL concentrations less than 100 ng/ml. Women present with the classic amenorrhea-galactorrhea syndrome, whereas men may ignore symptoms of erectile dysfunction, decreased libido, and gynecomastia caused by hyperprolactinemia. Menstrual abnormalities, libido, and erectile disorders are difficult to assess because of the high frequency of concomitant gonadotrophin deficiency. HYPOTHALAMIC HYPOGONADISM

This condition is commonly found in patients with organic hypothalamic lesions. Almost one-third of patients in Bauer’s series had hypogonadism, with lesions located in the floor of the third ventricle involving the tuberoinfundibular and more anterior regions of the hypothalamus [25,26]. The etiology is probably multifactorial in most instances. Mechanisms include destruction of GnRH-secreting neurons, disruption of the median eminence where the GnRH peptidergic axons converge, interference with the “pulse generator” responsible for normal pulsatile release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), damage to the hypothalamohypophyseal portal system, and/or hyperprolactinemia. LH deficiency may occur alone or together with FSH resulting in hypogonadotrophic hypogonadism, and gonadotrophin deficiency may be present as a component of multiple trophic hormone deficiencies seen in patients with idiopathic panhypopituitarism [1]. Kallmann syndrome is the most common form of congenital isolated gonadotrophin deficiency and can occur sporadically or in a familial setting as an Xlinked, autosomal-dominant, or autosomal recessive trait with incomplete penetrance and variable phenotypic expression [39]. The most frequently detected genetic mutation is deletion of KAL-1 gene whose product, anosmin-1, normally directs the migration of GnRH neurons from the olfactory placode to the hypothalamus. Genes also involved in the syndrome are NELF, CHD7, HS6ST1, FGF8/FGFR1, PROK2/ PROKR2, SEMA3E, and other genes encoding components of the FGF pathway [40,41], whilst mutations in KISS1R, TAC3, and TACR3 were identified as causes of normosmic hypothalamic hypogonadism [42]. The syndrome is characterized by a deficiency or absence of GnRH-secreting neurons in the hypothalamus, as well as agenesis or hypoplasia of the olfactory bulb, which is responsible for the hyposmia or anosmia. In boys, who are more often affected than girls, cryptorchidism and microphallus may be observed at birth (lack of

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fetal gonadotrophins which do not stimulate testosterone secretion from the fetal testes). However, clinical diagnosis does not occur until the time of expected puberty when there is a failure of gonadotrophins to increase testicular size, and development of secondary sexual characteristics. In addition to prepubertal levels of LH, FSH, and sex steroids elicited after a single bolus injection of GnRH, little or no increase in gonadotrophin levels occurs. After several weeks of priming pituitary gonadotrophs with pulsatile boluses of GnRH given at 90-minute intervals, the gonadotrophin response to a bolus injection of GnRH returns to normal, likely indicating that the initial inadequate gonadotroph response represented secondary atrophy of the gonadotroph due to the lack of endogenous GnRH secretion. Full virilization and fertility in these patients may be achieved with small doses of GnRH given by infusion pump every 90 minutes [39]. In girls, primary amenorrhea is observed together with absent mammary gland development. Since GH secretion is normal but the effect of sex steroids on growth cartilage is lacking, affected subjects may develop unusually long limbs and eunuchoid proportions in which the upper segment (crown to pubis) to lower segment (pubis to floor) ratio is less than 1, and the arm span exceeds total height by 5 cm or more. This occurs because long bone cartilaginous epiphyseal growth plates grow under the influence of GH and do not fuse, as fusion requires pubertal levels of androgens and estrogens. Other components of this syndrome include color blindness, nerve deafness, cleft palate, exostosis, and renal abnormalities [39]. Hypogonadotrophic hypogonadism has been observed with leptin and leptin receptor gene mutations, as well as GPR54 and DAX1 mutations [39]. Congenital gonadotrophin deficiency also occurs as a manifestation of panhypopituitarism (which may be on a hypothalamic basis), as well as with several complex hypothalamic disorders, including Prader Willi, Bardet Biedl, and Laurence Moon syndromes. CHARGE is an autosomal-dominant syndrome with a variable combination of coloboma of the eye, heart malformation, atresia of the choanae, retardation of growth and development, and genital and ear abnormalities. CHD7 is the predominant gene associated with the CHARGE syndrome which overlaps with Kallmann syndrome since affected patients do show anosmia and gonadotrophin deficiency [43]. Mutations of fibroblast growth factor-1 receptor gene occur in 7 10% of patients with autosomal-dominant congenital hypogonadotrophic hypogonadism and are associated with both anosmia and normosmia. Approximately 5% of patients with idiopathic hypogonadotrophic hypogonadism have a loss-of-function mutation in the GnRH-receptor gene [39].

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Acquired hypogonadism developing postpubertally results in amenorrhea, vaginal dryness, and some regression of glandular breast tissue in women. Men experience gradual loss of body and pubic hair, decreased muscular development, testicular atrophy, decreased libido, and erectile dysfunction. With longstanding hypogonadism, both sexes may develop fine wrinkling around the corners of the eyes and lips and osteopenia. Characteristic hormonal findings include low LH and FSH levels in association with low concentrations of gonadal steroids. Males have a low seminal plasma volume and oligo- or azoospermia [1]. GROWTH HORMONE DEFICIENCY

GH is produced and secreted primarily by somatotrophs in the anterior pituitary in a pulsatile manner through (long-loop) feedback involving GH and peripheral IGF-1. The hypothalamic hormones involved in GH regulation include GHRH and somatostatin, which stimulate and inhibit secretion, respectively. GH pulses are the result of a peak in hypothalamic GHRH and a trough in somatostatin. This sophisticated regulation is engendered by a crosstalk and reciprocal feedback regulation between GHRH- and somatostatin-producing neurons [10]. ACQUIRED GH DEFICIENCY Growth failure due to hypothalamic structural abnormalities is common, and may be multifactorial in origin, including deficiencies of GH, TSH, and gonadotrophins, as well as nutritional abnormalities. Growth retardation is present in about one-third of children with craniopharyngiomas [44,45], 40% of patients with the chronic disseminated form of histiocytosis [46], and 10 40% of individuals with suprasellar germinomas [38]. Due to the pulsatile nature of physiologic spontaneous GH secretion, formal testing for GH secretory response in patients with hypothalamic disease may lead to understanding of the true frequency of its abnormalities. For instance, 85 95% of patients with craniopharyngioma have an inadequate rise in serum GH following provocative stimuli [44]. In these patients, GH deficiency is presumed to be due to inadequate synthesis, release, or transmission of GHRH to the somatotrophs. Auxologic parameters are helpful in the diagnosis of acquired GH deficiency in children affected by a hypothalamic tumor or infiltrative process. In fact, relevant decreases in growth velocity or arrested growth can be observed (short stature is not a frequent finding due to the relatively acute onset of signs and symptoms) [47]. In the absence of auxologic signs, the clinical diagnosis of adult GH deficiency is more difficult and therefore its prevalence is likely underestimated, unless proactive screening with provocative testing reveals a high frequency of inadequate GH secretion in

patients with hypothalamic involvement. Adult GH deficiency contributions to the clinical picture of hypothalamic syndromes include reduced quality of life and physical fitness, osteopenia, osteoporosis, and increased cardiovascular risk. These manifestations are at least partially reversible by GH substitution [48,49], which however is contraindicated in the presence of an active neoplasm and should be carefully balanced in terms of risks and benefits in patients previously operated upon for a hypothalamic tumor, e.g., craniopharyngioma [50]. CONGENITAL GH DEFICIENCY Congenital absence of GH due to structural or functional hypothalamic abnormalities is observed in several midline developmental abnormalities including anencephaly, holoprosencephaly, transsphenoidal encephalocele, septooptic dysplasia, and some patients with simple cleft lip and palate [47]. In most affected patients, GH deficiency coexists with deficiencies of one or more of the other trophic hormones. Approximately one-third of these patients develop hormone abnormalities due to a traumatic transection of the pituitary stalk during delivery, while others have defective induction of mediobasal brain structures, which results in failure of pituitary lobes to fuse, and an absence or hypoplasia of the pituitary stalk [47,51]. GH deficiency occurs on a familial basis, either as part of familial panhypopituitarism or as an isolated defect. In panhypopituitarism, GH deficiency is the most common abnormality, followed by gonadotrophin, then ACTH, and finally TSH deficiency or deficiencies. Both autosomal recessive and X-linked recessive transmission have been described, although most cases appear to be sporadic [1]. Most of these patients exhibit a rise in anterior pituitary hormones following bolus injections of the releasing hormones, but not in response to provocative stimuli that work through the release of endogenous hypothalamic releasing hormones. Congenital GH deficiency is also due to defects in a variety of genes involved in pituicyte differentiation [52]. Monotrophic GH deficiency may be found in patients with abnormalities in the GH gene family found on chromosome 17. Most patients with monotrophic GH deficiency actually have a hypothalamic etiology, with an absence of appropriate secretion of GHRH. These patients have GHcontaining pituitary somatotrophs, but are unable to secrete adequate quantities because of inadequate stimulation from the hypothalamus. Most are capable of releasing GH following the exogenous GHRH administration [1]. Clinically, patients with congenital GH deficiency have normal birth length and weight. Males may exhibit micropenis, especially if gonadotrophin

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CLINICAL FEATURES OF HYPOTHALAMIC SYNDROMES

deficiency coexists. Growth retardation generally becomes apparent during the latter part of the first year, and both height age and bone age are delayed. Untreated patients develop proportional short stature, increased subcutaneous fat, “pinched facies” with a high forehead, and fine wrinkling of the skin around the corners of the mouth and eyes. Hypoglycemia may occur during infancy, since GH is an insulin antagonist, but later insulin deficiency may develop, resulting in abnormal glucose tolerance. Puberty may be delayed even in the absence of gonadotrophin deficiency. Exogenous GH replacement therapy stimulates linear growth, restores normal glucose tolerance, and allows puberty to progress normally [47]. HYPOTHALAMIC HYPOADRENALISM

Abnormalities of the hypothalamic pituitary adrenal axis are relatively common in patients with congenital or acquired structural hypothalamic disorders. Over half of patients with craniopharyngiomas, suprasellar germinomas and other hypothalamic and suprasellar lesions exhibit deficient cortisol levels or ACTH responses to stimuli that function via the hypothalamus [44]. Congenital monotrophic ACTH deficiency is rare [53]. Clinical manifestations of tertiary adrenal insufficiency include hypotension and hypoglycemia, especially if persistent organic GH deficiency coexists. In fact, functional GH deficiency can occur in patients with isolated ACTH deficiency due to the physiological stimulating role of cortisol on GH secretion with a complete recovery of GH reserve during glucocorticoid replacement therapy (Giustina effect) [54]. Acute adrenocortical insufficiency rarely occurs spontaneously, but may be precipitated with stresses such as surgery, infections, or trauma. In this situation nausea, vomiting, and hypotension may be found. Unlike patients with primary adrenocortical insufficiency, hyperpigmentation and electrolyte abnormalities that reflect aldosterone deficiency (hyponatremia and hyperkalemia) are not seen [1,9]. HYPOTHALAMIC HYPOTHYROIDISM

Tertiary hypothyroidism is found in one-third to one-half of patients with craniopharyngiomas, and other hypothalamic tumors [44]. Serum free-T4 levels are low, with serum TSH being inappropriately low or even slightly elevated, due to increased glycosylation of the TSH molecule, which reduces its biologic activity [55]. Differential diagnosis between secondary and tertiary (central) hypothyroidism is classically based on the evaluation of TSH secretory patterns after an intravenous bolus injection of TRH, which remain low and flat in the case of pituitary damage and undergo a delayed, prolonged and exaggerated response in those

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with hypothalamic damage. Normally, the highest concentrations of TSH are found between 15 and 30 minutes after TRH, while in patients with hypothalamic hypothyroidism, peak levels occur between 90 and 120 minutes [56]. Clinical manifestations of hypothalamic hypothyroidism are similar, but less severe than those found with primary hypothyroidism [1,9]. Disorders of Water Metabolism CENTRAL DIABETES INSIPIDUS

DI is characterized by excretion of copious volumes of dilute urine combined with persistent intake of abnormally large quantities of fluid, usually with excessive thirst. There are three general forms of the disease: central, cranial, neurogenic, or pituitary (vasopressin-deficient) DI; nephrogenic (vasopressin-resistant) DI; and primary polydipsia, in which vasopressin secretion is also suppressed due to excessive intake of fluids. Central DI may result from destruction of antidiuretic hormone (ADH)-producing magnocellular neurons in the supraoptic and paraventricular nuclei or interruption of the pituitary stalk [4]. Without sufficient AVP, renal distal tubules and collecting ducts are unable to adequately reabsorb water, leaving the urine inappropriately hypotonic relatively to the plasma osmolarity. Persistent diuresis leads to polyuria (up to 10 12 L/day) and nicturia, which in turn stimulates the thirst mechanism to bring about water-seeking behavior and polydipsia. If the osmoreceptor mechanism is intact and the patient is conscious and has access to fluids, plasma osmolarity may be maintained within the normal range. However, if the thirst center osmoreceptors are damaged, or if the patient is unable to ingest adequate quantities of water, hypernatremic dehydration may occur and result in rapid deterioration of the sensorium from lethargy to stupor to coma. Patients with lesser deficiencies of AVP may release sufficient hormone to maintain adequate water balance under basal conditions. Patients with partial DI may increase urine osmolarity to a level above plasma osmolarity during dehydration. However, in both conditions, administration of exogenous vasopressin to a dehydrated patient will result in a further increase in urine osmolarity, while dehydrated normal individuals will show little or no further increase in urine osmolarity after a standard dehydration test. DI is relatively common in patients with both acute (trauma) and chronic hypothalamic disorders [57]. Deficiency of ADH is frequently seen with hypothalamic syndromes [38,46,58]. DI may also occur in patients with other hypothalamic tumors [1,4,5]. Langerhans’ cell histiocytosis (LCH) is often associated with obesity and hypogonadism reflecting the anterior,

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302

9. THE HYPOTHALAMUS

TABLE 9.5 Causes of Diabetes Insipidus Etiology

Number of patients

Percentage

Idiopathic/familial

179

44

Primary intracranial

89

22

Metastatic

15

4

Lymphoma

3

1

Leukemia

3

1

Trauma

20

5

Histiocytosis

35

8

Neurosyphilis

9

2

Meningitis

3

1

Postencephalitic

5

1

Sarcoidosis

4

1

Other

46

11

Total

411

100

Neoplasm

Infectious

a

a

Cerebral atherosclerosis, birth injury, postvaccinal, giant cell granuloma, systemic illness, postirradiation, congenital malformation, and postoperative. Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary, 3rd ed. Boston: Academic Press; 2002. p. 303 41.

medial hypothalamic localization of lesions affecting the supraoptic and paraventricular nuclei (Table 9.2). Transient DI may occur in individuals with posterior pituitary or low pituitary stalk lesions, manipulation of the pituitary stalk during resection of a pituitary adenoma, or in patients with acute, reversible hypothalamic lesions. The spectrum of pathological lesions accounting for DI from two large series is shown in Table 9.5 [57]. The most common form of central DI is idiopathic, and can occur sporadically or in a familial setting as an autosomal-dominant trait, with nucleotide deletions or substitutions in the ADH gene on chromosome 20 [59], and may present in childhood. About one-third of “idiopathic” patients have detectable antiAVP cell antibodies [60]. Central DI is treated by desmopressin, the synthetic vasopressin analog, which reduces urine production and increases osmolarity. The medication is administered sublingually or by oral tablets, by nasal inhalation, or rarely by intramuscular injection [61]. ADIPSIC OR ESSENTIAL HYPERNATREMIA (CEREBRAL SALT RETENTION SYNDROME)

Adipsic hypernatremia occurs generally in patients with partial DI. Damage to the osmoreceptors in the anterior medial and anterior lateral preoptic

hypothalamic regions may lead to essential hypernatremia characterized by chronic, fluctuating elevations of serum sodium (and chloride), often to dangerously high levels, despite the spontaneous ingestion of amounts of fluid (1 2 L/day) capable of maintaining appropriate plasma osmolarity in otherwise normal adults [1]. Affected individuals have impaired thirst mechanism, demonstrating hypodipsia or adipsia despite marked serum sodium elevations. Nevertheless, these patients have normal extracellular fluid volume and are not dehydrated, and, therefore, maintain normal blood pressure, pulse rate, blood-urea nitrogen, serum creatinine, and creatinine clearance. Since vascular volume status also regulates AVP release, these patients can release AVP and concentrate urine with volume depletion. However, even while hypernatremic, an oral or intravenous intake of a large volume of water results in inhibition of AVP release due to increased volume, culminating in excretion of a dilute urine. Most of these patients do have partial DI, as their urine osmolarity increases with exogenous AVP administration [62]. Clinically, few symptoms reflecting hypernatremia are found with serum sodium concentrations below 160 mmol/L. Above this level, patients develop fatigue, lethargy, weakness, muscle tenderness and cramps, anorexia, depression, and irritability. Stupor and frank coma occur with sodium concentrations .180 mmol/L. Although the pituitary gland at autopsy is normal, anterior pituitary hormone deficiencies are found, reflecting a hypothalamic etiology for the hypopituitarism. Obesity and hypertriglyceridemia have also been reported [1]. Children have been described with essential hypernatremia but without a structural hypothalamic defect (Hayek Peake syndrome). They demonstrate recurrent hypernatremia, hypodipsia, obesity, hyperprolactinemia, hypothyroidism, hyperlipidemia, lethargy, increased perspiration and, in some cases, central hypoventilation. These findings suggest an involvement of the osmoreceptors and the ventral medial nucleus [63]. SYNDROME OF INAPPROPRIATE SECRETION OF ANTIDIURETIC HORMONE

This condition is characterized by serum hypoosmolarity (Posm ,275 mOsm/kg) and hyponatremia, an inappropriately concentrated urine (Uosm .100 mOsm/kg) for the low serum osmolarity, continued urinary excretion of sodium despite low serum sodium levels, and hypouricemia in a patient with normal renal, adrenal and thyroid function, and who does not exhibit findings of extracellular fluid volume expansion (i.e., no evidence of congestive heart failure, cirrhosis, or other edematous states) [64].

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CLINICAL FEATURES OF HYPOTHALAMIC SYNDROMES

The most frequent cause is ectopic ADH hypersecretion by tumors of neuroendocrine origin (lung microcytoma, Schwartz Bartter syndrome). Drug-induced (e.g., antidepressant) increased ADH production or activation of peripheral volume receptors may also occur. In some individuals the syndrome is due to a decreased set-point for serum osmolarity-mediated release of AVP. Indeed, SIADH may occur, usually transiently, following head trauma, intracranial bleeds, meningitis, encephalitis, transsphenoidal pituitary surgery, and other neurosurgical procedures. It has also been noted in some patients with hydrocephalus, craniopharyngiomas, germinomas, pinealomas, central pontine myelinolysis, and acute intermittent porphyria [64]. An idiopathic, cyclic form of the syndrome has been described in young women with menstrual irregularities and enlarged lateral ventricles [1]. Besides symptoms from the underlying disease, these patients demonstrate clinical findings of water intoxication. Dilution hyponatremia is the most relevant biochemical feature. In fact, clinical features depend on the rate of decrease of serum sodium, as well as absolute serum sodium concentration. At serum sodium levels ,120 mmol/L patients become symptomatic with anorexia, headache, weakness, lethargy, nausea, vomiting, and mental confusion; at very low levels, with seizure and coma [65]. Hyponatremia and other manifestations of SIADH also co-occur with the syndrome of cerebral saltwasting, primarily reported in postoperative neurosurgical patients treated for subarachnoid bleeding, intracranial aneurysms, or following head injury [66]. In contrast to SIADH these patients are hypovolemic due to renal salt (and water) loss which could be due to disrupted renal sympathetic nervous system input. Abnormal brain natriuretic peptide secretion is also implicated in this syndrome [65,66]. Disorders of Caloric Balance HYPOTHALAMIC OBESITY

Obesity is a common finding in patients with hypothalamic diseases, occurring in approximately 25% of individuals with anatomically proven lesions, although rarely is the initial manifestation of hypothalamic dysfunction [25,26]. Most patients with hypothalamic obesity have large lesions or extensive involvement of multiple areas of the hypothalamus. Bilateral destruction of the ventromedial nucleus results in obesity in humans, as it does in experimental studies in animals [6]. In patients with documented structural involvement, about 90% harbor a neoplasm, most often craniopharyngioma (approximately 60%) [67]. Approximately 6% are the result of inflammatory or

303

granulomatous processes including sarcoidosis, tuberculosis, arachnoiditis, and encephalitis, 5% are posttraumatic, and 2% are due to leukemic infiltration [1]. Defective hypothalamic leptin signal transduction may play a role in hypothalamic obesity. In fact, when circulating leptin derived from adipocyte energy storage transduces hypothalamic signals, anorexigenesis is achieved, which increases sympathetic tone, with resultant increases in energy expenditure and decreased vagal tone, appetite, and energy storage. Conversely, defective leptin signaling, or “leptin resistance,” leads to orexigenesis, with decreased sympathetic tone and increased vagal tone, and with resultant increased appetite and energy storage [3]. Leptin signaling may be disrupted by exceptionally rare leptin deficiency with obesity documented as early as 6 months of age and extremely low or not measurable serum leptin levels, which is reversible after treatment with recombinant leptin [68]. Genetic defects of leptin signal transduction, including leptin receptor mutation, POMC splicing mutation, prohormone convertase-1 (PC-1) deficiency, melanocortin-3 receptor (MC3R) mutation, MC4R mutation, and single-minded 1 (SIM-1) mutation, are generally characterized by very high leptin levels and have no known treatment [69]. Obesity is clearly the result of hyperphagia. In many instances, the abnormality appears to reflect resetting of the satiety set-point, especially observed in patients with obesity that develops following head trauma. Most affected individuals gain weight for approximately 6 months following trauma, followed by a period of stabilization as energy expenditure equals the caloric content of ingested food, with a subsequent gradual decrease in food intake and a loss of weight. Similarly, patients with tumor destruction of ventromedial nuclei may develop hyperphagia and rapid weight gain, followed by a plateau and then further weight gain as the neoplasm grows [1]. These patients have more extensive hyperinsulinemia than is observed in patients with essential obesity, which may be due to enhanced insulin secretion through vagal stimulation, as increased vagal firing occurs in animals with ventromedial nucleus lesions [67]. In addition to hyperphagia and hyperinsulinemia, lowered basal metabolic rate and deficient GH, TSH, and gonadotrophins may contribute to the weight gain [67]. DIENCEPHALIC SYNDROME OF INFANCY

Severe emaciation can be observed in infants with hypothalamic tumors despite an apparently good food intake, associated with an alert appearance and euphoric affect, and nystagmoid eye movements. The majority of these infants have been found to have lowgrade hypothalamic or optic nerve gliomas that

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304

9. THE HYPOTHALAMUS

destroy the ventromedial nuclei [70]. The infants appear normal at birth and demonstrate normal feeding and developmental parameters during the first 3 12 months. Towards the end of the first year of life, the infants begin to lose weight and subcutaneous fat, show signs of hyperactivity and a cheerful, happy affect, but continue to grow normally. They exhibit an alert appearance secondary to eyelid retraction (Collier sign) [70]. Other findings include nystagmus, pallor, vomiting, tremor, and optic atrophy. Endocrine evaluation is generally normal, although plasma cortisol diurnal variation is absent, insulin-like growth factor-1 (IGF-1) levels are low, and basal serum GH levels are elevated, with a paradoxical rise following a glucose load [71]. Elevated GH levels are not specific to these patients, since other illnesses associated with weight loss, such as anorexia nervosa, are also accompanied by such elevations [10]. Usually, the infants succumb to the tumor and emaciation by the age of 2 years. Paradoxically, infants who survive beyond age 2, either due to spontaneous stabilization or therapy, often become obese, develop irritability and rage, somnolence, and precocious puberty [70]. ANOREXIA NERVOSA

The typical patient with anorexia nervosa is a young, white female from a middle to upper socioeconomic background who inappropriately views herself as obese, and, therefore, severely restricts food intake, exercises excessively, and may engage in bulimic binges with self-induced vomiting, and diuretic and cathartic abuse. The typical age of onset is less than 25 years. Patients lose more than 15% of their weight, and are usually 25% below their ideal body weight. Amenorrhea is a characteristic finding and often precedes weight loss, and may persist even after weight is regained [72]. A number of functional hypothalamic endocrine abnormalities have been noted in patients with anorexia nervosa. A prepubertal pattern of gonadotrophin release is characteristically present with low basal LH and FSH concentrations, prepubertal, apulsatile 24-hour LH-secretory pattern, diminished LH response to GnRH, and loss of positive feedback effect of estrogen on LH secretion. With weight gain, patients enter a “second puberty,” developing nocturnal secretory pulses of LH, and eventually an adult pattern of pulsatile LH release throughout the day and night. The GnRH response also returns to normal [73]. Basal serum GH levels are normal or elevated, but rise paradoxically following a glucose load. The GH response to GHRH is normal, but the response to L-dopa and apomorphine is impaired. A rise in serum GH levels may also occur following an injection of TRH, a response also found in patients with acromegaly, depression, chronic renal failure, and cirrhosis [72].

IGF-1 is low, suggesting nutritionally acquired GH resistance. GH/IGF-1 abnormalities revert to normal with weight gain [72,73]. Patients with anorexia nervosa also exhibit abnormalities in the hypothalamic pituitary adrenal axis. CRH-mediated hypercortisolemia may contribute to maintenance of anorexia by suppression of appetite drive acting in food-motivating brain regions (amygdala, hippocampus, and insula) [74]. As it occurs in patients with Cushing’s syndrome, depression, and obesity, serum cortisol suppression is incomplete after low-dose dexamethasone. The abnormalities return to normal following weight gain. These individuals share clinical features with hypothyroid patients, including dry skin with a yellowish hue due to hypercarotemia, scalp hair loss, bradycardia, and hypothermia. Thyroid function tests are similar to those seen in patients with the “sick euthyroid syndrome.” Thus, serum thyroxine levels are in the low-normal or frankly low range, the triiodothyronine concentrations are low, the reverse triiodothyronine levels are elevated, and TSH concentrations are in the low-normal range. Serum TSH responses to TRH are either normal or show a delayed rise with a peak at 45 or 60 minutes, characteristic of hypothalamic hypothyroidism. As with the other hormonal abnormalities, thyroid dysfunction resolves with weight gain [73]. DIENCEPHALIC GLYCOSURIA

Transient hyperglycemia and glycosuria may occur following hypothalamic injury that results in lesion of the tuberoinfundibular region. This has been most commonly noted after basal skull fractures, intracranial hemorrhage, or surgery near the floor of the third ventricle. Although each of these entities is associated with elevated concentrations of ACTH, glucocorticoids, GH, and catecholamines, which have insulin-contraregulatory effects, the occurrence of hyperglycemia with injuries to the tuberoinfundibular region and not with injuries to other areas of the hypothalamus which may also be associated with elevations of the same hormones, suggests that other factors are involved [1].

Nonendocrine Deranged Control of Body Temperature HYPERTHERMIA

Acute injury to the anterior hypothalamic and preoptic areas from intracranial bleeds, neurosurgical procedures in the region of the floor of the third ventricle, or trauma may result in temperature elevations up to 41 C, tachycardia, and unconsciousness that generally lasts for less than 2 weeks if the patient survives. With such lesions, heat production continues, while the

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CLINICAL FEATURES OF HYPOTHALAMIC SYNDROMES

heat-dissipating mechanisms fail to respond appropriately. The pulse rate in patients with hyperthermia due to hypothalamic lesions is not increased to the same extent for a given elevation in temperature as is the pulse rate in patients with fever from infections or inflammatory processes [25,26]. Acute hyperthermia to 41 C or greater is a characteristic of the neuroleptic malignant syndrome (NMS). This syndrome develops in susceptible individuals over 24 72 hours following exposure to phenothiazines, butyrophenones, thioxanthenes, or ioxapine [75]. The potential for development of this syndrome roughly parallels the antidopaminergic D2 receptor potency of the neuroleptic drug. The pathophysiology of this syndrome is mainly explained by a central hypodopaminergic state with disruption of dopamine neurotransmission in the nigrostriatal system by neuroleptic-induced dopamine receptor blockade in susceptible individuals. This activates heat generation through muscle contraction, impairment of heat dissipation through hypothalamic injury, and inhibition of diaphoresis through a peripheral anticholinergic effect of the neuroleptics [76]. Autopsy studies have shown injury in the preoptic medial and tuberal nuclei [77]. Other clinical characteristics of the syndrome include hypertonicity of skeletal muscles with “lead-pipe” type of rigidity, fluctuating consciousness varying from agitation to stupor to coma, and instability of the autonomic nervous system reflected by pallor, diaphoresis, wide swings in blood pressure, tachycardia and arrhythmias, tremors, and akinesis. Leukocytosis, elevations of serum creatine phosphokinase, and nonspecific encephalopathic findings on electroencephalography (EEG) also occur. The syndrome lasts 5 10 days and is associated with a 20 30% mortality rate [76]. The serotonin syndrome is closely related to NMS and is becoming a more frequent diagnosis in intensive care units [78]. It presents with altered mental status (somnolence, confusion, agitation, seizures, and coma), autonomic instability (fever, diaphoresis, tachycardia, and mydriasis), and abnormal neuromuscular activity (myoclonus, rigidity, hyperreflexia) [78]. Hyper- (or hypo-) thermia is caused by either direct or indirect effects on hypothalamic thermogenesis [76]. Drugs or combinations of drugs that elevate the serotonin concentration in the central nervous system (CNS) can cause the syndrome. These include selective serotonin reuptake inhibitors, tricyclic antidepressants, monoamine oxidase inhibitors, cocaine, and amphetamines, opioids (fentanyl), and antiemetics [79]. Malignant hyperthermia also occurs in susceptible individuals during and following anesthesia. Patients with chronic hypothalamic hyperthermia do not exhibit the generalized malaise that accompanies elevated temperatures due to infections, and also

305

have paradoxical peripheral vasoconstriction with cold, clammy extremities. Hyperthermia may respond to sedatives or anticonvulsant medications, but not to salicylates [25,26]. HYPOTHERMIA

Chronic hypothermia with temperatures below 32 C is infrequently associated with large lesions involving the anterior and/or posterior hypothalamus. Destruction of the thermoregulatory mechanisms by such lesions results in an inability to generate heat through shivering and vasoconstriction. Hypothermia has been noted with third-ventricular and large hypothalamic neoplasms, poliomyelitis, neurosyphilis, sarcoidosis, gliosis of the anterior hypothalamus, multiple sclerosis [80], and other neurologic diseases involving the hypothalamus such as Parkinson disease and Wernicke’s encephalopathy [1]. Episodic or paroxysmal hypothermia, also known as diencephalic autonomic epilepsy, is a distinct syndrome in which body temperature abruptly decreases, often to 32 C or lower, over a period of minutes to days, associated with a variety of signs and symptoms of autonomic nervous system dysfunction [81]. The frequency of attacks varies from daily to decades apart. Patients experience flushing, diaphoresis, fatigue, hypotension, bradycardia, salivation, lacrimation, pupillary dilatation, Cheyne Stokes respirations, nausea, vomiting, asterixis, ataxia, and obtundation. Thus, during the episodes, heat generation is impaired and heat loss is increased due to vasodilatation and sweating. EEG slowing occurs during the episodes. Recovery occurs spontaneously over hours to days, and is associated with heat generation through shivering and vasoconstriction. Attacks often begin in the teenage years and the frequency and duration of attacks may increase as the patient ages, likely due to a resetting of the thermostat during the episodes [82]. Approximately half of the patients with episodic hypothermia have agenesis of the corpus callosum, a combination given the eponym “Shapiro’s syndrome.” Such patients may also have hypogonadism, precocious puberty, DI, reset osmostat, and GH deficiency [81,82]. POIKILOTHERMIA

This rare condition [25,26] results from loss of both heat conservation and heat-loss homeostatic mechanisms. Wide fluctuations of temperature are seen in patients generally affected by large lesions involving the posterior hypothalamus and rostral mesencephalon, as well as in patients with both anterior and posterior hypothalamic destruction, who do not experience thermal discomfort, or attempt to alter their environment to maintain their core body temperature [83].

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Poikilothermia may also be found in Wernicke’s encephalopathy and multiple sclerosis [1].

Sleep Wake Cycle and Circadian Abnormalities Suprachiasmatic nuclei are responsible for maintenance of many circadian rhythms, and lesions involving this region alter the sleep wake cycle. Somnolence may be the presenting sign of patients with hypothalamic tumors (craniopharyngioma or suprasellar germinoma) involving the posterior hypothalamus but more frequently it appears during the course of illness often in association with hypothalamic obesity. Narcolepsy, sudden episodes of sleep that last minutes to hours, may in some instances be due to hypothalamic involvement in patients with third ventricular tumors, multiple sclerosis, encephalitis, and after head injuries. Patients with lesions of the anterior and preoptic hypothalamic nuclei may exhibit hyperactivity and insomnia or, more commonly, alterations in the sleep wake cycle, with daytime sleepiness and nighttime hyperactivity [84]. Fatal familial insomnia is characterized by loss of sleep and delta sleep, oneiric stupor with autonomic/motor hyperactivity (agrypnia excitata) and pyramidal signs, myoclonus dysartria/dysphagia, and ataxia. Thalamic hypometabolism and severe neuronal loss in thalamic nuclei are observed. Genetic analysis has revealed a mutation in the PRNP (encoding the prion protein) gene classifying it as a prion disease [85].

Spontaneous resolution typically occurs in late adolescence or early adulthood. The syndrome also occurs in adolescent girls linked to the menstrual cycle [86].

Diencephalic Epilepsy Seizure activity arising from the hypothalamus is defined as diencephalic epilepsy, although the term was originally used to describe a patient with a third ventricular cholesteatoma and periodic hypothermia and associated autonomic discharge. Periodic hypothermia with absence of the corpus callosum (Shapiro’s syndrome) and periodic hyperthermia (Wolff’s syndrome) are also forms of diencephalic epilepsy [82]. Gelastic or laughing seizures are seen primarily in children with hamartomas of the tuber cinereum (50%) and other lesions near the floor of the third ventricle and extending to the mammillary region [87]. During a typical seizure the child stops activity, makes laughing, giggling, or bubbling noises, and develops a grimacing appearance from unilateral or bilateral clonic movements of the ocular, palpebral, and/or buccal muscles [87]. There also may be dacrystic seizures, characterized by a crying quality with grimacing, with no loss of consciousness, although gelastic seizures may be followed by a grand mal or petit mal seizure. The diagnosis is established by stereotyped recurrences, absence of precipitating factors, concomitance of other ictal manifestations, epileptiform abnormalities on EEG, and no other obvious cause for pathologic laughter [87].

Behavioral and Emotional Abnormalities Lesions involving ventromedial hypothalamic nuclei are associated with rage reactions with emotional lability, agitation, and aggressive and destructive behavior that occur spontaneously. During the episodes, there is usually activation of the autonomic nervous system with tachycardia, a rise in blood pressure, diaphoresis, and pupillary dilatation. Similar sham rage reactions are found with lesions of the medial temporal lobes or orbitofrontal cortex [1]. Hypersexual behavior may accompany lesions in the limbic system, medial temporal lobe, and the caudal hypothalamus. The Kline Levin syndrome appears to have a genetic predisposition with an environmental trigger, which may result in reduced hypothalamic dopaminergic tone [86]. The syndrome usually involves adolescent boys who exhibit recurrent episodes of somnolence, with periodic arousal associated with irritability, incoherent speech, hallucinations, forgetfulness, compulsive gorging of food (megaphagia), masturbation, and other sexual activity. Symptoms generally develop over a 2 4-day period with a vague sensation of malaise and headache. Episodes occur at 3 6-month intervals, and usually last 5 7 days or for several weeks.

SPECIFIC HYPOTHALAMIC DISORDERS Impairment of hypothalamic functions without any evident structural abnormality is typical of a series of congenital syndromes, such as PWS and most of the ciliopathies, characterized by defects that involve appetite control, hypogonadotrophic hypogonadism, mental retardation with personality anomalies and, less frequently, sleep wake cycle and temperature regulation problems. Other specific syndromes are caused by organic lesions such as hypothalamic tumors, trauma, and irradiation or occur during the course of systemic infiltrative or neoplastic diseases [1,4,5].

Prader Willi Syndrome PWS is a multisystemic complex genetic disorder caused by alterations of a series of paternal genes which are normally active and located in the chromosome 15q11.2 13 region. In particular, errors in genomic imprinting during gametogenesis are related to deletion of this region in 65 75% of patients, maternal uniparental disomy in 20 30% of subjects and, rarely,

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SPECIFIC HYPOTHALAMIC DISORDERS

307

TABLE 9.6 Clinical Features of Patients With the Prader Willi Syndrome Criteria

Abnormality

Percentage affected

Major

Neonatal hypotonia

93

Feeding problems in infancy

87

Excessive weight gain

82

Typical facial features

88

Hypogonadism

70

Developmental delay

98

Hyperphagia

91

Decreased fetal activity

71

Behavior problems

78

Sleep disturbances

76

Short stature

76

Hypopigmentation

73

Small hands and/or feet

89

Eye abnormalities

66

Thick saliva

89

Articulation defects

80

Skin-picking

83

Cryptorchidism

85

Delayed bone age

66

Minor

Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary, 3rd ed. Boston: Academic Press; 2002. p. 303 41.

imprinting defects (1 3%), chromosomal translocations, or rearrangements (,1%) [88]. Loss of multiple imprinted genes contributes to the phenotype of this syndrome, although SNORD116, a small nucleolar organizing RNA gene, may also be associated with hyperphagia and obesity [89]. Diagnosis can be confirmed and differentiated from Angelman syndrome with parent-specific DNA methylation analysis. Although a few familial cases have been reported, most patients develop this syndrome sporadically and the birth incidence has been estimated between 1:10,000 and 1:30,000. Autopsy studies have failed to demonstrate histopathologic hypothalamic abnormalities in the hypothalamus [90]. Major clinical features derived from several series of studied patients are depicted in Table 9.6. Clinical manifestations begin before birth with reduced fetal movements, increased incidence of breech presentation and prematurity, together with low birth weight. Once born, children show severe hypotonia with poor suckling and feeding difficulties, so that they often need gavage feeding [91] (Fig. 9.2).

FIGURE 9.2

Representative patients with Prader Willi syndrome across the life span. (A) An 8-month-old female; (B) a 19-yearold male with severe obesity and prevalent abdominal fat distribution; (C) a 34-year-old male with moderate overweight. Source: Reprinted with permission from Cassidy SB, Schwartz S, Miller JL, Driscoll DJ. Prader-Willi syndrome. Genet Med 2012;14:10 26, [90].

Affected infants exhibit multiple somatic anomalies, including a narrow bitemporal diameter of the cranium, strabismus, orbital hypertelorism, almondshaped eyes, upslanting palpebral fissure, low-set ears, micrognathism, ogival palate, clinodactyly, scoliosis, and small hands and feet with straight borders of the ulnar side and of the inner legs. Furthermore, sleep disturbances, high pain threshold, and impairment in temperature control have been reported in several cases [90,91]. Neuroradiologic abnormalities occur in most individuals, although clinical counterparts of these findings are unclear [92]. During late infancy and early childhood, mental and developmental retardation (average IQ of 65) becomes evident, with motor and language delay, while personality problems (i.e., stubbornness, manipulative and compulsive behavior) are commonly noted [93]. Subjects with PWS usually progress from birth through seven different nutritional phases [94]. When they are around 2 years old, weight starts to increase without a significant change in appetite or caloric intake, but at 4 5 years of age they develop hyperphagia with indiscriminate food-seeking behavior (eating garbage, frozen or inedible food, stealing food or money in order to buy food), resulting in severe obesity. Delayed meal termination, lack of satiety, early hunger after meal, extremely increased food ingestion (approximately three times more than controls food ingestion with relevant weight gain over a short period of time) are invariably observed in PWS patients [94]. These behaviors contribute to central obesity together with decreased caloric expenditure due to hypersomnolence, persistent poor muscle tone, low lean body mass, and reduced physical activity [95]. Even though it is likely that obesity is of hypothalamic origin, recent studies have observed that both hyperphagic children and adults affected by PWS have exaggerated levels of the orexigenic hormone ghrelin.

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Interestingly, this occurs before development of morbid obesity [96]. Early start of a low-calorie diet together with an appropriate exercise regimen is the mainstay of obesity therapy in PWS subjects, but it is seldom satisfactory if not associated with psychological and behavioral counseling of the patients and their families [97]. Although recent studies have shown that glucose intolerance and insulin resistance are similar to that found in weight-matched subjects with simple obesity, type 2 diabetes mellitus is quite frequent, with a very low mean age of onset (around 20 years) [98]. Some observational studies reported that around 25% of adults with PWS suffer from diabetes, whilst 38% are affected by hypertension [4]. Subcutaneous administration of GLP-1 agonists may be useful to achieve glycemic control due to their insulinotrophic effects and increased satiety [99]. However, chronic treatment with these drugs might be limited by their delaying effect on gastric emptying. Few reports on bariatric surgical intervention and their potential risk are available in this group of patients and results are contradictory [100,101]. There is currently little justification for exposing PWS patients to the potential risks of surgical interventions [102] although death in these patients is often related to obesity and its comorbidities (cardiorespiratory failure complicated by obstructive and central apnea, severe skin infections, pneumonia). In fact, death may also be caused by choking due to abnormal ingestion of food when unsupervised, with gastric necrosis and rupture [103,104]. Hypogonadism is a prominent feature, especially in males. At birth, these patients may present with unilateral or bilateral cryptorchidism and scrotal hypoplasia due to inadequate fetal testicular androgen production, with micropenis and immature testes [90]. Affected females usually show poor development of labia and clitoris [105]. During adolescence, they achieve variable degrees of sexual maturation, but precocious puberty occurs infrequently [106]. During adulthood, infertility is almost always observed: sex steroid hormones are invariably low in both males and females, with low basal serum LH and FSH levels and with a marked blunting of the gonadotrophin response to GnRH tests, reflecting a longstanding GnRH deficiency [105]. Chronic GnRH treatment amplifies the LH and FSH response to a bolus injection of GnRH and treatment with clomiphene citrate, a competitive inhibitor of estradiol, increases basal levels of both gonadotrophins and sex hormones and restores gonadotrophin responses to normal range [105]. Most reports confirm the hypothalamic origin of hypogonadism; however, evidence has emerged supporting a significant contribution from primary gonadal failure [107]. Generally, ACTH and cortisol dynamics as well as prolactin levels are normal [98]. Moreover, the

prevalence of hypothyroidism is variable and levotiroxine therapy should not be routinely prescribed unless confirmed by thyroid function testing [1]. Short stature, related to GH deficiency, is a common feature of patients affected by PWS [108]. GH responses to secretagogues are invariably blunted, with reduced IGF-1 levels [98]. Guidelines recommend that GH therapy, together with dietary, environmental and lifestyle interventions, should be considered for pediatric and adult subjects with PWS, as it may also exert beneficial cognitive effects and help reduce fat mass and increase lean mass without altering hyperphagia [109,110].

Ciliopathies Ciliopathies are rare genetic diseases due to defects in the immotile sensory cilia function [111] (Fig. 9.3). Bardet Biedl syndrome is the most frequent of these, with an expected prevalence of one case in 125,000 160,000 subjects. It is a rare highly pleiotropic autosomal recessive disorder, associated with a wide array of phenotypes [112]. At least 15 genes are involved, accounting for 80% of cases and ciliary defects are the predominant cause of this syndrome [113]. Although the clinical characteristics seem to be connected with the hypothalamus, specific histopathologic alterations in the region have not been demonstrated. The pathogenesis of the presumed hypothalamic dysfunction may be related to a transient internal hydrocephalus during the eighth and ninth embryonic weeks, with an enlarged third ventricle causing a diencephalic lesion [1]. The cardinal features of this syndrome are tapetoretinal degeneration, obesity, mental retardation, postaxial polydactyly, and hypogonadotrophic hypogonadism [114] (Table 9.7). More than 65% of the patients present an extra digit in one or more hands and/or feet (hexadactyly), but other digital abnormalities (e.g., syndactyly or brachydactyly) are found in 10 15% of affected subjects [111,114]. In early stages, the retinopathy may not be pigmentary, is associated with night blindness, decreased visual acuity, and an abnormal electroretinogram response. Around the age of 20, 75% of subjects affected by Bardet Biedl syndrome are blind or near blind [114]. Obesity and glycemic impairment start precociously, together with altered hypothalamic action of leptin [115]. Moreover, patients affected by Bardet Biedl syndrome might also present with nerve deafness, brachycephaly, and rarely DI and hyperlipidemia [111]. Hypertension and congenital heart disease are commonly encountered, as well as hepatic fibrosis. Hypothalamic hypogonadism occurs more frequently in men than in women [111,114]. Moreover, variable

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FIGURE 9.3 Analysis by transmission electron microscopy of ependymal cell cilia in the ventral portion of the third ventricle, adjacent to the arcuate nucleus of the hypothalamus. This analysis in wild-type (A) and Bardet Biedl syndrome M290R mutant (B) mice demonstrates enlargement of the distal region of cilia in Bardet Biedl syndrome mutant animals. Source: Reprinted with permission from Guo DF, Rahmouni K. Molecular basis of the obesity associated with Bardet-Biedl syndrome. Trend Endocrinol Metab 2011;22:286 93, [112].

TABLE 9.7 Clinical Features of the Bardet Biedl Syndrome Percentage affected Men (n 5 188)

Women (n 5 133)

Pigmentary retinopathy and other ocular manifestations

93.6

92.5

Obesity

89.4

94.7

Developmental delay

86.7

85.0

Polydactyly

76.1

73.7

Hypogonadism

76.1

51.9

Abnormality

Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary, 3rd ed. Boston: Academic Press; 2002. p. 303 41.

degrees of speech deficit and mental retardation are invariably present [116], while a variety of behavioral abnormalities have been described, including emotional lability and short attention span. Kidney disease is a cardinal feature of disease and a major cause of mortality; in fact, patients may present with glomerulosclerosis, mesangial proliferation, and/or renal cysts [117]. Several other ciliopathies that resemble Bardet Biedl syndrome have been recognized: • Laurence Moon syndrome includes retinal pigmentary degeneration, mental retardation, hypogonadism, progressive spastic paraparesis, distal muscle weakness but no polydactyly [1]; • Biemond syndrome consists of hypogonadotrophic hypogonadism, mental retardation, polydactyly or brachydactyly, obesity, and iris coloboma [118]; • Alstro¨m Hallgren syndrome is caused by an autosomal recessive defect of the ALMS1 gene, which is ubiquitously expressed and encodes for a

309

protein involved in ciliary function, intracellular transport, and cell cycle control [119]. Peculiar features include atypical retinal pigmentary dystrophy, nerve deafness, obesity, insulin resistance with hyperinsulinemia, glycemic abnormalities, acanthosis nigricans, hypothyroidism, and short stature due to GH deficiency [120]. In contrast to the Bardet Biedl syndrome, hypogonadism is suggested to be caused by primary gonadal failure, rather than hypothalamic dysfunction [111].

Optic Nerve Hypoplasia Optic nerve hypoplasia is a cause of congenital blindness in children. A population study in Minnesota reported an incidence of 1 in 2287 live births [121]. The etiology is unknown and is probably multifactorial, although rare cases have been described in patients with mutations in early developmental genes (HESX1, SOX2, SOX3, OTX2). Affected individuals are usually the first-born of a young woman whose pregnancy may have been complicated by toxemia [122]. Birth weight is normal, but the infants demonstrate poor feeding, vomiting, prolonged neonatal hyperbilirubinemia, and hypoglycemia. In half of the patients developmental and neurologic deficits were observed, and in one-fourth an endocrine abnormality was diagnosed [121]. The combination of optic nerve hypoplasia, midline neuroradiological abnormalities including agenesis of the corpus callosum and absence of the septum pellucidum and pituitary hypoplasia and consequent panhypopituitarism has long been known as septooptic dysplasia [122]. Other dysmorphic features such as cleft palate, syndactyly, low-set ears, misshapen pinnae, hypertelorism, mongoloid slants of the palpebral fissures, and agenesis of the olfactory nerves may be present. The optic nerve atrophy is manifest by a small optic disk, one-third to onehalf the normal size, and variable degrees of visual impairment [123]. Hypopituitarism in optic nerve hypoplasia may correlate with a hypoplasic anterior pituitary but it has also been shown to relate to hypothalamic dysfunction with undescended posterior pituitary and absent pituitary stalk on MRI [124]. Moreover, other associations with optic nerve hypoplasia such as developmental and cognitive delay and relational and communication difficulties or autism may occur independent of septum pellucidum development [125]. The most common endocrine abnormality is short stature associated with isolated GH deficiency. Other hormone deficiencies may occur over time [122], and ACTH deficiency, DI, and hypothyroidism may be observed during followup. The relatively low frequency of hypogonadism

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reported in these patients undoubtedly reflects the fact that the mean age of diagnosis of the disorder is around 5 years [126]. However, some children have been noted to have micropenis, which may be a reflection of prenatal fetal hypogonadotropism [126]. Examination of the hypothalamus at autopsy has demonstrated the absence of the supraoptic and paraventricular nuclei, hypoplasia of the posterior pituitary, ependymal scars around the third ventricle, and a normal anterior pituitary, supporting the hypothalamic origin of hypopituitarism [127]. FIGURE 9.4 Hypothalamic hamartoma: axial (A) and coronal (B)

Environmental Deprivation Syndrome (Psychosocial Short Stature) Children presenting with this unusual, reversible syndrome were reported over 40 years ago, with a constellation of signs and symptoms that suggest hypothalamic dysfunction. These include short stature, polydipsia, polyphagia, emotional or mental retardation, and bizarre behavior [128]. Most children have onset of the syndrome before the age of 2, and over half have a social history of divorced or separated parents, or disturbed parent child relationship. Males are more frequently affected than females. Short stature is generally the presenting complaint and is associated with a retarded bone age and inadequate GH response to insulin hypoglycemia [129]. Body weight is low for the chronological age and in most instances for the height age. Polydipsia and polyphagia are uniformly present. Bizarre behavior is a common concomitant of the syndrome. These patients often steal food and hide it around their homes, and some have been discovered to have eaten food retrieved from garbage cans. Emotional retardation is manifest by shyness and a tendency to play alone, temper tantrums, delayed toilet training, and retarded speech [128 130]. The syndrome has been reported in association with psychosis [131]. When these children are removed from their home environment, polydipsia, polyphagia, and foodstealing rapidly cease. They gain weight, and some even become obese. In addition, they exhibit rapid linear growth, showing a “catch-up” growth phenomenon, and return of the GH abnormalities to normal. If they are once again placed in their home environment, growth rapidly ceases [130].

Hypothalamic Hamartoma Hamartomas are benign heterotrophic hyperplastic malformations composed of a fibrous glial matrix with mature ganglion cells and occasional myelinated nerve fibers [132]. They range in size between a few

T2-weighted MRI study.

millimeters and 3.5 cm, with most ,1.5 cm, and are usually located in the posterior hypothalamus between the tuber cinereum and the mammillary bodies (Fig. 9.4) [132]. They likely represent a midline dysraphic syndrome with displacement of cells from the mammillary region as the infundibulum moves behind the notochord [132]. More than half of affected individuals are males, and most patients present before the age of 4 years. Close to 90% of patients develop precocious puberty with pubertal or adult concentrations of sex steroid hormones and gonadotrophins. Over half have neurodevelopmental delay with low intelligence quotient and seizures. Often the seizures are gelastic or laughing seizures with brief lapses without loss of consciousness [29,133]. Emotional lability and sham rage may also be present. Gross neurological abnormalities are usually absent, although large hamartomas may be associated with ataxia and nystagmus due to pontine compression. A tendency to obesity in late childhood and adolescence is reported [30]. Three hypotheses have been proposed to explain precocious puberty in these patients. First, the hamartoma may mechanically compress the median eminence to secrete GnRH. Support for this explanation is the finding that some hamartomas associated with precocious puberty are devoid of nerve cell bodies, and that precocious puberty also occurs in some patients with suprasellar arachnoid cysts (SACs) and craniopharyngiomas located in the same region, suggesting that median eminence compression is of etiologic significance. Alternatively, these lesions may interrupt interneuronal pathways that tonically inhibit the GnRH-secreting neurons in the median eminence, allowing disinhibition of these neurons with subsequent GnRH secretion [1,132]. The third hypothesis is that the hamartoma acts as an “accessory hypothalamus” directly secreting GnRH, which in turn stimulates gonadotrophs to secrete gonadotrophins. Indeed, hamartomas may contain GnRH,

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and cerebrospinal fluid (CSF) GnRH concentrations in patients with hypothalamic hamartoma-associated precocious puberty are elevated [1]. That GnRH is involved in the pathogenesis of the precocious puberty is evidenced by the pubertal or adult gonadotrophin response to a bolus injection of GnRH [134]. Moreover, the response to therapeutic administration of long-acting analogs of GnRH, which downregulate the GnRH receptors on gonadotrophs, results in a lowering of basal levels of sex steroid hormones and gonadotrophins, inhibition of the gonadotrophin response to a bolus injection of GnRH, cessation of menses, and deceleration of advancing bone age and growth rate [134]. Indeed, treatment of precocious puberty with GnRH analogs may be a therapy for this disorder [29]. Surgical approaches for management of the hamartoma depend upon site, type (sessile or pedunculated), and its extension [135]. About 76 90% of patients undergoing microsurgical resection of pedunculated lesions show resolution or significant lowering of seizure attacks with improvement of the cognitive status [135]. Microsurgical disconnection of the hamartoma leads to seizure control in 47 70% of patients [136]. Indications for surgery are increased intracranial pressure or neurologic deterioration from progressive growth of the hamartoma. Alternative approaches can be gamma-knife surgery or stereotactic laser ablation [136]. The Pallister Hall syndrome is a complex autosomal-dominant disorder associated with a frameshift mutation of the GLI-Kruppel family member 3 gene located on chromosome 7p13 [137]. It includes hypothalamic hamartoma, which often occupies the space between the optic chiasm and interpeduncular fossa, and pituitary dysplasia with hypopituitarism [138]. Hypopituitarism determines micropenis and cryptorchidism in affected males as well as associated hypoadrenalism. Craniofacial malformations (large fontaneles, a shortened midface, short nose with long philtrum, posteriorly rotated ears, cleft uvula and palate, microglossia, cleft larynx, hypoplasia of the epiglottis) and limb abnormalities (postaxial polydactyly, syndactyly, nail dysplasia, clinodactyly of the fifth fingers, shortened fourth metacarpals and/or metatarsals)

are commonly found. Other anomalies noted with this syndrome are congenital heart disease, hypoplastic renal dysplasia, abnormal lung segmentation, and imperforate anus [139].

Germ Cell Tumors GCTs are defined as extragonadal when there is no tumor evidence in the testicles or ovaries [140]. These are generally located at the midline in the pineal and suprasellar regions and the sacrum [140] (Fig. 9.5). In children these locations represent the most frequent extragonadal form [140]. GCTs are classified as germinomas (seminomas; 65% of intracranial germ cell neoplasms) and nongerminomatous germ cell tumors (NGGCTs) [140]. This group includes teratomas, embryonal carcinomas, endodermal sinus tumors, and choriocarcinomas [38]. Tumors are also classified according to the secretion of α-fetoprotein (AFP), β-hCG, placental alkaline phosphatase in the CSF and blood [140]. GCTs can also be classified as secreting and nonsecreting tumors, and secreting tumors are usually related to poor prognosis. Pure germinomas are generally associated with no β-hCG and AFP secretion. Germinomas containing syncytiotrophoblasts show high CSF level of β-hCG [140]. Differentiated GCTs, i.e., “poor prognosis” tumors (choriocarcinoma, yolk sac tumor, embryonal carcinoma, and mixed NGGCTs), are characterized by neoplastic involvement of the hypothalamus, third ventricle, or spinal cord. Pure germinomas and mature teratomas are considered “good-prognosis” tumors [38,140]. The incidence of these neoplasms in Western countries is between 0.5 and 3% of pediatric CNS tumors, while the incidence in Japan and Asian countries is 11% [140]. Asian and Pacific-originating patients show a higher incidence outside their respective countries, suggesting a genetic predisposition [140]. Germinomas and NGGCTs show a difference depending upon age of onset, sex ratio, site of primary neoplasm, and prognosis. The peak age at diagnosis is 10 12 years and NGCCTs tend to occur earlier [140,141]. The male/female sex ratio is 1.88:1 FIGURE 9.5 Suprasellar germinoma: coronal (A), axial (B), and sagittal (C) postcontrast administration MRI study.

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for patients with germinoma, and 3.25:1 for NGGCTs [38,140]. Suprasellar and pineal regions are affected in about 95% of cases, but lesions have also been reported in cerebral and cerebellar hemispheres, the thalamus, and basal ganglia [38]. Five to ten percent of patients present with synchronous pineal and suprasellar lesions. Tumor seeding is reported in about 10% of cases by direct hypothalamic invasion or spreading to the ventricles or subarachnoid pathway [38,140]. Most suprasellar lesions are germinomas, in contrast with pineal nongerminomatous tumors [38,140]. Symptoms are related to the tumor extension and location. Compared to pineal NCGGT lesions, suprasellar germinomas show more endocrinological symptoms than hydrocephalus [142]. The classic triad of DI, visual field abnormalities, and clinical and/or biochemical evidence of anterior pituitary hormone deficiency explain the involvement of the optic chiasm, median eminence, and region of the third ventricle [143]. Hypothalamic dysfunctions include abnormalities in appetite control with both emaciation and obesity being found, and the adipsia/ hypernatremia syndrome characterized by severe proximal muscle weakness: polyuria, adipsia or hypodipsia, hypernatremia, and hypertriglyceridemia [144]. Other neurological symptoms include hydrocephalus, pyramidal tract signs, ataxia, and, rarely, seizures, dementia, and psychosis [38]. In contrast, patients with NGGCTs, usually involving the pineal gland, exhibit more neurologic abnormalities, due to obstruction of the third ventricle and aqueduct of Sylvius and less endocrine disturbances. Thus, less than 20% have DI or evidence of hypothalamic anterior pituitary failure [142]. Precocious puberty may rarely occur with intracranial germ tumors, as a pressure effect of the neoplasm on the median eminence, or, alternatively, as a result of the production of β-hCG by the neoplasm [142], which directly stimulates the Leydig cells of the testes to produce androgens, resulting in sexual precocity. Testes of affected individuals show Leydig cell hyperplasia with no spermatogenesis since FSH levels are suppressed [142]. Intracranial germ cell neoplasms spread by direct invasion of the hypothalamus or through seeding into

the ventricles or subarachnoid pathways. Poor prognostic features include neoplastic involvement of the hypothalamus, third ventricle, or spinal cord, and histologic tumor type, with germinomas having the best prognosis, choriocarcinomas the worst, and the other varieties falling in between [140]. Histologic tumor examination is essential to determine treatment strategy. These tumors should be differentiated from other benign or malignant tumors involving the hypothalamic area, such as gliomas, LCH, and craniopharyngiomas [38,140,144]. Evaluation of CSF or serum secretion could help in the differential diagnosis. CSF cytology is important to determine the possible CSF spread of the tumor, especially in cases with a negative whole-spine MRI study [140,141]. Germinomas are highly curable tumors because of their radiosensitivity [140]. In fact, surgery is usually followed by radiotherapy and/or chemotherapy [140,145]. Gross total tumor resection can be definitively curative only in cases of mature teratomas with normal tumor markers [140 142].

Optic Chiasm and Hypothalamic Gliomas Gliomas of the optic pathways tend to be low-grade pilocytic astrocytomas, occurring primarily in children, with close to 40% being found in those under 2 years of age and 80% under 10 [146,147]. Approximately one-fourth of optic pathway gliomas are intraorbital, while three-fourths arise in the optic chiasm, optic tract, or hypothalamus. Tumors of the chiasm and hypothalamus are grouped together because they tend to infiltrate and involve both structures, and it is often not possible to differentiate between tumors originating in one structure or the other (Fig. 9.6). von Recklinghausen’s neurofibromatosis type 1 (NF1) is a major predisposing factor for development of these tumors and is diagnosed in 20 50% of patients with gliomas of the optic pathways, while the prevalence of these tumors in the NF1 population is about 1.5 15% [148]. Intraorbital optic nerve gliomas rarely involve the hypothalamus or pituitary. The natural history of optic FIGURE 9.6

Hypothalamic glioma: axial (A), coronal (B), and sagittal (C) postcontrast administration MRI study.

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glioma pathways is not clear [148]. Usually these tumors show an indolent clinical history, while in the symptomatic group the major clinical manifestations are unilateral visual loss, proptosis of the ipsilateral eye, papilledema, strabismus, and nystagmus [149]. In contrast, optic chiasm/hypothalamic gliomas are associated with endocrine disturbances, visual field abnormalities, DI, hydrocephalus, and the diencephalic syndrome of infancy, in addition to decreased visual acuity, optic atrophy, and papilledema [146,147]. Some cases of spontaneous tumor regression have also been reported [148]. Treatment is required for tumors causing symptoms or for those with potential adverse growth impact [150]. Patients older than 1 year, those with chiasmatic or hypothalamic extension, nonpilocytic optic-chiasm astrocytoma, no response to previous chemotherapy, presence of hydrocephalus at diagnosis, and absence of unidentified bright object lesions in NF1 patients have been associated with adverse progression-free survival, while optic pathways glioma in NF1 patients and tumors limited to the optic nerve are associated with longer survival [148]. NF1 is a well-known good prognostic factor, because many tumors in this group are located in the anterior visual pathway [151]. From a prognostic standpoint, patients with intraorbital gliomas have a better prognosis as compared to patients with chiasmatic and hypothalamic gliomas. There is also evidence that optic pathway gliomas in patients with neurofibromatosis behave in a more benign fashion than gliomas in patients with no neurofibromatosis [149]. Behavior of tumors in adults is mainly related to tumor histology, which usually shows malignant features [151]. Since these tumors are generally indolent and behave more like hamartomatous lesions than neoplasms and some spontaneously regress, they do not require therapy unless they continue to grow and cause neurologic dysfunction [152]. Chemotherapy is considered the first-line treatment, because of the high response rate in terms of delaying needs of radiation therapy and for lower rates of complications [148,152]. Approximately half of the tumors treated with firstline chemotherapy show progression and require further treatments, such as radiation and second-line chemotherapy [148,152]. Surgical treatment is considered for those tumors causing hydrocephalus [151,152], and tailored according to tumor extension and clinical setting, balancing surgery-related morbidity with the extent of tumor debulking [151,152]. Spontaneous resolution or tumor stability after surgical debulking has been reported [151,152]. Despite delayed radiation therapy, especially in younger children, the complete surgical resection does

313

not improve significantly the survival and the endocrine complication rates [151,152]. Radiation therapy has been advocated, citing improvement or stabilization of visual abnormalities and inhibition of tumor growth, or a delayed time to recurrence [146,147,151,152]. Radiotherapy positively affects survival rates [151], currently being considered for children older than 5 7 years with progressive tumors, because of the potential cognitive and endocrine damage to younger children [148,151].

Craniopharyngioma Craniopharyngiomas are rare (reported incidence 1.7 cases per 1,000,000 person-years) epithelial neoplasms originating along the path of the craniopharyngeal duct, that often show local growth and infiltration of surrounding tissue (Fig. 9.7) [44,45,153 155]. They account for 2 5% of brain tumors and approximately 5 10% of brain neoplasms in children [44,154,155]. The male/female sex ratio is 1.2 1.4:1 [154,156]. Almost half of the patients present before the age of 20, with a median age of 22 years. Most tumors are cystic or partially cystic, while 15% are solid [44,45]. Craniopharyngiomas show a bimodal age distribution with a peak incidence in the pediatric population from 5 to 14 years and from 54 to 74 years in adults [156]. Craniopharyngiomas are considered as WHO grade I tumors [154], and are differentiated into two histological subtypes, adamantinomatous and papillary types, with few descriptions of transitional or mixed variants [157]. The adamantinomatous variant is frequent in young patients and is characterized by both solid and cystic components with some calcifications. The cyst contains fluid generated by desquamation of epithelial cells that contain phospholipids and keratin [154]. Aberrant Wnt/β-catenin pathway signaling has been shown to be involved in its pathogenesis [158]. The papillary type is most frequent in adults, and is solid or mixed with rare calcifications [154]. This tumor shows a less invasive behavior as compared to the adamantinomatous type [44,154]. Results of surgical resection, radiosensitivity, and survival do not differ in the variant groups [44,154]. Clinical presentation and prognosis depend upon patient age as well as location of the neoplasm and its size [44]. The most frequent symptoms related to craniopharyngioma are headache (often occurring intermittently in the morning), nausea/vomiting, visual disturbances, papilledema, hydrocephalus, growth failure (in children), hypogonadism (in adults), and hypothalamic syndrome [44,154,155]. Hydrocephalus, resulting from

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FIGURE 9.7 Representative craniopharyngioma patients with postoperative panhypopituitarism. Normal weight patient (A) with small tumor removed transsphenoidally (B). Development of eating disorder and severe obesity in a patient (D) with a large tumor extending to the suprasellar region and infiltrating the hypothalamus (C). Source: Reprinted from Bereket A, et al. Hypothalamic obesity in children. Obes Rev 2012;13:780 98.

obstruction of CSF circulation, occurs in 6.8 69.4% of patients, is related to tumor size and location, and is more frequent in young children (41 54% of cases) [157,159]. Because of the proximity of the visual pathways, visual disturbances, as decreased visual acuity and visual field abnormalities, are also common (about half of cases), especially in the adult group [160]. Growth arrest due to GH deficiency is the most common endocrine disturbance, reported in 35 95% of children in the published series [44,161]. The presence of DI at the diagnosis ranges from 6% to 38% [44,154]. Features of hypopituitarism, weight gain, hypogonadism, and mental and cognitive changes or hypothalamic dysfunctions have also been reported both in children and in adults [44,154,155,157], although endocrine abnormalities are the presenting complaint in less than 15% of these patients [161]. Abnormalities of the sleep wake cycle and excessive somnolence occur more frequently in children than in adults. In contrast, visual abnormalities, especially progressive diminution of vision and an asymmetric bitemporal hemianopsia, are the most common presenting symptoms in adults [44,159]. Other prominent symptoms are headache, deterioration of cognitive abilities, personality change, vomiting, weight gain, and hypogonadism [44,159]. Lateral extension of the neoplasm into the cavernous sinus may damage cranial nerves III, IV,

and VI, which result in diplopia and abnormalities of the extraocular muscles, and a portion of cranial nerve V, which leads to facial pain. Temporal lobe involvement is associated with temporal lobe seizures, while posterior extension to the midbrain may give rise to cerebellar ataxia and pyramidal tract findings [44]. The clinical and biochemical features of craniopharyngiomas derived from several series of patients are summarized in Table 9.8 [44,159]. Children with craniopharyngiomas differ from adults in several other respects. They are more likely to have an enlarged sella turcica with calcifications, larger tumors, and better prognosis. Short-term survival rates are elevated ( . 85% at 3 years) [162] but long-term standardized overall mortality rate is increased (from 2.88 to 9.28 in cohort studies). Patients with craniopharyngioma (particularly women) have an increased cardiovascular mortality in comparison to the general population, in relationship with an adverse lipid profile driven by hyperinsulinemia [163]. On MRI, solid components appear iso- to hypointense on T1-weighted sequences and hypo- to hyperintense on T2-weighted acquisitions, whereas the cystic component is hypointense in T1-weighted and hyperintense in T2-weighted sequences [44,154]. Cyst walls enhance after contrast administration. MRI is also useful for assessing hypothalamic relationships and

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TABLE 9.8 Clinical and Biochemical Features of Patients With Craniopharyngioma Number abnormal/ number studied

Percentage

269/296

91

218/304

72

182/506

36

Growth hormone deficiency

189/268

70

Short stature/delayed bone age

418/1294

32

Adrenocorticotropic hormone deficiencya

185/480

38

Thyroid-stimulating hormone deficiency

169/522

32

Multiple hormone deficiency

246/296

83

Hyperprolactinemia

224/567

40

Galactorrhea

12/296

4

Precocious puberty

21/363

6

Decreased visual acuity/visual field defect

945/1390

68

Headache

836/1200

70

Obesity

228/685

33

Vomiting

312/922

34

Mental deterioration

221/866

26

Diabetes insipidus

316/1439

22

Papilledema

197/826

24

Hydrocephalus

16/102

15.7

Somnolence

69/383

18

Ataxia

48/280

17

Pyramidal tract signs

4/67

6

Cranial nerve palsy

29/379

8

Feature Anterior pituitary dysfunction a

Gonadotrophin deficiency Clinical hypogonadism

a

a

a

Results from biochemical testing. Reprinted from Braunstein GD. The hypothalamus. In: Melmed S, editor. The pituitary, 3rd ed. Boston: Academic Press; 2002. p. 303 41.

tumor invasion [154]. The degree of hypothalamic involvement on preoperative MRI correlates significantly with the rate of postoperative hypothalamic syndrome [153]. The main treatment strategy advocated for craniopharyngiomas is gross total tumor resection, which leads to the best results in terms of overall and progression-free survival rates [44,153 155,157,160]. However, potential morbidity and mortality should be considered as well as the possibility to maintain quality of life after surgery [153 155,157,160,164,165]. The results and extent of surgical resection depend

315

upon tumor location (sellar vs hypothalamic involvement), size and calcifications, its relations with the surrounding neural and vascular structures, preoperative neurological status, and tumor aggressive and infiltrative behaviors [153,154]. Radical surgical resection may also carry the risk of significant morbidity in terms of visual, hypothalamic, and endocrine complications [154]. Currently, the mean gross total tumor resection rate is around 60 65%. The main features that hamper complete tumor resection are hydrocephalus, infiltration or adherence to the adjacent nervous or vascular structure, and calcifications [44,154]. The mean reported visual improvement rate is 45% and the visual worsening rate is 13%. Visual deterioration is more common in patients with partial resection compared to the complete resection group (Table 9.9) [44,154,165 169]. Postoperative irreversible hypopituitarism is common and is not related to the treatment modality (range 21 100%) [44,154]. The reported rate of postoperative DI ranges from 14% to 94% (Table 9.9) [44,154,165 167,170 172]. Hypothalamic involvement of the tumor may result postoperatively in obesity and hyperphagia, cognitive impairment, water balance disorders, sleep disturbance, and loss of temperature control (Fig. 9.7) [154]. Postoperative obesity ranges from 26% to 61% and is related to the preoperative degree of hypothalamic wall involvement (Table 9.9) [153 155,161,164,168,170 175]. In fact, quality of life is very often reduced due to complications of aggressive surgery such as hypothalamic obesity [176], neurobehavioral (depression, irritability, impulsivity, aggressiveness, and emotional outbursts), social (withdrawal, internalizing behavior, school dysfunction) and emotional impairments which are highly prevalent in survivors of childhood craniopharyngioma [177]. There is an ongoing debate if a subtotal resection (hypothalamus-sparing) may attenuate the fall in quality of life and increased morbidity in long-term survivors [176]. A drawback of subtotal resection (even with negative postsurgical imaging) is a tendency for the mass to recur or progress [176]. The combination of hypothalamus-sparing surgery and radiotherapy may limit recurrence and reduce clinically relevant postsurgical complications [178]. For stereotactic radiotherapy, doses higher than 54 Gy are associated with improved progression-free survival rates [154]. Stereotactic gamma-knife radiosurgery series report an average control rate of 90% for solid tumors, 88% for cystic, and 60% for mixed tumors (Table 9.10) [179 181]. Patients presenting with hypopituitarism generally do not improve after surgery and exhibit permanent DI and GH deficiency [4]. Replacement

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TABLE 9.9 Craniopharyngioma Surgery: Literature Review of Published Series

Author

Follow-up Patients (years)

Visual improvement (%)

Visual worsening (%)

Postoperative hypopituitarism (%)

Postoperative diabetes Postoperative insipidus (%) obesity (%)

Major morbidity (%)

Surgical morbidity (%)

Yasargil (1990)

144

N/A

3636

13

79

90

N/A

N/A

16.7

Hoffman (1992)

50

4.9

36

41

N/A

93

53

6

2

De Vile (1996)

75

6.4

N/A

N/A

99

80

15

13

0

Duff (2000)

121

10

N/A

N/A

21

21

35.7

18.4

1.7

Kim (2001)

36

4.3

N/A

25

100

94

6

42

0

Van Effenterre (2002) 122

7

70

11

76

57

36

8

2.5

Merchant (2002)

30

6.1

N/A

17

97

50

N/A

10

0

Maira (2004)

57

6

N/A

0

32

14

N/A

0

0

Stripp (2004)

76

7.6

21

15

N/A

80

49

N/A

1

Gonc (2004)

66

5.1

N/A

N/A

100

52

N/A

10.6

2

Lena (2005)

47

9.5

16

16

89

86

48

N/A

2.4

Minamida (2005)

37

11.1

N/A

2.7

97

N/A

N/A

5.4

0

Shirane (2005)

42

5

N/A

N/A

81

52

N/A

6.7

0

Thompson (2005)

48

5.6

61

N/A

96

84

20

15

0

Tomita (2005)

54

N/A

43

13

93

87

28

9

0

Zuccaro (2005)

153

N/A

45

8.5

85

50

35

10

3

Lee (2008)

66

7.2

N/A

N/A

N/A

67

18

6

0

Shi (2008)

309

2.1

42

5.5

N/A

53

N/A

6

3.9

Zhang (2008)

202

N/A

42

5

N/A

81

22

5.4

1

Hofmann (2012)

73

2.1

65

5.7

N/A

52

N/A

13.8

0

Mortini (2012)

134

7.1

64

12

90

76

23

7

2.2

Cohen (2013)

33

4

N/A

N/A

58

55

58

N/A

0

Yu (2014)

24

3.5

29.1

0

33.3

62.5

8.3

8.3

0

Gerganov (2014)

16

N/A

37.5

6.25

37.5

75

N/A

12.5

N/A

treatment with recombinant GH has been reported to be safe and effective [182]. However, GH treatment is contraindicated in cases of growing lesions and treatment can be initiated only in case of volumetrically stable postsurgical residual tumor mass [183]. Obesity occurs in up to 75% of survivors and, characteristically, it occurs despite caloric restriction, and lifestyle modification can hardly be useful in its prevention and treatment (Fig. 9.7) [184]. Obesity in craniopharyngioma patients may be linked to neuroendocrine abnormalities such as hyperleptinemia and leptin resistance [3,184] with consequently reduced central sympathetic tone and decreased energy expenditure [185]. Derangements in α-MSH, ghrelin, and orexin secretion and action at the hypothalamic level have also been reported in obese craniopharyngioma patients with daytime sleepiness and disturbances of circadian rhythms [3,4].

Suprasellar Meningiomas Meningiomas arising from the tuberculum sellae, diaphragma sellae, and planum sphenoidale may encroach upon, but not invade, the hypothalamus (Fig. 9.8). These benign neoplasms have a male/female sex ratio of 1:2 3, and become symptomatic in adults. The peak incidence is between the ages of 40 55 years [186]. Most patients (more than 80%) present with a progressive unilateral or bilateral visual loss, and over 90% have objective evidence of decreased visual acuity [187,188]. Other neuro-ophthalmologic signs and symptoms such as headache and abnormal visual fields may coexist [186]. Deterioration of cognitive function, confusion, and memory loss may also be observed [186]. Preoperative endocrine metabolic abnormalities such as hypogonadism, hypothyroidism, and DI are relatively frequent [186,189].

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SPECIFIC HYPOTHALAMIC DISORDERS

TABLE 9.10

Craniopharyngioma Radiosurgery: Literature Review of Published Series Previous treatment (%)

Marginal dose (Gy)

Control rate (unchanged/ decreased volume) (%)

Author

Patients

Follow-up (months)

Prasad (1995)

9

N/A

67

12.9

63

Mokry (1999)

23

27

100

10.8

74

Chung (2000)

31

33

81

12.2

87

Yu (2000)

46

16

91

8 18

89

Chiou (2001)

10

63

100

16.4

58

Ulfarsson (2002)

21

144

73

5

36

Amendola (2003)

14

39

86

14 29

86

Barua (2003)

7

52

100

14.2

100

Albright (2005)

5

27

0

N/A

80

Yomo (2009)

18

26

100

11.5

94

Niranjan (2010)

46

62.2

93

13

68

Xu (2011)

37

50

74

14.5

68

Jeon (2011)

13

58

100

11

62

Mortini (2012)

32

52

97

13.8

90

Kobayashi (2012)

98

66

100

11.5

80

Lee (2014)

137

52

69

12

69

Notes

Combined 32P intracavitary radiation

Combined 90Y intracystic instillation

FIGURE 9.8 Tuberculum sellae meningioma: preoperative coronal (A), sagittal (B), and postoperative coronal (C) and sagittal (D) postcontrast administration MRI study, showing complete resection.

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318

9. THE HYPOTHALAMUS

Meningiomas generally contain estrogen receptors that may cause an increase in size during pregnancy or even during the menstrual cycle. The latter feature may account for some fluctuations in signs and symptoms described in patients with these tumors [189]. Surgical resection is the treatment of choice for these lesions [186,188]. After surgery, hypopituitarism and transient DI can occur [187]. Postoperative recovery of visual impairment is reported in 23 91.7% of patients [187]. In case of subtotal resection or grade II or III WHO tumors, adjuvant fractionated radiation therapy or gamma-knife radiosurgery is indicated, depending on tumor volume and the distance of the target from the optic pathways, with an overall tumor control rate of 90% [187,188].

Suprasellar Arachnoid Cyst SACs (Fig. 9.9) are a consequence of an anomalous development of the subarachnoid cistern due to an erroneous splitting of the arachnoid membrane or to an anomalous development of the Lillequist membrane [190]. These congenital fluid-filled closed cysts may cause symptoms through mass effect [190]. The majority (70%) of patients with this congenital anomaly present before 5 years of age [191]. Some cases of prenatal diagnosis have been described [190]. Symptoms and signs of SAC are related to their location and developmental pattern. Recently, three different types of SAC with distinct clinical setting, natural history, and treatment strategy were described [190]: type 1 SACs are purely suprasellar cysts that usually cause hydrocephalus by blocking the CSF outflow from the Monro foramens [191]; type 2 SACs are typically characterized by a dilated interpeduncular cistern. Usually these cysts are asymptomatic and remain stable without requiring treatment; type 3 SACs usually develop asymmetrically on contiguous subarachnoid spaces, causing macrocrania and hydrocephalus [190]. Signs and symptoms related to compression of the brain stem, thalamus and the optic tracts have been reported. GH and ACTH deficiency, and precocious puberty are primary endocrine problems encountered in children affected by this rare abnormality, especially in type 2 SAC lesions, as a consequence of a slit-valve mechanism of the cyst causing a mass effect on the pituitary [190]. The treatment of choice is surgical decompression. Ventriculocystostomy appears to be an effective technique with low morbidity [190]. Endoscopic fenestration of the cysts, to date, is considered a safe and effective option [191], and persistent hydrocephalus may require a CSF diversion through a shunt procedure [190].

FIGURE 9.9 Suprasellar arachnoid cyst: axial (A) and coronal (B) T2-weighted MRI study and coronal (C) and sagittal (D) postcontrast administration MRI study.

Colloid Cyst of the Third Ventricle Colloid cysts are usually located in the roof of the third ventricle or rarely in the area of the septum pellucidum, growing between the forniceal columns (Fig. 9.10) [192]. They represent about 0.5 2% of all intracranial tumors and occur between the ages of 30 and 60 years, with a male/female sex ratio of 2 3:1 [192,193]. They are composed of a collagen wall lined with a single layer of cuboidal epithelium [192]. Three major clinical presentations are encountered in these patients [192]. One-third of patients present with features of increased intracranial pressure, complaining of nonspecific headache and vomiting, and exhibiting papilledema [194]. Approximately 10 20% of patients develop fluctuating or progressive dementia with gait disturbance and urinary incontinence, a combination that closely resembles normal-pressure hydrocephalus. Another 20% present with a history of intermittent headache, vomiting, and visual disturbances, followed by a transient loss of consciousness. Headache appears suddenly and is localized in the frontal area, usually after head movements such as lying down. Pain intensity increases rapidly, and the

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SPECIFIC HYPOTHALAMIC DISORDERS

319

Lymphoma Non-Hodgkin’s lymphoma may involve the CNS either as a primary tumor or as secondary lesion of systemic lymphoma (5 29% of patients) generally associated with widespread disease [196]. Primary CNS lymphoma (PCNSL) is a less commonly encountered clinical entity and is limited to the cranial spinal axis without systemic disease. Pathological examination of brain tissue taken postmortem from patients with PCNSL showed pituitary gland involvement in about 25% of cases [197]. Sellar and parasellar lymphomas, similar to nonpituitary sellar and parasellar masses, and similar to pituitary adenomas, may present with symptoms of anterior pituitary hormone dysfunction. These symptoms include gonadal failure, secondary hypothyroidism, and, less often, clinical adrenal cortical insufficiency. Hyperprolactinemia, which may be asymptomatic or accompanied by hypogonadism, is often observed at presentation of sellar lesions. In patients with severe headache and coexisting signs of hypopituitarism with DI and/or cranial nerve involvement, diagnosis of a pituitary lymphoma should be considered [196,198].

Inflammatory lesions FIGURE 9.10 Colloid cyst of the third ventricle: axial CT scan study (A); axial (B), coronal (C), and sagittal (D) MRI study.

patient develops nausea and vomiting until loss of consciousness occurs [192 194]. This “classical” presentation is due to the cyst acting as a ball valve obstructing the foramen of Monro or the aqueduct of Sylvius. A similar mechanism is probably responsible for drop attacks that can occur abruptly. In approximately 10 20% of patients, sudden death may occur, presumably from acute obstruction to the CSF flow [194]. The goal of surgical treatment is to completely remove the cyst, to restore CSF flow, and to prevent recurrence [192,194]. Both microsurgical and endoscopic techniques are effective in treating these cysts [192,194].

Hematologic malignancies Leukemia DI due to leukemic infiltration or thrombosis of the small vessels of the hypothalamus or posterior pituitary is a rare manifestation of acute leukemia. Most of these patients have acute nonlymphoblastic leukemia, while others have acute lymphoblastic leukemia or chronic myelocytic leukemia and rarely chronic lymphocytic leukemia [195]. Antileukemic therapy generally fails to resolve DI.

Lymphocytic hypophysitis is a focal or diffuse inflammatory infiltration of the pituitary with varying degrees of gland damage. Laboratory and clinical findings point towards an autoimmune pathogenesis [199]. When the inflammatory lesion involves the neurohypophysis and the pituitary stalk it is referred to as lymphocytic infundibuloneurohypophysitis (LINH) [199]. LINH exhibits a balanced sex distribution, with a mean age at diagnosis of about 47 years although it can also occur in children [199,200]. General and ophthalmologic symptoms of LINH include headache, visual disturbances, nausea or vomiting, fatigue, weakness, and anorexia [200]. Endocrine symptoms may include partial or total hypopituitarism, hyperprolactinemia and, almost invariably, DI [199,201]. In LINH, together with diffuse thickening of the pituitary stalk, with or without enhancement after gadolinium, loss of the normal posterior “bright spot” on T1-weighted images is observed on MRI [199]. Glucocorticoids are usually effective in LIHN, although inflammatory lesions can be self-limited (spontaneous remission can be observed within 2 years); however, DI may be permanent [200,201].

Infiltrative Disorders Hypothalamic Pituitary Sarcoidosis Sarcoidosis is a chronic disease with multiorgan involvement characterized by immune granulomas

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320

9. THE HYPOTHALAMUS

which mainly affects young and middle-aged adults [199]. The more frequent target organs are the lungs, skin, and lymph nodes. Sarcoidosis involves the intracranial CNS in a minority of patients (5 15%) and very rarely affects the hypothalamic pituitary region (,1% of cases). When this is the case, sarcoid granuloma is generally localized in the basal hypothalamus and floor of the third ventricle, as well as the posterior, but not the anterior, pituitary. Both sexes are equally involved, and over 80% of patients have evidence of systemic involvement with sarcoidosis, especially hilar adenopathy, which is present on chest X-ray in twothirds of cases [202]. Endocrine complications are rare in sarcoidosis, but the hypothalamus and pituitary are the glands most commonly affected [58,199,203]. The most common anterior pituitary abnormalities are GH and gonadotrophin deficiency [204], followed by central hypothyroidism and hypoadrenalism and hyperprolactinemia [202]. Interestingly, hypothalamic pituitary involvement may precede the diagnosis in more than half of patients [202]. Involvement of the basal hypothalamus and floor of the third ventricle may lead to diminished visual acuity, visual field abnormalities, dysfunction of other cranial nerves (especially VII, I, V, and VIII), and evidence of other CNS involvement [204]. In addition to DI, hypothalamic involvement can lead to thermal dysregulation, somnolence, personality changes, abnormalities of thirst with resetting of the osmostat, and obesity [205]. Pituitary stalk thickening is a frequent finding on MRI [202,203]. Most patients with sarcoidosis involving the CNS, including the hypothalamus, receive therapeutic doses of glucocorticoids, which may improve some clinical manifestations, especially visual dysfunction, although the response is less frequent for longstanding abnormalities, especially DI [204]. Neuroradiologic abnormalities likely respond better than endocrine dysfunctions to corticosteroid treatment [202]. Langerhans’ Cell Histiocytosis LCH is a rare disease characterized by aberrant proliferation and tissue infiltration of specific clonal Langerhans’ dendritic cells deriving from a myeloidderived precursor. Frequent finding of activating somatic BRAF (encoding the serine/threonine-protein kinase B-Raf) mutations in affected tissue specimens, led to the inclusion of LCH as a myeloid neoplasm [206]. The hypothalamo pituitary system is a target of the disease together with bone, lung, and skin. The natural history of the disease varies from spontaneous resolution to progression and dissemination associated with relevant mortality risk [206]. LCH is more often encountered in children, with an incidence of 3 5 cases per million per year, peak age 1 4 years, and a male-to-female ratio of 2:1. LCH may also more rarely

occur in young adults [207]. Early diagnosis is important because multisystem LCH is associated with a 20% mortality rate. The hypothalamic pituitary system is involved in up to 50% of children with LCH who prevalently develop DI either during the course of the disease (often within the first year) or as the presenting feature of disease [208]. These latter patients quite rapidly (within 1 year) develop other LCH localizations [208]. Once established, DI is generally permanent and, if not remitting after treatment for the underlying disease, requires substitutive treatment [209]. Hypothalamic involvement in these patients may also cause sleep disturbances, hyperphagia with obesity, temperature dysregulation, and behavioral abnormalities [209]. Anterior pituitary dysfunction occurs in up to 20% of patients with LCH and is almost invariably associated with DI [208,209]. Once established, anterior pituitary deficiencies seem to be marginally responsive to treatment of LCH [209]. Growth retardation is found in 40% of patients with prepubertal onset of disease and is caused by GH deficiency which is the most frequent and earlier anterior pituitary hormone abnormality [210] due to altered formation and/or release of GHRH by the hypothalamus. Gonadotrophin deficiency is also a common hormone defect observed in adults with LCH [209]. Mild hyperprolactinemia due either to infundibular infiltration or concomitant gonadotrophin deficiency may be in some instances observed in adult LCH patients [210]. Endocrine deficiencies may evolve during the course of the disease [209]. Therefore, patients with isolated or partial pituitary hormone deficiency should be monitored at regular intervals [211]. On MRI, pituitary stalk thickening ( . 3.5 mm) and absence of the normal hyperintense signal of the posterior pituitary (bright spot) are the most common findings [212]. Prolonged treatment with vinblastine and prednisone improves patient survival and reactivation rates in multisystem LCH, but is rarely successful in reversing the DI or growth retardation [209].

Brain Irradiation Patients who receive therapeutic irradiation to the pituitary hypothalamic region for treatment of pituitary adenomas or primary suprasellar neoplasms may develop hypopituitarism. Nevertheless, it is commonly accepted that normal hypothalamus and pituitary are relatively radioresistant. Moreover, brain irradiation techniques have significantly improved in the last two decades, achieving enhanced efficacy and safety [213]. However, histologic studies have shown areas of necrosis in the hypothalamus following cranial irradiation and it is increasingly recognized that pituitary and especially hypothalamic dysfunction may be a

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TRAUMATIC BRAIN INJURY

long-term, adverse consequence of irradiation to the head and neck for disease outside of the sellar region [214]. Whole-brain irradiation widely used in patients older than 2 years for acute lymphoblastic leukemia, primary or metastatic brain tumors, total body irradiation (e.g., in patients undergoing hematopoietic stem cell transplantation for acute hematologic malignancies), or more localized radiation used for treatment of nasopharyngeal cancer, paranasal sinus tumors, and other head and neck neoplasms has been found to be associated with hypothalamic and pituitary dysfunction and decreased pituitary gland height on MRI [214 217]. Risk factors include the dose of irradiation, with higher doses being associated with a higher risk, the interval over which radiotherapy is delivered, the age of the patient, with children and adolescents being more susceptible than adults, and the interval following completion of radiation therapy and the time that the patient is specifically tested for damage [216,218]. Many of the early reports comprise series referred for evaluation of short stature or hypothalamic dysfunction; objective endocrine abnormalities were commonly observed in these cases [216]. Endocrine deficiencies secondary to brain irradiation are insidious, progressive, and generally irreversible; their onset may be delayed up to 10 years after the exposure to radiation [214]. They are often associated with a negative impact on growth, skeletal health, fertility, sexual function, and physical and psychological health. Whether endocrine disturbances may derive from hypothalamic damage and secondary pituitary insufficiency or from direct pituitary damage is still a matter of debate [219]. Samaan and colleagues studied 166 patients who received a median of 5000 rads to the hypothalamus and 5700 rads to the pituitary during treatment of nasopharyngeal carcinoma and paranasal sinus tumors [220]. Most patients had one or more hormonal abnormalities that suggested a hypothalamic lesion, while others had evidence of primary pituitary dysfunction. The prevalence of the various anterior pituitary hormone abnormalities was directly related to the number of years between completion of therapy and evaluation. The earliest abnormality was found to be GH dysfunction, documented a mean of 2.6 years following therapy. In fact, GH deficiency is the most common radiation-induced endocrine abnormality especially after low doses (18 24 Gy) [213]. The last abnormality to be detected was ACTH secretion, which occurred 6 years following the therapy [220]. Secondary neoplasms or relapse should be considered when treating endocrine deficiencies in cancer survivors. The risk of secondary neoplasia is increased following radiation exposure and certain malignancies. Treatment with GH does not increase cancer recurrence or death in survivors of childhood cancer but survivors

are at increased risk of secondary solid neoplasia [221]. Nonendocrinological manifestations of hypothalamic damage are thirst disorder, mental and personality changes, obesity, and sleep disorders [219]. Radiation therapy can also produce visual symptoms, including decreased visual acuity and visual field defects, by vasa nervorum obliteration of optic pathways, occurring from 3 months to 8 years after treatment [219].

TRAUMATIC BRAIN INJURY Traumatic brain injury (TBI) is the most common cause of death in young adults. Patients who survive the acute phase suffer from long-term sequelae with both physical and neuropsychological disabilities [222]. TBI may be caused by motor vehicle accidents, unintentional falls, and intentional physical abuse. Lesions due to falls during velocity sport competitions or to heavy and repeated concussions in contact sports are emerging causes of TBI [222,223]. Hypopituitarism is among the chronic (5 months or more after the event) consequences of TBI survivors [222]. Manifestations range from a single hypothalamo-pituitary axis defect to panhypopituitarism, and occur in a high percentage of injured subjects if they are proactively screened with dynamic pituitary function testing [224]. The reported prevalence of hypopituitarism after TBI in children and adolescents is similar to the prevalence in adults, but in early childhood, the reported prevalence is lower. Differences in reported post-TBI prevalence of hypopituitarism are likely due to selection criteria, age, and associated comorbidities [224]. Variable methodological approaches (baseline hormone assays vs dynamic testing) may also explain these differences [225]. Conflicting results have also been reported on hypopituitarism in the acute post-TBI phase [226]. TBI may induce hypothalamic pituitary damage via direct or indirect mechanisms such as hypotension (decrease in cerebral blood flow and hypoxia), increased intracranial pressure, and autoimmunity [222]. GH deficiency is the most common hormonal deficiency reported after TBI and should be proactively evaluated [222,224]. Development of GH deficiency may complicate the neuropsychological conditions (decreased quality of life, impaired cognitive performance) in TBI survivors [227]. This clinical picture appears to be at least partially reversible with GH treatment [228]. Hypopituitarism in TBI adults is also linked to several metabolic alterations, such as altered glucose levels, insulin resistance, and hypertriglyceridemia [229] likely due, at least in part, to GH deficiency [49]. Other epidemiologically relevant anterior pituitary dysfunctions include secondary

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hypoadrenalism [226] and hypogonadism [222]. DI is also a well-recognized complication in these patients, with a reported incidence up to about 25% in the acute phase and in association with more severe head injury and cerebral edema, and with a higher mortality rate. Acute-phase DI as well as acute hypocortisolemia are associated with a poor prognosis and long-term pituitary deficits [222].

Acknowledgments Authors are grateful to Dr Glenn Braunstein, author of the previous version of this chapter for enabling use of prior edition material.

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[157] Muller HL. Craniopharyngioma. Endocr Rev 2014;35:513 43. [158] Hussain I, Eloy JA, Carmel PW, Liu JK. Molecular oncogenesis of craniopharyngioma: current and future strategies for the development of targeted therapies. J Neurosurg 2013;119: 106 12. [159] Sanford RA, Muhlbauer MS. Craniopharyngioma in children. Neurol Clin 1991;9:453 65. [160] Mortini P, Gagliardi F, Boari N, Roberti F, Caputy AJ. The combined interhemispheric subcommissural translaminaterminalis approach for large craniopharyngiomas. World Neurosurg 2013;80:160 6. [161] Sklar CA. Craniopharyngioma: endocrine abnormalities at presentation. Pediatr Neurosurg 1994;21(Suppl. 1):18 20. [162] Zacharia BE, Bruce SS, Goldstein H, Malone HR, Neugut AI, Bruce JN. Incidence, treatment and survival of patients with craniopharyngioma in the surveillance, epidemiology and results program. Neuro Oncol 2012;14:1070 8. [163] Erfurth EM, Holmer H, Fjalldal SB. Mortality and morbidity in adult craniopharyngioma. Pituitary 2013;16:46 55. [164] Yano S, Kudo M, Hide T, Shinojima N, Makino K, Nakamura H, et al. Quality of life and clinical features of long-term survivors surgically treated for pediatric craniopharyngioma. World Neurosurg 2016;85:153 62. [165] Hofmann BM, Ho¨llig A, Strauss C, Buslei R, Buchfelder M, Fahlbusch R. Results after treatment of craniopharyngiomas: further experiences with 73 patients since 1997. J Neurosurg 2012;116:373 84. [166] Duff J, Meyer FB, Ilstrup DM, Laws Jr ER, Schleck CD, Scheithauer BW. Long-term outcomes for surgically resected craniopharyngiomas. Neurosurgery 2000;46:291 305. [167] Lena G, Paz Paredes A, Scavarda D, Giusiano B. Craniopharyngioma in children: marseille experience. Childs Nerv Syst 2005;21:778 84. [168] Minamida Y, Mikami T, Hashi K, Houkin K. Surgical management of the recurrence and regrowth of craniopharyngiomas. J Neurosurg 2005;103:224 32. [169] Shi XE, Wu B, Fan T, Zhou ZQ, Zhang YL. Craniopharyngioma: surgical experience of 309 cases in China. Clin Neurol Neurosurg 2008;110:151 9. [170] Zuccaro G. Radical resection of craniopharyngioma. Childs Nerv Syst 2005;21:679 90. [171] Gerganov V, Metwali H, Samii A, Fahlbusch R, Samii M. Microsurgical resection of extensive craniopharyngiomas using a frontolateral approach: operative technique and outcome. J Neurosurg 2014;120:559 70. [172] Yu T, Sun X, Ren X, Cui X, Wang J, Lin S. Intraventricular craniopharyngiomas: surgical management and outcome analyses in 24 cases. World Neurosurg 2014;82:1209 15. [173] Cohen M, Bartels U, Branson H, Kulkarni AV, Hamilton J. Trends in treatment and outcomes of pediatric craniopharyngioma, 1975-2011. Neuro Oncol 2013;15:767 74. [174] Bomer I, Saure C, Caminiti C, Ramos JG, Zuccaro G, Brea M, et al. Comparison of energy expenditure, body composition, metabolic disorders, and energy intake between obese children with a history of craniopharyngioma and children with multifactorial obesity. J Pediatr Endocrinol Metab 2015;28:1305 12. [175] Sterkenburg AS, Hoffmann A, Gebhardt U, Warmuth-Metz M, Daubenbu¨chel AM, Mu¨ller HL. Survival, hypothalamic obesity, and neuropsychological/psychosocial status after childhood-onset craniopharyngioma: newly reported longterm outcomes. Neuro Oncol 2015;17:1029 38. [176] Mu¨ller HL. Paediatrics: surgical strategy and quality of life in craniopharyngioma. Nat Rev Endocrinol 2013;9:447 9. [177] Zada G, Kintz N, Pulido M, Amezcua L. Prevalence of neurobehavioral, social, and emotional dysfunction in patients

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C H A P T E R

10 Anterior Pituitary Failure John D. Carmichael

INTRODUCTION Hypopituitarism results from the failure of one or more pituitary hormones to be produced or secreted from the anterior pituitary. The anterior lobe of the pituitary is responsible for the production and secretion of hormones that affect specific peripheral glandular tissues. The anterior pituitary is under the control of the hypothalamus via hypophysiotrophic neurohormonal regulation, which integrates feedback mechanisms that govern secretion of hypothalamic factors, pituitary hormones, and peripheral hormones. Peripheral stimuli from a wide array of sources prompt modulation of the neurohormonal control of anterior pituitary function, including production and secretion of pituitary hormones. These hormones are released into the circulation, resulting in specific systemic effects. Anterior pituitary failure can result from the disruption of any step in the production, stimulation, secretion, and regulation of these hormones.

treatment for hormone replacement and comorbidities such as hypertension and hyperlipidemia [2]. These later studies devoted to assessment of mortality risk in patients with hypopituitarism treated with hormonal replacement suggest a normalization of mortality when growth hormone (GH) is replaced in men, and an improved, but not normalized mortality in women compared to those not receiving GH treatment [3
5]. While GH deficiency has been implicated as a cause for increased mortality in subjects with hypopituitarism, long-term controlled studies are not available to adequately point toward this deficiency as the sole mediator of increased mortality. Imprecise and excessive glucocorticoid replacement carries with it increased morbidity, but this has not been adequately studied to determine its exact role in excess mortality in hypopituitarism. The underlying etiology, age of onset, and aggressiveness of the cause of hypopituitarism remain the most important predictors of mortality, usually due to cerebrovascular events, respiratory failure, or infection in adrenal-insufficient patients [2].

MORTALITY ETIOLOGY Hypopituitarism is associated with increased mortality compared to the general population. Early epidemiologic studies of patients with hypopituitarism demonstrated an excess standardized mortality ratio (SMR) of 1.2
2.2 years, often ascribed to a higher incidence of cardiovascular and cerebrovascular events. Meta-analysis of studies of hypopituitarism demonstrates increased mortality in men (SMR 2.06; 95% CI 1.94
2.2) and a more pronounced increase in women (SMR 2.80; 95% CI 2.59
3.02) [1]. Risk attributable specifically to hormonal replacement remains a controversial issue, however updated studies reflect more contemporary replacement regimens, refinement of radiation and surgical treatment, and availability of more widespread and effective The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00010-6

The etiology of anterior pituitary failure (Table 10.1) can be broadly divided into structural and functional causes. Normal physiologic secretion of pituitary hormones relies on intact hypothalamic control of pituitary function, transport of hypophysiotrophic hormones from the hypothalamus to the pituitary via the portal blood supply, and normal functioning of the anterior pituitary hormone-secreting cells. Mass lesions arising from the pituitary or hypothalamus can affect normal pituitary or hypothalamic function and cause deficiency of one or more pituitary hormones. Structural abnormalities rooted in the embryologic development of the adenohypophysis can lead to deficiencies in one or more pituitary hormones. These and other structural

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330 TABLE 10.1

10. ANTERIOR PITUITARY FAILURE

Causes of Anterior Pituitary Failure

Neoplastic Pituitary adenoma Pituitary carcinoma Craniopharyngioma Pituicytoma Fibroma Glioma Meningioma Paraganglioma Teratoma Chordoma Angioma Sarcoma Ependymoma Germinoma Cysts Rathke’s cleft, arachnoid, epidermoid, dermoid Ganglioneuroma Astrocytoma Metastatic Breast, lung, colon, prostate Treatment of sellar, parasellar, and hypothalamic disease Surgery Radiotherapy Radiosurgery Infiltrative disease Autoimmune Lymphocytic hypophysitis Anti-PIT-1 antibody syndrome Granulomatous Sarcoidosis Langerhans cell histiocytosis Giant cell granuloma Granulomatous hypophysitis Xanthomatous hypophysitis Wegener’s granulomatosis Hemochromatosis Empty sella Idiopathic

Vascular Pituitary tumor apoplexy Sheehan’s syndrome Intrasellar carotid artery aneurysm Subarachnoid hemorrhage Genetic (see Table 10.2) Combined pituitary hormone deficiencies Isolated pituitary hormone deficiencies Developmental Midline cerebral and cranial malformations Pituitary hypoplasia or aplasia Ectopic pituitary Basal encephalocele Traumatic Head injury Perinatal trauma Infectious Bacterial Viral Fungal Tuberculosis Syphilis Medications Opiates Glucocorticoid therapy Megestrol acetate Suppressive thyroxine treatment Dopamine Sex steroid treatment GnRH agonists Antiseizure medications Systemic disease Obesity Anorexia nervosa Chronic illness

causes are generally not reversible without invasive intervention. Functional causes of pituitary deficiency are present without extrinsic or intrinsic mass and are generally reversible, once the underlying etiology is discovered and treated. In contrast, genetic or congenital causes may or may not have structural manifestations, can cause failure of one or more pituitary hormones, and are not reversible.

Structural Causes of Pituitary Failure Mass Lesions PITUITARY TUMORS

Pituitary adenomas are the most common cause of hypopituitarism. These lesions arising from monoclonal cells of pituitary origin are the most common

sellar lesions, account for approximately 15% of all intracranial lesions, and are classified by cell type and size. Microadenomas are tumors with a largest diameter of less than 10 mm. These masses are common, with an estimated incidence ranging from 1.5 to 27% based on autopsy series [6,7]. Nonsecretory microadenomas are rarely associated with hypopituitarism and have a benign natural course. Macroadenomas are less common but are frequently associated with hypopituitarism. The mechanism responsible for diminished pituitary function appears to be increased intrasellar pressure causing compression of the portal vessel blood supply to the normal pituitary, or compression of the pituitary stalk, interrupting hypothalamic control over pituitary secretion [8]. Hyperprolactinemia is commonly seen in patients with nonprolactinoma tumors due to interruption of the normal suppressive effects of dopaminergic tone from the hypothalamus [9]. The resultant compression can lead to decreased secretion of one or many pituitary hormones. Hyperprolactinemia alone can cause hypopituitarism, specifically isolated hypogonadotrophic hypogonadism, via a short feedback loop interrupting gonadotrophin-releasing hormone (GnRH) pulse regulation in the hypothalamus, with resultant decreases in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Since the mechanism of pituitary dysfunction appears to be related to pressuremediated effects, it is not surprising that pituitary recovery is anticipated in some, but not all, cases of pituitary mass lesions after surgical or medical decompression. PITUITARY SURGERY Hypopituitarism is a complication of pituitary surgery. Factors contributing to the risk of hypopituitarism following surgery on a sellar mass include tumor size and degree of invasiveness, and experience of the surgeon. New-onset hypopituitarism is reported in 3
14% of surgical series with most complication rates ranging between 5 and 7% [10]. Details regarding surgical outcomes are found in Chapter 24, Pituitary Surgery. NONPITUITARY NEOPLASMS

A number of nonpituitary neoplastic lesions can cause hypopituitarism due to their proximity to the pituitary gland and hypothalamus [11,12]. Craniopharyngiomas arise from the remnants of Rathke’s pouch and may be cystic, solid, or mixed tumors. Due to the involvement of the pituitary stalk and hypothalamus, craniopharyngiomas frequently present with diabetes insipidus (up to 38%), in addition to some degree of anterior pituitary failure (up to 95%) [13].

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PITUITARY DISORDERS

ETIOLOGY

Metastatic lesions from breast, lung, colon, and prostate primary tumors have been described in the sella [14]. These tumors generally spread to the hypothalamus or posterior pituitary prior to invasion of the anterior pituitary. Diabetes insipidus may result from metastatic lesions, however other manifestations of pituitary failure are rare, especially since the time to develop symptomatic hypopituitarism is lengthy and patients may not live long enough for symptoms to be apparent. Other rare solid tumors in the parasellar area can cause hypopituitarism. These include pituicytomas, fibromas, parasellar meningiomas, paragangliomas, teratomas and germ cell tumors, chordomas, and angiomas. Sarcomas are a rare cause of hypopituitarism, which arise from structures adjacent to the pituitary and cause compression of the adenohypophysis. These include fibrosarcomas, osteosarcomas, and undifferentiated sarcomas. CYSTIC LESIONS

Cystic lesions arising in the sellar region may cause pituitary dysfunction and these include cystic adenomas, Rathke’s cleft cysts, arachnoid cysts, epidermoid cysts, and dermoid cysts [15]. Rathke’s cleft cysts arise from cystic remnants during the formation of Rathke’s pouch. They are thin-walled cysts lined by cuboidal or columnar ciliated epithelium. The size and growth rate of these lesions is variable. They may be intrasellar, intrasellar with extrasellar extension, or rarely entirely suprasellar. Arachnoid cysts are derived from the arachnoid membrane and are usually located in the suprasellar subarachnoid space. Epidermoid cysts are rare causes of hypopituitarism. These unilocular cysts are slow-growing and lined by laminated squamous epithelium, and rarely become malignant. Dermoid cysts have a firm fibrous capsule, lined by keratinized squamous epithelium. Other tissue types may be present in the cyst wall. ANEURYSMS

Aneurysms of the cavernous portion of the carotid artery or branches from the circle of Willis may compress the hypothalamic/pituitary unit resulting in hypopituitarism [16]. The appearance of an aneurysm in the parasellar area may resemble a pituitary adenoma, and careful consideration of this entity is required prior to invasive interventions. INFILTRATIVE LESIONS HYPOPHYSITIS Hypophysitis is characterized by immune cell infiltration and destruction of the pituitary resulting in various degrees of hypopituitarism. It is commonly seen in the postpartum period and

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during pregnancy and is most likely due to an autoimmune process, although subtle variations in histopathology may indicate two separate underlying immune processes [17]. The lymphocytic subtype is predominantly seen in women; however, a growing number of reports in children and men have been described. Infiltration may affect the anterior pituitary (hypophysitis), the infundibulum and posterior pituitary (infundibuloneurohypophysitis), or both (panhypophysitis). The anterior pituitary is most commonly affected resulting in pituitary hormone deficits, without diabetes insipidus. Infiltration of the posterior pituitary and infundibulum may present with diabetes insipidus and hyperprolactinemia, and infiltration of the entire hypophysis is rarely seen but presents with both anterior and posterior pituitary dysfunction. Three main forms of hypophysitis include: lymphocytic, granulomatous, and xanthomatous. More rare forms have been described including IgG4-related hypophysitis, necrotizing hypophysitis, and mixed forms [18]. Lymphocytic infiltration is mostly due to cytotoxic T-lymphocyte cells that cause anterior pituicyte destruction and fibrotic replacement with fibrosis. The presence of giant cells and granulomatous formation suggests the more rare entity, granulomatous hypophysitis. Granulomatous hypophysitis occurs in equal frequency in men and women and has a reported incidence of 1:1,000,000 [19]. Xanthomatous hypophysitis is exceedingly rare and is characterized by the presence of foamy macrophages, lymphocytes, and plasma cells [20]. IgG4-related hypophysitis is due to infiltration of IgG-4 staining plasmacytes, while necrotizing hypophysitis is characterized by necrosis [21,22]. Hypophysitis is generally a primary disease of the pituitary, however it has been associated with infectious etiologies, Langerhans cell histiocytosis, sarcoidosis, Wegener’s granulomatosis, Crohn disease, Takayasu arteritis, and ruptured cysts [19]. Anticytotoxic T-lymphocyte antigen-4 antibodies used in cancer treatments have an adverse effect of hypophysitis and pituitary dysfunction in up to 17% of patients [23,24]. Patients generally present with symptoms of a pituitary mass, either with headache or visual disturbances, and symptoms of pituitary insufficiency. Since it is commonly associated with pregnancy and the postpartum period, failure of lactation and lack of menses may be one of the first signs of disease. Symptoms of multiple pituitary deficiencies occur in 75% of cases [20]. Thyrotroph and corticotroph cells appear to be affected more frequently in lymphocytic hypophysitis with sparing of the gonadal axis. Prolactin levels may range from undetectable to elevated, with low levels of prolactin attributable to destruction of lactotrophs, an uncommon finding in

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other pituitary masses. Elevated levels of prolactin may be expected during pregnancy and the postpartum period, but have been reported in men and nonpregnant women [19]. Characteristically, magnetic resonance imaging (MRI) shows an enlarged pituitary gland, and suprasellar extension is common [20]. The findings are usually distinguishable from pituitary adenomas. With hypophysitis, the gland is symmetrically enlarged with uniform enhancement with gadolinium. The gland is usually low signal on T1-weighted images, and high signal on T2-weighted images. Images obtained later in the disease process may demonstrate shrinkage, fibrosis, and an empty sella [18]. The diagnosis of hypophysitis should be strongly considered in female patients who are pregnant or who have recently delivered, and have symptoms of pituitary insufficiency. A heightened index of suspicion should be present in patients with other autoimmune disease processes. Biopsy of the mass is necessary to definitively make the diagnosis; however, a presumptive diagnosis can be made in the proper clinical setting with typical imaging characteristics, rapid development of symptoms, and hypopituitarism affecting adrenocorticotrophic hormone (ACTH) secretion and thyroid-stimulating hormone (TSH) secretion with a serum prolactin level below the normal range. Measurement of pituitary antibodies remains beyond the scope of standard practice due to a lack of sufficient sensitivity or specificity; recent discovery of anti-PIT-1 antibodies suggest a role of this transcription factor antibody in the pathogenesis of autoimmune-related hypopituitarism [25]. The natural course of history of hypophysitis is variable. The disease is marked by initial inflammation, infiltration, and enlargement of the gland, accompanied by mass effects. Destruction of the gland follows with resultant hypopituitarism of varying degrees. Cases of spontaneous partial and complete recovery have been documented, with complete resolution of the pituitary mass. Recurrence is rare after remission, but has been reported. Management of lymphocytic hypophysitis ranges from conservative management to more aggressive resection of the mass [24]. Corticosteroid therapy has been advocated, but the effects are variable [19]. All patients require appropriate replacement of deficient pituitary hormones, with attempts to withdraw replacement after the acute phase to assess for recovery of pituitary function. SARCOIDOSIS AND OTHER GRANULOMATOUS DISEASES Central nervous system involvement

occurs in approximately 5
15% of patients with sarcoidosis. These granulomatous lesions of the hypothalamus commonly result in hypopituitarism and

diabetes insipidus [26]. Sarcoidosis is generally not seen in the hypothalamic region without evidence of systemic involvement. Infiltration can be visualized on MRI in the hypothalamus, infundibulum, or pituitary. Corticosteroid therapy often improves the appearance of the lesion, but deficits in pituitary function usually remain [26]. Giant cell granuloma is a rare cause of pituitary failure, affecting primarily the anterior pituitary [27]. Langerhans cell histiocytosis is a rare proliferative disease characterized by infiltration of organs by abnormal dendritic cells [27]. Hypopituitarism generally is caused by infiltration of the hypothalamus and is found in 20% of cases. Wegener’s granulomatosis is a rare cause of pituitary insufficiency, with partial or panhypopituitarism [28]. Other forms of granulomatous disease have been associated with failing pituitary function including idiopathic giant cell hypophysitis, Takayasu disease, Cogan syndrome, and Crohn disease [27]. HEMOCHROMATOSIS Hereditary hemochromatosis is an autosomal recessive disorder and is a rare cause of hypopituitarism [29]. Excessive absorption of dietary iron leads to iron overload. Hypopituitarism is the result if iron infiltration of the anterior pituitary. Hypogonadism is the most common pituitary manifestation, with preferred uptake of iron by gonadotroph cells [30]. Secondary hypogonadism is also reported, and infiltration at multiple areas may be seen in more severe disease. TSH and ACTH deficiencies have been described but are less common.

Pituitary Irradiation Hypopituitarism is a well-known complication of radiation treatment of pituitary tumors and other masses and malignancies and has been extensively documented and reviewed [31,32]. Advances in stereotactic techniques and delivery systems providing focused beams of various types of radiation have the theoretical benefit of precise treatment of the disease without adverse effects on normal surrounding tissue. Despite these advances, the pituitary tissue often receives significant doses of radiation during therapy with the propensity to develop hypopituitarism over the years following treatment [33
37]. As an example, 76 patients with secretory adenomas were treated with stereotactic radiosurgery and observed for a mean of 96 months. Remission of secretory function was achieved in 45% of these patients with 23% experiencing new hypopituitarism [33]. In comparison, prior long-term evaluation of hypopituitarism following radiation therapy demonstrates up to 80% prevalence of new deficits in gonadotroph, thyrotroph, or adrenocorticotroph function [38
40]. Whether newer modalities utilizing stereotactic gamma knife radiosurgery are

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less prone to complications of hypopituitarism is yet to be determined, as few studies have been published with large numbers of treated patients and sufficient follow-up duration. Pituitary adenomas are the most common sellar masses treated with radiation therapy. Residual secretory tumors and nonfunctioning adenomas are treated for control of hormonal activity and tumor growth. Multiple adverse effects of radiation therapy include optic neuropathy, second brain tumors, vascular injury, as well as hypopituitarism, necessitating lifelong monitoring [32]. Radiotherapy is commonly delivered in fractionated doses of 1.8 Gy over 25 fractions, for a total dose of 45 Gy. Radiosurgery, utilizing doses of 15
30 Gy during a single session, has been used more frequently in the last decade. The incidence of radiation-induced hypopituitarism varies from study to study and reaches 100% in some series [31]. A number of effects of radiation on normal and neoplastic tissue may be responsible for hypopituitarism seen after treatment. DNA damage induced by ionizing radiation may have acute or delayed effects on cell replication, possibly explaining the delayed onset of hypopituitarism. Degenerative changes in glial cells may lead to damage to the hypothalamic supportive structures causing chronic and subacute neural damage. Vascular damage leads to long-term damage to pituitary and parasellar tissues [31]. Hypothalamic damage is likely responsible for early radiation-induced hypopituitarism, whereas pituitary damage is more associated with late-onset hypopituitarism. Typically, GH and gonadotrophin secretion are more susceptible to effects of radiation than TSH or ACTH secretion. The mechanism underlying this expected progression of deficiencies is unknown. Most patients present with a typical progression of deficiencies starting with GH deficiency, gonadotrophin deficiency, ACTH deficiency, progressing to TSH deficiency [41]. However, any deficiency can arise at any time, even years from the radiation treatment. Infectious Etiologies A wide array of infectious agents has been described as etiologies of pituitary insufficiency. These are, however, all rare causes of pituitary failure. Most infectious etiologies spread via hematogenous spread or by direct extension. Bacterial infections with abscess formation have been described, with a variety of pathogens as a bacterial source: Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas, Klebsiella ozaenae, Bacteroides, Gram-positive Coryneform rods, have all been associated with abscess formation [42]. Fungal infections caused by Aspergillus, Coccidioides, Candida albicans, and Histoplasma have been described [27]. Granulomatous infections from Mycobacterium

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tuberculosis have been described. Syphilitic gummatous lesions have been described. Hypopituitarism in the setting of HIV infection has been described, as well as other viral pathogens [43]. Pituitary Hemorrhage APOPLEXY

Pituitary tumor apoplexy is a rare event resulting in spontaneous hemorrhage or infarction of a pituitary adenoma. Sudden hemorrhage increases the intrasellar volume and pressure compressing surrounding structures, portal vessels, and normal pituitary tissue. The increase in pressure results in sudden onset of headache, visual disturbance, and acute pituitary insufficiency. Subclinical hemorrhage may be present in existing pituitary adenomas; however, this entity is distinctly separate from the syndrome of acutely deteriorating pituitary function in the setting of hemorrhage into the pituitary adenoma. Spontaneous hemorrhage may be present without clinically significant symptoms in up to 25% of patients [44]. The true incidence of pituitary tumor apoplexy or subclinical hemorrhage is difficult to establish. It is a rare event, with reported incidences ranging from 0.6 to 9%, but inclusion of subclinical cases brings the estimate to as high as 15% [45]. Nearly 50% of cases occur in patients without prior knowledge of a pituitary adenoma. All types of pituitary tumors are susceptible to apoplectic events, with a higher prevalence in nonfunctioning adenomas, and men appear to be affected more commonly than women. Most cases present in the fifth or sixth decade of life, but cases have spanned the first through eighth decades. Patients present with acute onset of headache and visual disturbance, including cranial nerve palsies resulting in extraocular muscle defects. Neurological signs may be present including an altered level of consciousness and meningismus. Generalized symptoms are not uncommon with nausea and vomiting, malaise, and lethargy frequently reported. Endocrine dysfunction contributes to the morbidity and mortality associated with pituitary tumor apoplexy with acute ACTH deficiency having a central role. The extent of pituitary dysfunction varies with adrenal insufficiency occurring in 50
100% of cases, thyroid dysfunction present in 25
75% of cases, and gonadal function impaired in 60
100% of cases [45]. Noncontrast CT scan of the sella turcica reveals acute hemorrhage, and MRI of the sella may be helpful to elucidate the extent of parasellar involvement. Hemorrhage is more evident on MRI in the subacute phase than during the acute phase. SHEEHAN SYNDROME

The pituitary gland normally enlarges during pregnancy. Postpartum pituitary necrosis occurs in women

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who suffer large-volume hemorrhage during delivery, resulting in hypovolemic shock and ischemia. Usual causes of hypovolemia include placenta previa or retained placenta, leading to blood loss, hypovolemia, and necrosis of the enlarged pituitary due to the limited blood supply. Loss of pituitary function may be partial or complete. Due to advances in obstetric care and peripartum supportive measures, Sheehan syndrome is not commonly encountered. Congenital and Inherited Pituitary Insufficiency DEVELOPMENTAL PITUITARY DYSFUNCTION

Pituitary development occurs after midline cell migration from Rathke’s pouch. Structural pituitary abnormalities are seen with midline anomalies such as corpus callosum and anterior commissure defects. Congenital absence of the pituitary and ectopic pituitary tissue is rarely encountered, although functional ectopic posterior pituitary tissue is sometimes incidentally noted on brain or pituitary imaging. Craniofacial anomalies such as cleft lip and palate, septooptic dysplasia, and basal encephalocele often present with varying degrees of pituitary dysfunction and hypoplasia. Children with congenital malformations and structural defects impairing pituitary function require lifelong replacement of pituitary deficiencies. Improvements in imaging techniques over the years have led to improved detection of structural abnormalities accompanying pituitary and hypothalamic deficits. Patients may present with a small pituitary, complete or partial empty sella, ectopic posterior pituitary, or pituitary stalk abnormalities. GENETIC FACTORS

Mutations of several genes including those responsible for pituitary hormone production, hormone receptors, and pituitary development can cause hypopituitarism (Table 10.2) [46]. Several transcription factors involved in pituitary development and function have been identified in recent years, and mutations in any of these factors may lead to multiple or isolated pituitary deficiencies [48,49]. Mutations in genes specific for single pituitary hormones and receptors give rise to isolated hormonal deficiencies. Morphological changes, often but not always limited to the pituitary and parasellar region, may be evident. The roles of these transcriptional factors are complex and interdependent upon each other in many circumstances. The embryogenesis and development of the pituitary and the roles that each of these factors play in development of the adenohypophysis is addressed elsewhere in this text (see chapter: Pituitary Development). Prop-1 (OMIM601538) is the most common factor associated with multiple pituitary deficiencies, occurring

in as many as 50% of patients with combined pituitary deficits. The gene, located on chromosome 5q, is required for subsequent Pit-1 activation. Several human mutations have been described associated with deficiencies in GH, TSH, PRL, and gonadotrophins. Human Prop-1 mutations are associated with deficiencies in Pit1-dependent cell lines (GH, PRL, TSH) and impaired gonadotroph and corticotroph reserve, despite these latter cell lines not being Pit-1-dependent. Several mutations have been associated with the phenotype of multiple pituitary deficiencies. The inheritance mode is autosomal recessive; thus, patients are homozygous for either deletion or missense frameshift mutations. The resulting protein product is truncated and nonfunctional. GA or AG deletions in a Prop-1 hotspot on exon 2 result in a coding frameshift and termination at codon 109. Siblings that are unaffected are either heterozygous or homozygous for a normal Prop-1 sequence on both alleles. Prop-1 gene mutations are frequently found in patients with combined pituitary hormone deficiencies occurring in as many as 30
50% of affected subjects [50]. Clinical features most often include a presentation of hypogonadism, with delayed or absent puberty. Some patients will enter puberty spontaneously and present with late-onset hypogonadotrophic hypogonadism, similar to patients with acquired disease [51]. Patients may develop or present with adrenal insufficiency or panhypopituitarism [52]. The appearance of the pituitary on MRI may be hypoplastic or normal. Cystic changes may be present with the appearance of empty sella. Linear growth arrest becomes evident after the age of 3, with height severely lower-than-expected height. Eunicoid habitus and reduced upper to lower body proportions may be seen. Affected adults usually have short stature and lack secondary sexual characteristics [53]. The Pou1F1 gene (formerly known as Pit-1) (OMIM173110) is located on chromosome 3p11 and contains two protein domains, the POU-specific- and the POU-homeodomain [48]. Both of these domains are necessary for DNA binding, transcription of the GH and PRL genes and regulating PRL, TSH-β, and Pit-1 genes. The Pit-1 nuclear protein activates the transcription of the PRL, GH, TSH, and growth hormone releasing hormone (GHRH) receptor genes. Pit-1 acts with coactivator proteins such as cyclic AMP response element binding cAMP response elementbinding protein, P-Lim, Ptx-1, HESX-1, and Zn-15. Pit-1 expression is autoregulated and is restricted to the anterior pituitary. Its expression is necessary for the development of lactotrophs, somatotrophs, and thyrotrophs. Most mutations in Pou1F1 are recessive, but autosomal dominant mutations have been described. The most common dominant mutation is the R271W mutation [54]. Inactivating mutations of the gene result in varied pituitary hormone deficiencies [55].

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TABLE 10.2

Genetic Disorders of Hypothalamo-Pituitary Development in Humans

Gene

Phenotype

Inheritance

ISOLATED HORMONE ABNORMALITIES GH1 Isolated GH deficiency

AR, AD

GHRHR

Isolated GH deficiency

AR

TSH-β

Isolated TSH deficiency

AR

TRHR

Isolated TSH deficiency

AR

TBX19 (TPIT)

Isolated ACTH deficiency

AR

GnRHR

HH

AR

PCI

ACTH deficiency, hypoglycemia, HH, obesity

AR

POMC

ACTH deficiency, obesity, red hair

AR

DAX1

Adrenal hypoplasia congenital and HH

XL

CRH

CRH deficiency

AR

KAL1

Kallmann syndrome, renal agenesis, synkinesia

XL

FGFR1

Kallmann syndrome, cleft lip and palate, facial dysmorphism

AD, AR

FGF8

HH, variable midline defects, rare multiple hormone deficiencies

AR

Leptin

HH, obesity

AR

Leptin-R

HH, obesity

AR

GPR54

HH

AR

Kisspeptin

HH

AR

FSH-β

Primary amenorrhea, defective spermatogenesis

AR

LH-β

Delayed puberty

AR

PROK2

Kallmann syndrome, severe sleep disorder, obesity

AD

PROKR2

Kallmann syndrome

AD, AR

AVP-NP11

Diabetes insipidus

AR, AD

COMBINED PITUITARY HORMONE DEFICIENCY POU1F1 GH, TSH, and prolactin deficiencies

AR, AD

PROP1

GH, TSH, LH, FSH, prolactin, and evolving ACTH deficiencies

AR

SPECIFIC SYNDROME HESX1

Septooptic dysplasia

AR, AD

LHX3

GH, TSH, LH, FSH, prolactin deficiencies, limited neck rotation

AR

LHX4

GH, TSH, ACTH deficiencies with cerebellar abnormalities

AD

SOX3

Hypopituitarism and mental retardation

XL

GLI2

Holoprosencephaly and multiple midline defects

AD

SOX2

Anophthalmia, hypopituitarism, esophageal atresia

AD

GLI3

Pallister
Hall syndrome

AD

PITX2

Rieger syndrome

AD

CHD7

CHARGE syndrome

AD

WFS1

Wolfram syndrome

AR

Adapted from [46] and [47]. R, receptor; GH, growth hormone; AR, autosomal recessive; AD, autosomal dominant; TSH, thyroid-stimulating hormone; ACTH, adrenocorticotrophic hormone; HH, hypogonadotrophic hypogonadism; XL, X-linked; CRH, corticotrophin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone, CHARGE, coloboma of the eye, heart malformations, atresia of the choanae, retardation of growth, ear abnormalities.

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However, several Pou1F1 mutations have characteristic clinical phenotypes [50]. In general, GH and prolactin deficiencies are the first to become apparent clinically, with TSH deficiency presenting later in childhood [56]. The anterior pituitary may be small or normal on MRI, with normal posterior pituitary. HESX1 (Rpx) is the earliest known transcriptional marker of the developing pituitary and is limited to Rathke’s pouch. The expression of HESX1 is essential for normal forebrain and pituitary formation. HESX1 expression declines as specific pituitary cell types develop and is longer expressed in the mature pituitary. The gene is located on chromosome 3p212 and encodes a 185-amino-acid protein that competes with Prop-1 protein for DNA binding. Septooptic dysplasia is associated with a homozygous Arg53Cys homeodomain mutation, but only accounts for approximately 1% of subjects affected by septooptic dysplasia. Affected patients have panhypopituitarism, possibly related to the anatomical and structural anomalies associated with the syndrome. Pitx1 and Pitx2 are pair-like homeodomain transcription factors related to Otx genes. No mutations have been describes for Pitx1, but Pitx2 mutations are associated with Axenfeld
Rieger syndrome, an autosomal dominant condition manifested by eye anomalies, dental hypoplasia, and pituitary deficiencies [49]. Lhx3 and Lhx4 are members of the LIM homeodomain protein family and mutations of each have been described with combined pituitary hormone deficiencies. LHX3 is expressed in the anterior and intermediate lobes of the pituitary, spinal cord, and medulla. Three LHX3 isoforms have been identified in humans, including hLHX3a, hLHX3b, and hM2-LHX3. Patients with mutations in LHX3 have deficiencies in GH, PRL, TSH, and gonadotrophins [57]. They also demonstrate abnormal pituitary morphology and a rigid cervical spine limiting rotation [58]. Homozygous mutations are a rare form of hypopituitarism. Mutations in the LHX4 gene result in the arrest of the formation of Rathke’s pouch and a hypoplastic pituitary. Mutations in the LHX4 gene prohibit activation of both Prop-1 and Pou1F1, resulting in pituitary failure [59]. In addition to pituitary deficiencies, findings of ectopic posterior pituitary, cerebellar abnormalities, Chiari malformation, and poorly developed sella turcica have been described [56]. OTX2 is a transcription factor required for formation of forebrain structures. Mutations have been implicated in syndromes of anophthalmia and microphthalmia in humans. Heterozygous mutations have been associated with eye abnormalities and variable pituitary defects [60,61]. MRI appearance of the pituitary is small or normal with an ectopic posterior pituitary. OTX2 mutations have also rarely been reported in patients with pituitary deficits without ocular anomalies [62].

Isolated hormone deficiencies also occur due to genetic mutations in genes encoding hormones and receptors. Congenital isolated GH deficiency is mostly sporadic, with an incidence of approximately 1:4000 to 1:10,000 live births. Four familial forms comprise 5
30% of cases [63,64]. Isolated growth hormone deficiency (GHD) may be autosomal dominant, autosomal recessive, or X-linked. Autosomal recessive forms are associated with mutations in GH1 and GHRH receptor genes. Autosomal dominant forms have been described due to mutations in GH1 gene. An X-linked form is associated with agammaglobulinemia or hypogammaglobulinemia, but no gene has been found associated with this syndrome [56]. Autosomal recessive mutations in TBX19 (TPIT) are the primary cause of congenital isolated ACTH deficiency. [65]. Mutations result in profound hypoglycemia, seizures, and cholestatic jaundice and may be an underappreciated cause of neonatal mortality. Basal plasma levels of ACTH and cortisol are very low, with lack of ACTH stimulation after CRH administration. Proopiomelanocortin (POMC) mutations are also associated with isolated ACTH deficiency [66]. Factors arising from the ventral diencephalon are associated with hypopituitarism. Fibroblast growth factor 8 (Fgf8) inhibits anterior pituitary development and maintains proliferative factors such as Lhx3 and Lhx4. Mutations in Fgf8 have been associated with hypogonadotrophic hypogonadism [49]. GLI2 is a transcription factor that mediates Sonic hedgehog signaling, an important regulator of early Rathke’s pouch development, and proliferation of pituitary progenitors. Mutations in GLI2 have been found in patients with holoprosencephaly both with and without pituitary deficiencies [49]. Septooptic dysplasia (de Morsier syndrome) is a heterogeneous syndrome. Diagnosis relies upon the presence of two of the three following features: optic nerve hypoplasia, midline forebrain defects (agenesis of the corpus callosum and absent septum pellucidum), and pituitary hypoplasia. Pituitary insufficiency is variable in this condition. Approximately 30% of patients have all three features, with hypopituitarism evident in approximately 60%, and in 60% with absent septum pellucidum. Optic nerve abnormalities may be unilateral or bilateral, and this may be the initial presenting feature. Neurological symptoms are common, ranging from focal deficits to developmental delay. Hypopituitarism ranges from isolated GH deficiency to panhypopituitarism. Gonadotrophin secretion may be retained. Pituitary deficiencies may develop over time and usually do not include posterior pituitary deficits. The etiology is multifactorial, with cases usually being sporadic, and multiple factors including genetic and environmental exposures being implicated.

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Mutations in HESX1 and SOX2 have been implicated, but the majority of cases do not have an identifiable mutation [67]. Prader
Willi syndrome is a genetic disease characterized by hypotonia, short stature, hyperphagia, mild mental retardation, obesity, and hypogonadotrophic hypogonadism. The defects indicate a hypothalamic source for dysfunction in growth, sexual development, hunger, and satiety. Details regarding Prader
Willi syndrome are addressed in Chapter 9, The Hypothalamus. Kallmann syndrome is an X-linked recessive syndrome caused by mutations in the KAL1 gene on the Xp22.3 region of the X chromosome coding for anosmin-1. The syndrome consists of hypogonadotrophic hypogonadism accompanied by one or more congenital abnormalities including anosmia, midline facial anomalies, renal agenesis, neurologic abnormalities, deafness, and color blindness. Several other mutations have been defined that lead to hypogonadotrophic hypogonadism including fibroblast growth factor receptor 1, PROKR2, GnRH receptor, and CHD7 [47]. Rarely, mutations in FEZF1, NROB1, NSMF, WDR11, SEMA3A, HS6ST, and SOX10 have been associated with central gonadal deficiencies [47]. Traumatic Brain Injury The incidence of posttraumatic hypopituitarism was thought to be rare until formal studies demonstrated the high prevalence after head trauma [68,69]. In 2000, a retrospective review characterized 367 patients with hypopituitarism following traumatic brain injury (TBI) [69]. Patients in this retrospective study suffered from deficiencies in all pituitary axes. Since then, a number of prospective and cross-sectional studies have examined the effects of TBI on pituitary function. Disparate methods of assessment of pituitary function, degrees of TBI, and varying definitions of posttraumatic hypopituitarism have contributed to a wide variation in incidence and prevalence of hypopituitarism following TBI [70]. While hypopituitarism has been shown to be a consequence of TBI, the incidence, methods of testing, and timing of testing are controversial [68,71]. Hypopituitarism results from local trauma to the vasculature supplying the anterior pituitary. The major blood supply is from the hypophyseal portal circulation. The inferior hypophyseal artery branches off from the internal carotid artery, supplying a small portion of the adenohypophysis and posterior pituitary. Edema, hemorrhage, increased cranial pressure, and skull fracture can compress the normal pituitary and vasculature, causing damage. Mechanical injury to the stalk and infundibulum also may contribute to insufficient pituitary function. Hypopituitarism may manifest in the acute setting following head trauma, with assessment and treatment of ACTH deficiency and

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diabetes insipidus of paramount importance. After recovery, patients may have persistent deficiencies, or may develop late onset of pituitary deficiencies during the first year after trauma. A systematic review of hypopituitarism following TBI demonstrated a prevalence of 15
50% in patients with a history of head trauma, and a prevalence of 38
55% in patients with subarachnoid hemorrhage [72]. A total of 809 patients with TBI were studied, with GH deficiency seen in 6
33%, gonadotrophin deficiency in 2
20%, ACTH deficiency in 0
19%, TSH deficiency in 1
10%, and multiple deficiencies seen in 4
12% [72]. With the number of hospitalizations and deaths from traumatic brain injuries occurring each year totaling approximately 180
250 persons per 100,000 population, it is clear that a large segment of the population has either been overlooked in the diagnosis and treatment of hypopituitarism or testing of deficiencies in these patients overestimates the incidence of hypopituitarism [71,73,74]. In either case, increased awareness is necessary at the primary care level and in subspecialty practice to fully address this cause of hypopituitarism. Additionally, further prospective studies are needed to elaborate the optimal timing of assessment and benefit of replacement in these patients. Empty Sella Syndrome Replacement of sellar contents with cerebrospinal fluid (CSF) can impact pituitary function. Empty sella syndrome can occur as a primary event, theoretically due to weakness of the diaphragma sella and herniation of the arachnoid space into the sella, or can be secondary to surgery, radiation, or infarction of a preexisting adenoma or other mass. Primary empty sella may be associated with increasing intracranial pressure. The appearance of an empty sella on MRI usually consists of either a complete or partial filling of the sellar space with CSF. Pituitary tissue may be visibly compressed against the floor of the sella. In most circumstances, an empty sella is an incidental finding with normal pituitary function. However, one large case series reported endocrine abnormalities in approximately 20% of patients with partial or complete empty sella [75]. The most common endocrine abnormality reported was hyperprolactinemia (10%) and panhypopituitarism was present in 4%. Isolated GH deficiency was noted in 4% of patients.

Functional Causes of Pituitary Failure Multiple disorders associated with functional disruption of pituitary hormone secretion are usually limited to an isolated pituitary hormone and are generally

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reversible when the underlying cause is corrected. Critical illness can have a profound impact on pituitary function and lead to low levels of multiple pituitary hormones and target hormones. These abnormalities, as well as other functional causes of pituitary failure, are covered in detail in other chapters of this book. Functional Central Adrenal Insufficiency Central adrenal insufficiency due to suppression of the hypothalamic
pituitary
adrenal (HPA) axis after glucocorticoid administration is the most common cause of adrenal insufficiency. The dose and duration of therapy are directly associated with risk for adrenal insufficiency once the treatment is stopped. Glucocorticoid administration suppresses both ACTH and CRH secretion. This results in eventual adrenal cortex atrophy and failure to maintain normal cortisol levels. Multiple sources of exogenous glucocorticoids have been implicated in causing adrenal insufficiency including oral, inhaled, intranasal, topical, and intraarticular injections [76]. Concomitant retroviral therapy for HIV infection may exacerbate adrenal suppression [77]. Recovery from adrenal insufficiency during glucocorticoid withdrawal can be prolonged with some cases extending as long as 18 months after discontinuing the therapeutic regimen. During this period, replacement doses are given to avoid symptomatic adrenal insufficiency, stress doses are advised during illness or physiologic stress, and medic alert bracelets should be worn. During the recovery period of the HPA axis, corticotroph activity recovers prior to adequate adrenal cortex recovery. Periodic assessment of adrenal activity with synthetic ACTH stimulation, coordinated with appropriate temporary holding of replacement glucocorticoids, will assist in the management of adrenal insufficiency and establish HPA axis recovery. In addition to exogenous glucocorticoid therapy, longstanding endogenous glucocorticoid excess has a suppressive effect on the normal secretion of CRH, pituitary corticotroph activity, and ACTH secretion. Cure of Cushing’s syndrome from an adrenal source or an ACTH-dependent source can render the patient adrenally insufficient. Similar to assessment and treatment of patients with adrenal insufficiency due to exogenous steroid excess, patients recovering from endogenous glucocorticoid excess should be treated with replacement glucocorticoids, given stress doses when appropriate, and advised to wear a medic alert bracelet. Recovery of normal adrenal function can be months after successful treatment of endogenous Cushing’s syndrome. Treatment with megestrol acetate, a medication used primarily to increase appetite in patients with

wasting and failure to maintain normal weight, can cause adrenal insufficiency when withdrawn. Megestrol acetate is a progestin with glucocorticoid activity, and while symptoms of adrenal insufficiency are rare during therapy, treatment with stress doses of glucocorticoids is necessary in times of physiologic stress to avoid adrenal crisis. Functional Central Hypothyroidism Central hypothyroidism occurs in a few settings due to functional causes that are reversible with time. Recovery of thyrotroph activity after longstanding hyperthyroidism occurs in both exogenous administration of thyroid hormone and after cure of endogenous hyperthyroidism. Treatment of hyperthyroid states with antithyroid drugs, radioactive iodine, and surgery may render the patient hypothyroid for a number of weeks. Prolonged exogenous thyroid hormone treatment of thyroid nodules, goiter, or thyroid cancer, with the intent to suppress TSH, can lead to central hypothyroidism for a short duration after discontinuation of therapy. In addition to suppression by thyroid hormone, other medications and hormones can have suppressive effects on TSH secretion. Dopamine, glucocorticoids, somatostatin analogues, rexinoids, and oxcarbazepine have all been reported to suppress TSH secretion [78,79]. These medications rarely lead to clinically significant or prolonged hypothyroidism. Nonthyroidal illness may also have a transient, nonclinically significant phase of hypothyroidism, which recovers during the recovery phase of the nonthyroidal illness. Functional Hypogonadotrophic Hypogonadism Failure of the gonadal axis can result from functional defects that occur in the absence of structural or genetic causes. In females, a common cause of amenorrhea is referred to as hypothalamic amenorrhea or functional amenorrhea. Functional amenorrhea may result from weight loss, hypocaloric diet, exercise, or stress. Similarly in some, but not all men, dietary restriction or extreme exercise may cause a hypogonadotrophic hypogonadism [80]. Endocrine abnormalities associated with anorexia nervosa are discussed in Chapter 9, The Hypothalamus. Hyperprolactinemia causes impairment of the gonadal axis in both men and women. Any cause of hyperprolactinemia can interfere with GnRH pulse generation in the hypothalamus via a short feedback loop causing decreased gonadotrophin secretion and a reduction in sex steroid secretion. Treatment of hyperprolactinemia results in normalization of the gonadal axis in most cases, with resolution of the hypogonadotrophic hypogonadism.

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Prolonged use of testosterone can result in suppression of the gonadal axis with variable time to recover after cessation of therapy. Testosterone can also have a suppressive effect on residual spermatogenesis during treatment. Similarly, GnRH analogues, commonly used in treatment of prostate cancer, results in suppression of gonadotroph secretion and hypogonadism, which may persist after completion of therapy. Obesity is associated with lower levels of testosterone in men. Hypogonadism in obesity is predominantly primary, however in cases of morbid obesity the etiology can be central in origin, with laboratory findings consistent with hypogonadotrophic hypogonadism [81]. Functional Growth Hormone Deficiency Multiple factors negatively impact GH secretion, with obesity and visceral adiposity being the most common causes of low levels of GH, both during 24-hour sampling and after stimulation with secretagogues [82]. Low levels of GH observed in obese individuals improve with weight loss, either from diet and exercise or from bariatric surgery. Children can have transient growth failure due to nonGH endocrine abnormalities, psychosocial causes, and idiopathic isolated GH deficiencies. GH secretion is impaired in the setting of hypothyroidism, Cushing’s syndrome, delayed puberty, and isolated ACTH deficiency. Approximately 40% of children diagnosed with idiopathic GH deficiency in childhood are found to be GH-sufficient when retested as adults. Psychosocial dwarfism is covered in Chapter 9, The Hypothalamus.

CLINICAL MANIFESTATIONS The clinical manifestations of anterior pituitary failure are variable depending upon the age and gender of onset, the pituitary axes affected, and the underlying disease. The presentation and findings associated with each individual pituitary hormone will be described here, with the associated manifestations specific to gender and age.

Manifestations of Secondary Adrenal Insufficiency Symptoms of secondary adrenal insufficiency can present insidiously with a gradual onset or abruptly with symptoms of adrenal crisis [83]. Chronic symptoms are largely nonspecific and consist of weakness, lethargy, malaise, nausea or abdominal pain, loss of appetite, arthralgias, and myalgias. Presenting features

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are often associated with hypoglycemia, hypotension, or hyponatremia. Hypoglycemia stems from impaired gluconeogenesis and hyponatremia from SIADH associated with low levels of cortisol and the impaired excretion of free water with adrenal insufficiency. Adrenal crisis usually presents with hypotension in the setting of concomitant illness and can be fatal if untreated. Presentation of secondary insufficiency is similar to that of primary adrenal insufficiency with a few exceptions that are characteristic of ACTH deficiency with intact mineralocorticoid secretion. Unique to secondary adrenal insufficiency is the sparing of aldosterone secretion due to the preservation of renin and angiotensin control of aldosterone production and secretion by the adrenal cortex. With mineralocorticoid activity, severe hypotension is rare unless acute stress is present. Hyperkalemia, volume depletion, and dehydration are not usually part of the clinical presentation. Secondly, the characteristic pigmentation of primary adrenal insufficiency is absent in ACTH deficiency due to the absence of the increase in POMC-derived peptides. In fact, due to a deficiency of melanocytestimulating hormone, patients with prolonged secondary adrenal insufficiency may be pale and experience diminished capacity to tan after exposure to sunlight. Secondary adrenal insufficiency can be partial in its impairment, and symptoms may only manifest in times of physiologic stress. Symptomatic manifestations may be slow and insidious, especially if the impairment is not noticeably progressive. Patients presenting with acute ACTH deficiency may become symptomatic from either acute loss of ACTH, as in pituitary apoplexy, or from a failure to increase exogenous steroid dosing in the face of acute physiologic stress. Cardiovascular collapse can be fatal but can be responsive to steroid administration, if administered promptly. Patents who present with secondary adrenal insufficiency usually have signs and symptoms of the underlying cause or concomitant findings suggestive of central ACTH deficiency. They may have a history of glucocorticoid use and may appear cushingoid. Loss of other pituitary function may be present, especially the presence of central hypothyroidism. Patients presenting with endocrine active pituitary tumors, nonfunctioning pituitary tumors, other sellar masses, or signs of congenital malformations and midline defects should be evaluated for adrenal insufficiency. Laboratory evaluation may reveal mild anemia, neutropenia, hypoglycemia, or hyponatremia. Adrenal Androgen Insufficiency Hypopituitarism and secondary adrenal insufficiency may result in low serum levels of adrenal androgens,

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including androstenedione, DHEA, DHEA-S, and testosterone. Absence of adrenal androgens is of minimal consequence to males who retain testosterone secretion or are treated with testosterone replacement. Women who lack adrenal androgens may exhibit hair loss, unexplained fatigue, depressed mood, reduced sense of wellbeing, reduced stamina, and loss of libido [84].

Manifestations of Thyrotrophin Deficiency Clinical features of central hypothyroidism are virtually indistinguishable from that of primary hypothyroidism [85]. Fatigue, lethargy, cold intolerance, constipation, dry skin, brittle hair, bradycardia, facial edema, and normocytic anemia are common findings in patients with hypothyroidism. Physical examination findings may include thinning of the lateral portion of the eyebrows, a sallow appearance to the skin, and a delayed relaxation phase of deep tendon reflexes. In addition to characteristic thyroid function studies, laboratory evaluation may reveal lipid abnormalities with a high total cholesterol and low- high-density lipoprotein (HDL) cholesterol. The presentation is variable, with some patients being asymptomatic or unaware of symptoms and some patients having symptoms of profound hypothyroidism. Goiter is not a feature of secondary hypothyroidism.

Manifestations of Hypogonadotrophic Hypogonadism

FIGURE 10.1

Clinical staging of male genital development. Tanner stages 1
5 depicted. Used with permission [98].

Adult Males The presenting features of men with hypogonadotrophic hypogonadism are variable and do not necessarily correlate to levels of circulating testosterone [86]. Adult males presenting with new-onset hypogonadotrophic hypogonadism may present with impaired libido, impotence, decreased body and facial hair, decreased muscle mass, osteopenia or osteoporosis, and atrophy of the testes. Laboratory findings may include hypercholesterolemia and decreased sperm count. Prepubertal Males Manifestations of hypogonadism prior to puberty may go unnoticed until puberty is delayed, defined as the absence of secondary sexual characteristics at an age more than two standard deviations above the population mean for the onset of puberty. The clinical staging of puberty has been established by James Tanner and is widely used (Figs. 10.1
10.4) [88,89]. The average age for the first signs of puberty in boys is 14, with increase in testicular size being the first sign. Boys with prepubertal hypogonadism have a small penis, small testes

FIGURE 10.2 Clinical staging of male pubic hair development. Tanner stages 2
5 depicted. Used with permission [98].

and prostate, and scant pubic and axillary hair. They may exhibit long arms and legs with a eunicoid habitus if epiphyseal closure has been delayed. Their voices may remain high in pitch and they may have gynecomastia.

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FIGURE 10.4 Clinical staging of female pubic hair development. Tanner stages 2
5 depicted. Used with Permission [87].

method to accurately differentiate between these two entities [90]. Serial measurements of height, weight, arm span, and testicular size may help clarify the diagnosis over a few years. Stimulation testing can be performed but often does not clearly distinguish between these two diagnoses. Neonatal Males Infants presenting with hypogonadotrophic hypogonadism may have the presence of a micropenis and cryptorchidism [91]. It is critical to assess these patients for concomitant pituitary deficiencies, as GH and ACTH deficiencies are associated with a high mortality risk due to hypoglycemia or cortisol deficiency [92].

Female Hypogonadotrophic Hypogonadism FIGURE 10.3 Clinical staging of female pubertal breast development. Tanner stages 1
5 depicted. Used with permission [87].

Males with pubertal delay due to congenital loss of GnRH, LH, or FSH secretion may exhibit congenital abnormalities consistent with abnormal development of the hypothalamic
pituitary axis. Patients may have midline defects, cryptorchidism, and cleft lip or palate. Headache, visual disturbance, anosmia, or mental retardation suggest a congenital gonadotrophin deficiency. Hypogonadotrophic hypogonadism must be differentiated from constitutional pubertal delay, and a family history may help in making this diagnosis. However, other than expectant observation, there is no

Adult Females (Secondary Amenorrhea) The hallmark of hypogonadotrophic hypogonadism occurring after puberty in premenopausal females is amenorrhea occurring for at least 3 consecutive months. Common causes of secondary amenorrhea in women are pregnancy, functional hypothalamic amenorrhea, hyperprolactinemia, and polycystic ovarian syndrome. Women with hypogonadotrophic hypogonadism are infertile, since they lack the hormones responsible for the usual regulation of follicle stimulation and ovulation. Decreased estrogen production results in symptoms similar to menopause: hypoestrogenic symptoms such as osteoporosis, decreased vaginal secretion, dyspareunia, decreased libido, and atrophy of breast tissue. Postmenopausal women with hypogonadotrophic hypogonadism do not manifest clinical symptoms due to deficiencies of

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gonadotrophins, but may manifest symptoms due to other pituitary hormone deficiencies. Primary Amenorrhea/Pubertal Delay Hypogonadotrophic hypogonadism in young females may manifest itself as an absence of menses and a complete lack of development of secondary sexual characteristics, with or without normal growth. Staging of pubertal development in girls has been established by Tanner (Figs. 10.3 and 10.4) [89]. Occasionally, patients may have partial pubertal development with pubertal arrest; however, complete absence of pubertal development is more common.

Growth Hormone Deficiency Adult-Onset GHD The features of GH deficiency in the adult include abnormal body composition with increased visceral adiposity and decreased lean muscle mass. Patients may exhibit psychological impairment with reduced energy, social isolation, emotional lability, anxiety, and depressed mood. They are typically overweight with increased central adiposity. Their skin may be cool to the touch, dry, and thin. Patients have reduced muscle strength, reduction in exercise performance, and may exhibit a depressed affect [93]. Unfortunately, none of these symptoms are specific to GH deficiency and there is a great deal of symptomatic overlap with the general population.

Inflammatory markers such as CRP, interleukin-6 (IL-6), and homocysteine, which are associated with increased cardiovascular risk, are elevated in subjects with GHD [100,101], while left ventricular function appears to be diminished in GHD subjects [87]. Childhood-onset GHD Patients with congenital or childhood-onset GHD have short stature and fail to grow at a normal rate. Children with congenital GHD are born with normal height and weight, but serial measurements of height and weight demonstrated a failure to progress normally. The growth curve of acquired GH deficiency is characteristic of growth failure with normal growth for a period of time and progressive fall in height away from the mean. Height tends to be affected more than weight and they appear overweight for their height, with a propensity for central weight gain in the abdomen and chest. Arm span is normal and they are normally proportioned, in regard to upper and lower segment ratios. Dentition may be delayed, and the voice high-pitched. When GHD is concomitant with ACTH deficiency, children may present with hypoglycemia. This is most commonly seen in abnormal development of the anterior pituitary or with idiopathic hypopituitarism. Midline developmental abnormalities such as cleft lip or palate, septooptic dysplasia, a single incisor, and congenital roaming nystagmus are associated with congenital GHD.

Prolactin Deficiency

CARDIOVASCULAR RISK

Retrospective studies have demonstrated that patients with hypopituitarism who have not been treated for GHD have a higher mortality than patients receiving conventional replacement therapy [94], largely due to cerebrovascular and cardiovascular disease [95]. Lipid profiles of GHD subjects are variable in reported retrospective studies and database reviews [96]. Subjects with GHD have higher total cholesterol levels and higher low-density lipoprotein (LDL) levels [97]. Triglyceride levels are elevated [98]. Measurements of HDL levels in GHD subjects have varied. The severity and duration of GHD may be associated with severity of the cardiovascular risk, as insulin-like growth factor 1 (IGF-1) levels are inversely associated with LDL levels [87]. Some studies have reported higher prevalence of hypertension, while others have not found elevated blood pressures in GHD subjects [87]. The KIMS database reports a prevalence of 15% of GHD adults with hypertension [97]. Other cardiovascular risk factors found to be elevated in patients with GHD include carotid intima media thickness [99]. Higher levels of fibrinogen, PAI-1, and tPA are also reported in GHD subjects [99].

Prolactin deficiency manifests itself solely with the inability to lactate in the postpartum time period. Absence of lactation may portend deficiencies in other pituitary hormones, warranting investigation.

DIAGNOSTIC TESTING The diagnosis of anterior pituitary failure requires the assessment of the integrity of the various stimulatory hormones secreted by the pituitary (Table 10.3). For the assessment of certain axes, dynamic testing is required due to the pulsatility of the pituitary hormones, and in others, dual assessment of the trophic hormone and target gland hormone is sufficient. The selection of the appropriate test can differ depending upon the age of the patient. Over the years, various tests have been developed and have either fallen out of favor due to advances in other testing methodologies, the convenience of other methods of testing, or the discontinuation of key components of the tests themselves.

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TABLE 10.3

Pituitary Hormone Diagnostic Testing

Pituitary axis Test

Method

Expected results

Corticotroph assessment

Basal testing (morning, 0700
0900 h)

Normal: serum cortisol level $ 18 μg/dL

Serum cortisol

Insufficient: ,3 μg/dL Indeterminate: 3
18 μg/dL

ITT

Overnight metyrapone test

Insulin (0.05
0.15 U/kg IV)

Symptomatic hypoglycemia, blood glucose ,40 mg/dL required for interpretation.

Serum measurements of cortisol at 0, 30, 60 min

Normal: peak cortisol $ 18 μg/dL

Metyrapone 30 mg/kg administered at midnight

Serum cortisol ,7 μg/dL confirms enzymatic blockade

Serum measurements of cortisol and 11-deoxycortisol at 0800 the following morning

Normal: serum 11-deoxycortisol .7 μg/dL

Insufficient: serum cortisol level ,18 μg/dL

Insufficient: serum 11-deoxycortisol ,7 μg/dL Caveat: adrenal crisis possible is basal cortisol # 7 μg/kg

Synthetic 1
24 ACTH (250 μg IV or IM or 1 μg IV)

Normal: serum cortisol level $ 18 μg/dL

Serum measurements of cortisol at 0, 30, 60 min

Caveat: false-negative results possible in acute or subacute time frame

Thyroid-stimulating hormone (TSH), free thyroxine index (FTI), or free thyroxine (fT4)

Basal testing

Normal: normal serum levels of fT4 or FTI, combined with normal serum TSH

Gonadotroph assessment (males)

Serum testosterone, luteinizing hormone (LH), and folliclestimulating hormone (FSH)

Basal testing (morning, 0700
0900 h)

Gonadotroph assessment (females)

Serum estradiol, LH, FSH

Basal testing

ACTH stimulation test

Thyrotroph assessment

Insufficient: serum cortisol level , 18 μg/dL

Central hypothyroidism: low levels of fT4 or FTI; low, normal, or high serum level of TSH Normal: serum testosterone level within the normal range, normal LH, FSH Hypogonadotropic hypogonadism: serum testosterone level below lower limits of normal, LH and FSH low or normal Normal: results are variable in menstruating women Normal postmenopausal women: elevated LH, FSH; low serum estradiol Normal prepubertal females: low LH, FSH, estradiol Gonadotroph failure: low levels of LH, FSH, estradiol (primary amenorrhea, secondary central amenorrhea, postmenopausal gonadotroph deficiency)

Somatotroph assessment

Serum IGF-I, IGFBP-3 (children)

Basal testing

Normal: serum IGF-1 within the age- and genderadjusted normal range Serum IGFBP-3 within the age- and gender-adjusted normal range Growth hormone (GH)-deficient: serum IGF-1 levels below 84 μg/dL (assay specific) with three or more additional pituitary deficits Serum IGFBP-3 below lower limits of normal

ITT

Insulin (0.05
0.15 U/kg IV)

Symptomatic hypoglycemia, blood glucose ,40 mg/dL required for interpretation

Serum measurements of GH at 0, 30, 60 min

Normal: peak GH response .5.1 μg/L Severe GH deficiency: peak GH ,3 μg/L GH-deficient: peak GH # 5.1 μg/L (Continued)

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(Continued)

Pituitary axis Test GHRH
arginine stimulation test

Method

Expected results

GHRH (1 μg/kg IV bolus); arginine 30 g IV infusion over 30 min

Responses variable

Serum measurement of GH at 0, 30, 60, 90, 120, 150, 180 min

Normal: peak GH $ 9 μg/L (ideal body weight 6 15%) GH deficiency: BMI , 25: peak GH # 11.5 μg/L BMI 25
30: peak GH # 8.0 μg/L BMI $ 30: peak GH # 4.2 μg/L

Glucagon stimulation test

Glucagon 1 mg IM; 1.5 mg IM if weight .90 kg

Normal: peak GH .3 μg/L GH deficiency: peak GH # 3 μg/L

Serum measurement of GH and glucose at 0, 30, 60, 90, 120, 150, 180, 210, and 240 min

Assay Variability In general, assessment of pituitary function is reliant on the measurement of hormones in serum. Distinguishing normal responses to stimulation testing, or normal basal serum levels from those of patients with pituitary insufficiency is also dependent upon establishing generalizable cutoff points that can be applied to clinical practice. It is well understood that as the field of clinical biochemistry advances, the methodology for assays will evolve. Techniques change and require validation, and therefore, cutoff points established under prior conditions require reevaluation. A great deal of variability is inherent in the various methodologies employed by laboratories to measure hormones and establish normative data. It should therefore be recognized that cutoff points established with one technique might not apply to other techniques. The interassay and intraassay variation of laboratory measurement is largely unrecognized, but various quality assessment studies have demonstrated certain vulnerabilities in our generalized use of cutoff points established with one technique [102
105]. The focus of this section is testing of anterior pituitary function. However, diagnostic testing of genetic causes of combined pituitary hormonal deficits is commercially available for testing POU1F1 and PROP1 genetic defects. Specific recommendations for testing other pituitary deficiencies in certain circumstances (i.e., TBI) are addressed in the following section.

Assessment of Pituitary Function Corticotroph Assessment An intact HPA axis is reflected by normal secretion of cortisol by the adrenal cortex. Cortisol secretion is

pulsatile and follows a diurnal pattern with serum levels highest in the early morning and lowest at midnight. Due to the pulsatile secretion pattern, there is a great deal of overlap in serum cortisol levels between patients with adrenal insufficiency and intact HPA function. It is for this reason that dynamic testing, either with direct stimulation of the adrenal cortex or corticotrophs or through an integrated stimulus such as hypoglycemia, is usually necessary to assess corticotroph function. BASAL TESTING

Laboratory measurement of serum cortisol reflects total cortisol levels, and as such, is subject to fluctuations in cortisol binding globulin. Measurement of random basal serum cortisol or ACTH is generally not helpful in the assessment of adrenal insufficiency due to the pulsatile nature of secretion and the extensive overlap of values obtained from healthy control subjects. When performed, they are most helpful when obtained in the morning (0700
0900 hours). An intact HPA axis can be inferred by a serum cortisol level .18 ng/mL [103]. Serum cortisol levels below 3 ng/mL are highly suggestive of adrenal insufficiency in morning samples, but can be found in normal subjects during other times of the day. Values between these levels usually require dynamic testing for a definitive diagnosis. DYNAMIC TESTING INSULIN TOLERANCE TESTING The insulin tolerance test (ITT) is widely regarded as the gold standard for testing for adrenal insufficiency. It is particularly useful for detecting secondary adrenal insufficiency. Intravenous insulin is administered (0.1 units/kg) with the intention of inducing hypoglycemia. Symptomatic hypoglycemia, with blood glucose values below

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40 mg/dL, is required to evoke a reliable central stress response with activation of the HPA axis. Higher doses may be required in patients with insulin resistance or acromegaly [106]. The test is usually contraindicated in patients older than 60 years and in those with a risk of seizures, or suspicion or history of active cardiovascular disease. Accordingly, the procedure requires close supervision throughout the duration of testing and should be performed in specialized centers with precautions to avoid consequences of potential adrenal crisis. Measurements are obtained at 0, 30, and 60 minutes with serum cortisol levels greater than 18 ng/mL indicating a normal response [106]. The ITT elicits few false negatives and some false positives [107]. The test is not frequently performed, but remains a valuable tool in the assessment of adrenal insufficiency, especially following recent pituitary surgery [103,108]. OVERNIGHT METYRAPONE TEST Metyrapone inhibits the enzyme 11β-hydroxylase, preventing the conversion of 11-deoxycortisol to cortisol in the adrenal cortex. For the overnight metyrapone test, 30 mg/kg of metyrapone is administered at midnight with measurement of cortisol and 11-deoxycortisol at 0800 the following morning. Serum cortisol levels below 7 μg/ dL confirm adequate enzyme inhibition [103,109]. After metyrapone, the normal response is a significant rise in serum 11-deoxycortisol. Serum levels below 7 μg/dL are consistent with adrenal insufficiency [103,109]. The metyrapone test does not induce hypoglycemia as the ITT does, but relies on an intact HPA axis to elicit the response to abrupt lowering of cortisol levels. The test can be administered on an outpatient basis. However, there is a risk for adrenal crisis, and metyrapone should not be administered to subjects with baseline morning cortisol values less than 7 μg/ dL. The metyrapone test is not frequently performed due to the limited availability of the drug, but is an effective method for testing the integrity of the entire HPA axis. ACTH STIMULATION TESTING

The most common test to assess adrenal insufficiency is the administration of synthetic ACTH (1
24 ACTH 250 μg). Synthetic preparations maintain the full potency of endogenous ACTH. The injection can be administered intravenously or intramuscularly, and both methods give equivalent results [103,108]. Injection of 250 μg of synthetic ACTH leads to supraphysiological levels approximately 1000-fold higher than the normal physiological peak, which provides ample stimulation of the adrenal cortex [103]. Baseline serum cortisol levels are obtained, and measurements of the serum cortisol response at 30 and 60 minutes are subsequently obtained. The test can be performed at any time of day

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and need not be done in the fasting state. This short test has largely replaced an 8-hour infusion test, due to its ease of use and similar cortisol responses. Peak serum cortisol levels above 18 ng/dL are regarded as a sufficient response to stimulation, indicating normal adrenal reserve. The ACTH stimulation is especially useful for diagnosing cases of chronic adrenal insufficiency or primary adrenal insufficiency. The ACTH stimulation test is also helpful in the diagnosis of secondary adrenal insufficiency because of adrenal atrophy caused by long-term deficiency of ACTH. The duration and extent of ACTH deficiency are directly related to the degree of adrenal atrophy and the utility of the ACTH stimulation test in making the diagnosis of secondary adrenal insufficiency. Patients with partial ACTH deficiency or complete ACTH deficiency due to recent trauma or surgery may have false-negative test results with the use of ACTH stimulation. The time necessary to develop adrenal atrophy in the absence of ACTH is variable, and the ACTH stimulation test is not recommended within 2 weeks of a known insult to the pituitary or hypothalamus. A low-dose ACTH stimulation test (1
24 ACTH 1 μg) has been developed to address the issue of poor sensitivity in the standard ACTH stimulation test. A 1-μg dose is not commercially prepared and must be diluted by injecting a 250-μg dose into 249 mL of normal saline. One milliliter is withdrawn from the mixture and administered intravenously. Initial studies demonstrated improved sensitivity for the diagnosis of secondary adrenal insufficiency [110,111]. Performance of the test may be inaccurate due to techniques involving dilution and administration of the low-dose test. Although the dose is reduced from the standard 250-μg dose, the 1-μg dose still delivers a supraphysiologic concentration to the adrenal cortex and similar results of the two concentrations have been demonstrated by multiple investigators [108]. CRH STIMULATION The CRH stimulation test is performed by administration of ovine or human corticotrophin-releasing hormone (100 μg IV) with subsequent measurement of cortisol and ACTH at 0, 15, 30, 45, 60, 90, and 120 minutes. Normal response is a two- to fourfold increase in ACTH from baseline, peaking at 15
30 minutes. Peak cortisol levels follow at 60 minutes after CRH injection. COMPARISON OF TESTS

The ITT has generally been regarded as the gold standard for the diagnosis of adrenal insufficiency and it has been validated against the cortisol response to surgical stress. When comparing the ITT to ACTH stimulation tests, performance is excellent for the diagnosis

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of primary adrenal insufficiency [108]. Discrepancies have been found, however, when comparing the ITT to the ACTH stimulation test in secondary insufficiency. Results are often concordant, but investigators have found a number of subjects who have normal ACTH stimulated responses who later fail the ITT. While the ITT is considered the gold standard, it is not infallible and the majority of conflicting results often are in subjects whose results are near established cutoff points for diagnosis [103]. A meta-analysis explored the utility of the 250-μg ACTH stimulation test for the diagnosis of primary adrenal insufficiency and secondary adrenal insufficiency and the 1-μg ACTH stimulation test for secondary adrenal insufficiency. Using receiver operator characteristics (ROC) analysis, the standard ACTH stimulation test performed well in the diagnosis of primary adrenal insufficiency with a sensitivity of 97% at a specificity of 95%. Sensitivities for the 250-μg test and the 1-μg test in the diagnosis of secondary adrenal insufficiency were 57% and 61% for summary ROC curves, set at a specificity of 95%. The standard ACTH and low-dose ACTH tests performed similar to each other in the diagnosis of secondary insufficiency [108]. A later meta-analysis of patient-level data found superiority of the low-dose ACTH stimulation test when assay methodology, sample size, and control subjects were taken into consideration [112]. Differences in methodology of the two analyses, and selection of published papers may account for the different conclusions and may indicate the usefulness of the low-dose test. The CRH test has been compared to basal cortisol, low-dose ACTH stimulation test, standard ACTH stimulation, and ITT for assessment of adrenal function in patients with suspected HPA insufficiency [113,114]. One study determined that serum cortisol levels # 13 μg/dL had a 96% specificity and a 76% sensitivity [113]. Basal cortisol levels in this study had 100% sensitivity and 61% specificity for a cutoff point of 10.3 μg/dL. Cortisol results after CRH stimulation in normal subjects have been highly variable, but the CRH test does seem to agree with results of ITT in patients with severe adrenal insufficiency [114]. Early use of the CRH test after transsphenoidal surgery has been applied to detect adrenal insufficiency, but requires retesting for confirmation as up to 30% of patients may be misclassified [115]. TESTING AFTER PITUITARY SURGERY

The ITT is also regarded as the gold standard for assessing adrenal reserve after recent pituitary surgery. The ACTH stimulation test is being performed in some centers due to its ease of performance [116]. A falsenegative rate of 3% in postsurgical patients has been demonstrated with a predictive value of 97% [117].

However, others have found higher false-negative rates, especially when performed within 1 week of surgery [107]. During the first 4
6 weeks of the postoperative period, ACTH testing has been found to be more unreliable and should be utilized after the immediate postoperative period has passed. DIAGNOSIS OF ADRENAL ANDROGEN DEFICIENCY IN WOMEN

Making the diagnosis of androgen insufficiency in women rests on three essential criteria [118]. First, the woman must present with clear evidence of clinical symptoms compatible with androgen deficiency: diminished sense of wellbeing, persistent fatigue, and changes in sexual function. Secondly, effects of estrogen deficiency should be isolated from effects of androgen deficiency by only considering the diagnosis in the setting of adequate serum estrogen levels. This can be accomplished by endogenous production or replacement. Thirdly, free testosterone values should be at or below the lowest quartile of the normal range for women in their reproductive years, bearing in mind that there is not a sufficiently sensitive assay or threshold for women with androgen insensitivity [118]. Thyrotroph Assessment The assessment of thyrotroph function is measured with basal sampling of TSH and concomitant measurement of free thyroxine (fT4). The diagnosis is made when TSH levels are inappropriately low or normal in conjunction with low levels of fT4, and the appropriate clinical findings suggestive of central hypothyroidism are present. TSH testing is widely accepted as the test of choice for the initial evaluation of hypothyroidism in the general population, and it is useful in the diagnosis of primary hypothyroidism. However, one cannot make the diagnosis of central hypothyroidism with the TSH alone, and therefore clinical suspicion must prompt the evaluation of the peripheral hormone (fT4). TSH values are reportedly normal in 84% of subjects with central hypothyroidism, elevated in 8% and low in only 8% of patients with central hypothyroidism [85]. Serum levels of fT4 provide the highest accuracy for the diagnosis of central hypothyroidism, whereas other hormonal measurements are neither sensitive nor specific enough for diagnosis [119,120]. Combined pituitary hormone deficiencies may affect the diagnostic workup of central hypothyroidism, with GH deficiency altering thyroid hormone metabolism [121]. TRH TESTING

Dynamic testing of thyrotroph reserve may be helpful in differentiating hypothalamic causes of central hypothyroidism from pituitary causes. Thyrotropinreleasing hormone (TRH) is no longer commercially

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available in the United States and its diagnostic utility in thyroid disease has largely been supplanted by the current sensitive TSH assay. The test is performed by measuring TSH, fT4, and free triiodothyronine (fT3) after an intravenous injection of TRH. The responses provide information regarding the integrity of the thyrotroph cells and the biologic activity of the TSH secreted by the pituitary. Dynamic testing is rarely indicated as it does not improve the diagnostic reliability above standard nondynamic testing in patients with pituitary adenomas [122]. It may be helpful in the evaluation of patients with low-normal serum TSH and low-normal serum fT4 levels in the setting of known hypothalamic
pituitary disease, but the limited availability of TRH limits the practical application of such testing [123]. Gonadotroph Assessment ADULT MALES

The initial biochemical assessment of central or hypogonadotrophic hypogonadism in adult males is performed by the measurement of morning levels of testosterone in conjunction with measurement of the gonadotrophins: LH and FSH. Testosterone levels vary widely due to circadian rhythms, seasonal variation, and episodic secretion. Results may be altered by concurrent illness, medications, and changes in sex hormone-binding globulin levels. Repeating the measurement of testosterone after initial low testosterone values found is recommended [124]. LH and FSH have short circulatory half-lives in serum, and FSH may yield more accurate results in a single serum sample due to its longer half-life. Pooling of LH in 2
3 samples taken 20
30 minutes apart may improve the accuracy of the specimen [125]. Dynamic testing is usually not necessary in adults, and imaging studies are more practical for the assessment of hypothalamic and pituitary disease in adults [125]. ADOLESCENT MALES

In addition to the assessment of gonadotrophins and testosterone, the assessment of hypogonadism in adolescent males may require stimulation testing. GnRH is not currently available in the United States, yet its use and the use of other dynamic tests remain as diagnostic tools in distinguishing constitutional delay of puberty (CDP) from hypogonadotrophic hypogonadism. Basal total testosterone levels of .1.7 nmol/L may suggest CDP [126,127], but further dynamic testing may be necessary. Measurement of a single inhibin B level may be of some use, but its use has not been well validated [128]. As previously discussed, the diagnosis of hypogonadotrophic hypogonadism versus CDP is often not resolved with

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stimulation testing, and the diagnosis is most reliably made with expectant observation. However, the following stimulation tests are discussed for reference. GnRH STIMULATION TESTING GnRH 100 μg is administered intravenously with the measurement of LH and FSH taken at 0, 15, 30, 45, 60, and 90 minutes [125]. Priming with GnRH may be necessary with GnRH delivered via infusion pump every 90 minutes for 36 hours prior to the GnRH stimulation test. Testing can be performed with GnRH analogues with measurement of gonadotrophins at 0 and 4 hours after a single subcutaneous injection [125]. Analogues provide the benefit of longer duration of action and a priming effect with the one injection. Normal response for the classic GnRH test is a rise of LH and FSH with a peak between 15 and 60 minutes. When performed with GnRH priming, the LH peak is lower than the standard GnRH test. Normal response with GnRH analogues is an LH peak 3
4 hours after injection and an FSH peak 3
6 hours after injection. A blunted rise in gonadotrophins is usually seen in pituitary disease or longstanding hypothalamic disease. CLOMIPHENE STIMULATION TEST The administration of clomiphene inhibits estrogen feedback centrally, interrupting the negative feedback of estrogen GnRH release and causing a rise in LH and FSH [125]. The test is cumbersome with 100 mg of clomiphene citrate administered for 5
7 days with sampling of LH and FSH at days 0, 4, 7, and 10. A doubling of LH and a 20
50% rise in FSH indicate a normal hypothalamic
pituitary response. The test does not distinguish between hypothalamic and pituitary disease, nor does it assist in the differentiation of constitutional pubertal delay and hypogonadotrophic hypogonadism. The test is therefore not recommended for differential diagnosis of delayed puberty. hCG STIMULATION TEST The human CG (hCG) stimulation test is utilized to distinguish between testicular agenesis and cryptorchidism in infants and can be used to diagnose hypogonadism versus delayed puberty in peripubertal males. Two different protocols are in use. In pubertal boys, a single dose of hCG (5000 IU intramuscularly) is administered. Serum testosterone is measured at baseline and every 24 hours for 5 days. Other protocols suggest repeated hCG injections, 1000 IU daily for 3 days or 2000 IU daily on days 0 and 2. Testosterone is sampled at 0, 48, and 72 hours. In infants, a flat response to hCG is consistent with testicular agenesis. Increased levels of testosterone suggest undescended testes, which should be localized. In peripubertal males, a 2
3-fold increase in testosterone is seen more commonly in CDP, whereas

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patients with hypogonadotrophic hypogonadism do not have a dramatic rise in testosterone. Studies have shown that testosterone levels above 8 nmol/L are consistent with CDP, whereas values less than 3 are seen in hypogonadotrophic hypogonadism [127,129]. These criteria result in a positive predictive value of 100% and a negative predictive value of 80%. Intermediate values are inconclusive and 29% of these patients remain unclassified after further testing [125]. ADULT FEMALES

The evaluation of gonadotroph function in postmenopausal females entails basal measurement of LH and FSH. As gonadotroph function in postmenopausal females does not involve stimulation of ovarian secretion of estrogen, menstruation, or ovulation, the levels of LH and FSH may be used indirectly to assess the overall functioning of the pituitary when damage is suspected. Normal postmenopausal elevations imply normal pituitary function, and lower levels may indicate pituitary dysfunction. Postmenopausal elevation of gonadotrophins may decline with advancing age and may not be a specific indicator of pituitary dysfunction. The evaluation of premenopausal females who have gone through puberty relies initially on the assessment of regular menses. Regular menstrual cycles imply, but do not guarantee, normal function of the gonadotrophic axis. If secondary amenorrhea is present, a workup of underlying causes is initiated (including measurement of hCG, TSH, LH, FSH, and PRL). Elevated serum FSH levels indicate primary ovarian failure, while normal or low serum levels of LH or FSH indicate a central cause that may be genetic, structural, or functional. A thorough evaluation of underlying causes should be pursued. PRIMARY AMENORRHEA

As with adolescent males with a delay in onset of puberty, females with delayed puberty should undergo an evaluation of the underlying cause. No test will reliably differentiate between CDP and hypogonadotrophic hypogonadism. Expectant observation is the only reliable way to distinguish between these two entities. Once a careful history and physical have been performed, imaging studies and biochemical testing are performed. Testing of pituitary function in females with suspected hypothalamic or pituitary function usually begins with a baseline measurement of LH, FSH, and estradiol. Levels of estradiol, LH, and FSH are typically low in patients with CDP and hypogonadotrophic hypogonadism. GnRH testing is not recommended since it may not reliably distinguish between CDP and GnRH deficiency.

Somatotroph Assessment ADULTS

The diagnosis of GH deficiency in adults relies on biochemical testing, due to the absence of specific signs and symptoms. Further, there are no auxological criteria associated with GHD in adults as there are in children who display a lack of growth. Diagnostic testing should be performed in the appropriate clinical context. Patients who should be considered for testing include those with known hypothalamic
pituitary disease or dysfunction, a history of cranial irradiation, or patients with a history of childhood-onset GH deficiency. Recent consensus guidelines have emphasized the importance of including patients with a history of TBI or subarachnoid hemorrhage [93,130]. In most circumstances, making the diagnosis of GHD in adults requires dynamic stimulation testing. Individuals with known genetic causes of GHD, childhood-onset GHD due to irreversible structural causes of GHD, or GHD due to embryological developmental causes need not be retested if they were found to be GH-deficient in childhood. Patients who have childhood-onset GHD should be retested as adults if they do not have one of these genetic or structural afflictions, since approximately 40% of children with GHD are found to be GH-sufficient when retested as adults [131]. Other patients with adult-onset GH deficiency do not require stimulation testing to make a diagnosis. Patients with multiple pituitary hormone deficits and low serum IGF-1 levels have a high likelihood of having GHD. A review of data from the US Hypopituitary Control and Complications Study (HypoCCS) revealed that of the 817 subjects included in the study at that time, a variety of stimulation tests had been performed on these subjects [132]. The percentage of subjects with severe GH deficiency defined by peak response to stimulation testing was 41%, 67%, 83%, 96%, and 99% for subjects with zero, one, two, three, and four other pituitary hormone deficits, respectively. The positive predictive value of having three or four pituitary hormone deficits (TSH deficiency, ACTH deficiency, gonadotrophins deficiency with LH and FSH considered together, or central diabetes insipidus) was 96% and 99%, respectively. Serum IGF-1 levels, measured by Esoterix Endocrinology Laboratories, had a positive predictive value of 96% for the diagnosis of adult GHD when serum levels were less than 84 μg/L. The presence of either a serum IGF-1 level below 84 μg/L or three or more pituitary hormone deficiencies provided a positive predictive value of 95% for adult GHD, with a sensitivity of 89% and sensitivity of 69%, respectively. When combined, the PPV and specificity increased to 100% when three or more pituitary hormone deficiencies and

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a low serum IGF-1 level were present. The combination of two or more pituitary hormone deficiencies and a low IGF-1 yields a PPV and specificity of 99% [132]. Many clinicians and insurers have accepted these criteria as being sufficient evidence for the diagnosis of GHD, without the need for stimulation testing. Stimulation testing is required for all adults not meeting the above criteria. There are multiple stimulation tests available for use in the diagnosis of GHD. While the ITT had widely been regarded as the test of choice for the diagnosis of GHD in adults [93], the test was rarely performed in the US due to the intensive nature of the test, contraindications for use in some patients, and the potential risks of performing the test. Alternative tests have been used, such as the combined use of GHRH and arginine with success in separating GHD subjects from healthy controls [133]. Cutoff points for diagnosis of GHD vary depending upon the test performed, the assay employed, and multiple physiologic factors influencing the GH response to secretagogues. Known physiologic factors affecting the dynamic testing of GH secretion include gender, body mass index (BMI), visceral adiposity, sex steroid status, and age [134]. Responses to some secretagogues do not appear to attenuate with age [135]. A multicenter study evaluated the sensitivity and specificity of six tests for the diagnosis of GH deficiency in adults [136]. The ITT, combined GHRH
arginine test, combined arginine and L-dopa, arginine alone, L-dopa alone, and serum IGF-1 were compared. Subjects were determined to be GHD based on multiple pituitary hormone deficits, and controls were matched for BMI, gender, age, and estrogen status. Of the six tests performed, the ITT and combination of GHRH and arginine performed with the greatest accuracy. With GHRH in limited supply, alternative tests to the GHRH
arginine and ITT are required. Injection of glucagon elicits pituitary GH secretion, and stimulation testing has been validated against the ITT [137,138]. Comparison of glucagon stimulation testing to the ITT has demonstrated that a cutoff of 3 μg/L provides 100% sensitivity and 100% specificity in diagnosing adults with GH deficiency [138]. Other investigators have found 97% and 88% sensitivity and specificity, respectively, using ROC curve analysis [139]. The standard cutoff point of 3 μg/L may overdiagnose patients with GHD, due to the known influence of BMI and visceral adiposity on GH secretion. Attempts to improve the accuracy of the glucagon stimulation test demonstrated that a cutoff point of 1 μg/L greatly improved sensitivity and specificity [140], which is safe and well tolerated, with no hypoglycemia. Nausea is the most common side effect.

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Alternative tests have been used in the past including arginine alone, L-dopa, clonidine, and the combination of L-dopa and arginine. These tests do not attain adequate accuracy and are not recommended as alternative tests to the ITT or GHRH
arginine stimulation test [130,136]. Promising alternatives include investigational compounds such as synthetic GH secretagogues, GHRP-2 and GHRP-6; however, these secretagogues have been utilized in combination with GHRH, which currently limits their use. THE ITT Hypoglycemia is a potent stimulus for GH secretion. GH levels generally peak 40
90 minutes after insulin administration, and only after a sufficient stimulus of hypoglycemia is attained. The test is performed in the fasting state, and baseline measurements of glucose and GH are obtained. If desired, assessment of the HPA axis can be performed simultaneously, with measurement of cortisol after hypoglycemia is attained. Bedside glucose monitoring is required. Regular insulin is injected at a dose of 0.1 units/kg intravenously. Higher doses of 0.15
0.2 units/kg may be necessary in patients with insulin-resistant states such as obesity, Cushing’s syndrome, or acromegaly. Lower doses (0.05 units/kg) are advisable if panhypopituitarism is suspected. The ITT is contraindicated in patients with ischemic cardiovascular disease, seizure disorders, patients with TBI, and the elderly. Severe GH deficiency has been defined as a peak response to the ITT of 3 μg/L and more recent data demonstrate that this cutoff represents the first percentile of responses in normal lean individuals [93,141]. A cutoff of 5.1 μg/L provides the best sensitivity and specificity by ROC curve analysis [136]. GHRH
ARGININE STIMULATION TEST The combination of GHRH and arginine has been touted as the most appealing alternative to the ITT in the diagnosis of adults with suspected GH deficiency [93,130]. The GHRH
arginine test has become a well-validated test for the diagnosis of GH deficiency; however, GHRH is in limited supply and is currently not available in the United States, making its widespread use challenging. The peak response to the test is clearly higher than those of other provocative tests with normal lean subjects having peaks greater than 9 μg/L [142]. The test is performed by infusing GHRH, 1 μg/kg by IV bolus, followed by a 30 g infusion of arginine over 30 minutes. GH is measured every half hour for 3 hours. Based on ROC curve analysis, a cutoff point of 4.1 μg/L provides 95% sensitivity and 91% specificity [136]. One study provided weight-based cutoff points in three BMI categories: lean (BMI , 25; # 11.5 μg/L; 99% sensitivity, 84% specificity), overweight (BMI $ 25 and ,30; # 8.0 μg/L 97% sensitivity, 76% specificity), and obese (BMI $ 30; # 4.2 μg/L; 94% sensitivity, 78% specificity) [143].

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GLUCAGON STIMULATION TEST With the limited availability of GHRH and the contraindications and safety issues related to hypoglycemia with the ITT, the glucagon stimulation test has emerged as a viable alternative test for GH deficiency in the adult. GH peaks approximately 120
180 minutes after glucagon stimulation. The glucagon stimulation test is performed in the fasting state, with baseline values for glucose and GH obtained prior to administration of 1 mg intramuscularly of glucagon. The dose is increased to 1.5 mg for patients weighing greater than 90 kg. Glucose and GH are measured serially every 30 minutes for 4 hours. A normal GH response has been determined to be greater than 3 μg/L, but as discussed above, a cutoff of 1 μg/L may be more accurate and requires validation before widespread acceptability [140]. Glucose measurements are not necessary for interpretation, but are obtained for safety. Hypoglycemia during the test is uncommon, however late hypoglycemia may occur and patients are instructed to eat small, frequent meals after completion of the test. Patients may feel nausea during and after the test. Antiemetics may be considered during the test. RECOMMENDATIONS Current recommendations for the diagnosis of GH deficiency include the consideration of the diagnosis in the appropriate clinical context. Evaluation proceeds with investigation of other pituitary hormone deficits and IGF-1 secretion. If three or more pituitary deficiencies are present, and the serum IGF-1 level is low compared to age- and gender-matched populations, the diagnosis of GHD is almost certain, and stimulation testing may not be necessary. If suspicion remains and there are two or fewer pituitary deficits or a normal IGF-1, stimulation testing should be performed after replacement of other pituitary hormone deficits is completed. Recommended stimulation tests include the ITT (if no contraindications are present), the GHRH
arginine stimulation test, or the glucagon stimulation test. Cutoff points specific to the test, and to the BMI for GHRH
arginine test, should be employed to diagnose GH deficiency in the adult. SOMATOTROPH ASSESSMENT IN CHILDREN

The diagnosis of GHD in children is based on auxological signs of growth failure. In contrast to the diagnosis of GHD in adults, auxological signs are the most important tool in the diagnostic workup [144]. Serial length or height data are plotted on growth charts, along with weight measurements (Fig. 10.5A
D) [146]. Height velocity can be determined with serial measurements using velocity charts. Diagnostic testing begins by ruling out other causes of growth failure, such as hypothyroidism, chronic disease, and skeletal disorders. In contrast to adults, the

measurement of serum IGF-1 levels is an excellent screening tool for the investigation of GHD. Measured with IGFBP-3, and bone age, these tests provide an initial step in the diagnosis of GH deficiency in children. IGF-1 and IGFBP-3 reflect an integrated assessment of GH secretion. Unlike the pulsatile secretion of GH, IGF-1 and IGFBP-3 have long half-lives in serum of 12 and 16 hours, respectively. While IGF-1 and IGFBP-3 are excellent screening tests, there are caveats that prevent their exclusive use. Serum IGF-1 values are low in early life, with a overlap between GHD patients and normal children. IGF-1 production is affected by nutritional status in adults and children and may not accurately reflect GH secretion. IGF-1 levels are also affected by comorbidities such as renal failure, hepatic failure, diabetes, and hypothyroidism, limiting its usefulness in these settings. IGF-1 levels do not distinguish between GH insensitivity and GH deficiency. IGF-1 and IGFBP-3 measurement should be interpreted in relation to gender- and age-related normative data. IGFBP-3 is the major carrier protein of the IGFbinding protein family. Normative data for IGFBP-3 are less related to age, and it is therefore a good test for young children and infants. GH STIMULATION TESTING IN CHILDREN As in adults, GH stimulation testing is an important part of the diagnostic workup of children with suspected GH deficiency. Also as in adults, there are a number of limitations to their use. GH stimulation testing is not physiological in that the secretagogues used do not reflect normal GH secretion in an individual. The cutoff points determined for each test are arbitrary, as there is no true gold standard stimulation test for GH deficiency in children. Measurement of GH secretion relies upon the use of pharmacological secretagogues and physiological stimuli. Physiologic causes of GH secretion include exercise, sleep, and fasting. There are a number of pharmacologic secretagogues used in the testing of GH secretion including L-dopa, clonidine, propranolol, glucagon, arginine, and the ITT. The diagnosis of GHD in children requires subnormal responses to two secretagogues, unless there is a known genetic defect or multiple pituitary deficits. GH stimulation testing is performed in the fasting state. Serial measurements of GH are obtained after the administration of one or a combination of secretagogues. The following tests are utilized in the diagnosis of children. Details regarding tests that are used in the diagnosis of both adults and children are provided in the above section on testing adults. Agents Used for GH Stimulation Testing in Children Arginine is infused at a dose of 0.5 g/kg body weight (up to a maximum of 40 g) over 30

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FIGURE 10.5 (A
D) Height and growth velocity curves for American boys and girls. (A) Height, American boys. Height depicted for the 50th percentile (solid black line) with 95th and 5th percentiles depicted (dashed lines). Early maturers (12 SD) depicted in red (50th percentile, solid; 95th percentile, dotted) and late maturers (
2S D) depicted in green (50th percentile, solid; 5th percentile, dotted). (B) Height velocity, American boys. Height velocity depicted for average males (black) (50th percentile, solid; 3rd percentile and 97th percentile, dashed). Early maturers depicted in red. Late maturers depicted in green. (C) Height, American girls. Height depicted for the 50th percentile (solid black line) with 95th and 5th percentiles depicted (dashed lines). Early maturers (12 SD) depicted in red (50th percentile, solid; 95th percentile, dotted) and late maturers (
2 SD) depicted in green (50th percentile, solid; 5th percentile, dotted). (D) Height velocity, American girls. Height velocity depicted for average males (black) (50th percentile, solid; 3rd percentile and 97th percentile, dashed). Early maturers depicted in red. Late maturers depicted in green. Used with permission [145].

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minutes. Serum GH is measured at baseline and at 30minute intervals for 2 hours [147]. Clonidine is administered at a dose of 5 μg/kg (maximum dose of 250 μg), and peaks approximately 1 hour after baseline. Clonidine may cause hypotension and hypoglycemia, and monitoring is performed during the test. GHRH is not commonly recommended for use in children because it may yield a false-negative result, since the defect in children is usually in the hypothalamic regulation of GH release. The combination of arginine and GHRH is commonly used in adults and adolescents transitioning to adult care. Insulin-induced hypoglycemia is a potent stimulant of GH reserve, however it is not commonly used in children due to the safety concerns of inducing hypoglycemia. Glucagon causes transient hyperglycemia and consequent insulin and GH secretion. It is safer than ITT. Glucagon is administered at a dose of 0.03 mg/kg subcutaneously in children (maximum 1 mg) and serum samples are obtained at 30-minute intervals for 3 hours after the stimulus. More details regarding the use of GH stimulation testing are provided in the section regarding diagnosis of adults with GH deficiency. Lactotroph Assessment The measurement of prolactin is performed more commonly for assessment of hypersecretion than in the workup of pituitary failure. Prolactin is the most resilient of anterior pituitary hormone to local damage, and usually remains normal or slightly elevated in the setting of other anterior pituitary hormone deficiencies. Basal measurement of prolactin with a current two-site immunometric sandwich assay is adequate to assess for deficiency, however dynamic testing with TRH stimulation has been performed in the past to assess pituitary reserve. This approach has been supplanted by more specific assessments of individual pituitary axes. TRH would be injected intravenously with an expected increase in prolactin of 2.5-fold from baseline 15
30 minutes after injection. Response times are variable, and many normal subjects have been found to have blunted responses. Special Considerations EVALUATION OF PATIENTS AFTER TBI

Due to the high prevalence of hypopituitarism after TBI and the high incidence of TBI, a systematic approach toward the evaluation of hypopituitarism in subjects at risk after TBI is required. Despite the reported high prevalence of hypopituitarism due to TBI, and a consensus statement regarding the testing of hypopituitarism in this setting, there remains controversy regarding the appropriate workup of hypopituitarism in these patients [72]. Patients who have

suffered TBI or subarachnoid hemorrhage should be evaluated immediately to assess for any acute loss of anterior or posterior pituitary function. Deficiencies of vasopressin and ACTH can be life-threatening and should be immediately addressed. Deficiencies in gonadal steroids, thyroid hormone, and GH are not necessary in the acute phase. Patients who have a history of TBI should be assessed periodically within the first 12 months after the initial insult, as hypopituitarism can be transient, or develop late after recovery. Testing of the HPA axis requires stimulation testing, but may be suspected with an assessment of a morning serum cortisol. Basal testing of thyroid function and the gonadal axis is sufficient for appraisal of these hormones. GH deficiency should be assessed with stimulation testing in most circumstances, unless multiple pituitary deficiencies are present with a low IGF-1, and not before 12 months after the inciting event [93]. EVALUATION OF PITUITARY FUNCTION IN CRITICAL ILLNESS

The effects of critical illness on pituitary function are addressed in Chapter 11, Pituitary Dysfunction in Systemic Disorders. The evaluation of pituitary function is challenging in the acute care setting. Changes in hypothalamic and pituitary function are evident in acute illness, and may be adaptive responses to critical illness. Diagnosing adrenal insufficiency in the ICU setting is challenging due to multiple factors including a lack of uniform criteria for the diagnosis, physiological factors affecting free and total serum cortisol levels, and the lack of an appropriate test, and the lack of reliable methods to measure free cortisol levels in serum [148]. Basal levels of total serum cortisol may indicate adrenal insufficiency, but cutoff levels vary widely in the literature. A cutoff of 15 μg/dL has been proposed as a level that best identifies patients who would benefit from glucocorticoid therapy during critical illness [149]. However, concomitant hypoalbuminemia may indicate lower levels of cortisol binding globulin, and therefore lower levels of total serum cortisol. Patients with albumin levels less than 2.5 g/dL may be best identified as adrenally insufficient with a cutoff level of 10 μg/dL [149
151]. Measurements of serum free cortisol, either directly or through the use of a calculated index, are appealing but not widely available, rapidly performed, or presented with validated normative data [148,152]. Dynamic tests carry undue risk to critically ill patients (ITT, metyrapone), are not validated in this setting (CRH stimulation, glucagon, low-dose ACTH stimulation), or do not reflect pituitary insufficiency (all ACTH stimulation tests). The 250-μg ACTH stimulation test may have prognostic value with a rise in serum cortisol levels of ,9 μg/dL portending an increased risk of

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mortality in critically ill patients, but may not necessarily reflect true adrenal insufficiency.

TREATMENT OF HYPOPITUITARISM The treatment of hypopituitarism is directed toward removal of the underlying cause if possible, with a potential for recovery of normal pituitary function and replacement of deficient hormones. Hormone replacement for pituitary insufficiency can be implemented in three ways depending upon the hormonal axis involved and the desired outcome: (1) replacement of the hormone secreted by the target gland, (2) replacement of an analogue of the deficient pituitary hormone, and (3) administration of an analogue of a hypothalamic releasing factor.

Recovery of Pituitary Function After Neurosurgical Treatment Recovery of pituitary function in subjects with some degree of preoperative hypopituitarism has been reported by a number of investigators [153
155]. Improvement in pituitary function can occur in the immediate postoperative period. Transsphenoidal resection of pituitary tumors was accompanied by improvement in pituitary function in 36
65% [153]. Deterioration of pituitary function after surgery appears to be low with reported values of 1.4
32% and no change in pituitary function noted in 24
54% [153]. Smaller preoperative tumor size and higher prolactin levels were found to be positively related to pituitary hormone recovery, whereas patient age did not correlate to recovery of pituitary function, in one study [153]. Other investigators have demonstrated higher chances of pituitary recovery in younger, nonhypertensive patients, and in patients without the complication of CSF leak [154].

Hormonal Replacement Glucocorticoid Replacement ACTH deficiency is potentially life-threatening and its replacement is the highest priority when considering hormonal replacement for hypopituitary patients. Initiation of other hormonal replacement, especially thyroid hormone or GH, can lead to an adrenal crisis. It is crucial to either ensure an intact HPA axis, or treat ACTH deficiency prior to initiating other hormonal therapies. Treatment of ACTH deficiency is accomplished by replacing endogenous adrenal cortisol secretion with a glucocorticoid equivalent. Optimal therapy would closely mimic the normal diurnal variation of cortisol, although this goal is often met with difficulty due to the complexity of administration

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required to replicate normal human physiology. In contrast to primary adrenal failure, wherein glucocorticoid and mineralocorticoid replacement are necessary, secondary adrenal insufficiency requires only glucocorticoid replacement. Mineralocorticoid secretion is preserved, since it is under the control of the renin
angiotensin system. There is no consensus on the best method for adrenal glucocorticoid replacement, nor is there an accepted method to test for optimal dosing or longterm monitoring of replacement therapy. Dosing has shifted away from the 12
15 mg/m2 dosing that was previously believed to be necessary, to 10
12 mg/m2 based on isotope studies that demonstrated a lower daily cortisol production rate than was previously determined. Replacement of glucocorticoids can be accomplished using multiple dosing regimens, but administration of hydrocortisone of 15
20 mg per day, in divided doses, has been the standard approach toward replacement. Twice-daily dosing (with 10
15 mg of hydrocortisone in the morning and 5 mg in the early afternoon) is one method of replacing glucocorticoids. Early afternoon doses provide the patient with less sleep interference compared to doses given later in the day. Thrice-daily dosing (10 mg in the morning, 5 mg at lunchtime, and 5 mg in the afternoon) has also been advocated and has been shown to more closely mimic the normal diurnal variation in cortisol production during the day than conventional twice-daily dosing [156]. Monitoring of glucocorticoid replacement is done solely on clinical grounds, as there has not been an accurate method to determine the appropriate dose, and interindividual variation is common. Some clinicians rely on weight-based dosing, surface area-based dosing, or fixed dosages, yet there have not been adequate randomized, powered studies to establish a best method to determine appropriate dosing. Biochemical markers of appropriate dosing are not helpful. ACTH is not helpful in either primary or secondary adrenal insufficiency [157]. Measurement of 24-hour urinary free cortisol is not helpful as there is a great degree of interindividual variation in cortisol excretion after glucocorticoid absorption, renal excretion is transiently elevated immediately after absorption and saturation of cortisol binding globulin, and normative data from patients with intact HPA activity is not applicable to patients on replacement dosing [157]. Cortisol day curves have been used, but are not validated by controlled studies. In the absence of biochemical markers, clinical judgment must be used to avoid symptoms associated with under- or overreplacement. Under-replacement may be associated with weight loss, fatigue, afternoon headaches, nausea, abdominal pain, and myalgias. Over-replacement

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may be associated with weight gain, obesity, hypertension, hyperglycemia, osteoporosis, and possibly cushingoid features. Osteoporosis has been reported in patients taking doses of 30 mg of hydrocortisone daily or greater, but lower doses have not been associated with osteoporosis [158]. Instructions regarding dosing for sick days and other times of physiologic stress need to be given to patients receiving glucocorticoid replacement. Patients dependent upon glucocorticoid medication do not respond to physiologic stress with increased levels of corticosteroids and therefore require increased dosing. Doubling or tripling an oral dose of glucocorticoid therapy for febrile illnesses or administration of 100
150 mg per day of hydrocortisone for other serious physiologic stressors for 2
3 days with a tapering dose as indicated is required depending upon the severity of the illness or surgical intervention. Patients should be instructed to wear a bracelet or other form of identification to alert others to their steroid dependence in the case of emergencies. Despite the fact that treatment of adrenal insufficiency is clearly beneficial, increased mortality in patients with pituitary disease has been noted in relation to treatment of infectious disease and overtreatment of adrenal insufficiency in patients with treated acromegaly [159]. Androgen Replacement in Women Adult women with hypopituitarism affecting adrenal androgen production have a deficiency in androgen production, with decreased levels of DHEA, DHEA-S, androstenedione, and testosterone [160]. DHEA replacement has been shown in controlled studies to improve symptoms of androgen deficiency in women with primary and secondary adrenal insufficiency [161]. DHEA is not available in an FDAapproved form, but is available as a dietary supplement. These supplements are not controlled and the quality and content of DHEA-S preparations that are available over the counter demonstrate a high degree of variability, and unreliable amounts of drug provided in each dose [162]. Large, prospective, randomized studies of DHEA supplementation in women are lacking but studies using 50 mg of daily DHEA have demonstrated increased levels of DHEA, DHEA-S, and testosterone. Side effects such as hirsutism, increased sweating, facial acne, and alopecia are infrequent and are reversible with discontinuation of DHEA. Transdermal testosterone replacement in women has been studied in several placebo-controlled phase III clinical trials. A 300-μg daily patch was investigated in women who are surgically postmenopausal and in female patients with hypopituitarism. Improvements in mood, sexual function, and testosterone levels were seen in the treatment group [163,164]. Long-term safety

data are lacking however, and therefore there is no FDA-approved formulation providing this dose. Thyroid Hormone Replacement Replacement of the thyroid axis in secondary, tertiary, or central hypothyroidism is usually done with synthetic L-thyroxine (LT4). Nonsynthetic preparations of thyroid hormone replacement are available, but synthetic formulations are preferred due to their uniform potency. As stated above, it is necessary to exclude adrenal insufficiency prior to the initiation of thyroxine replacement due to the potential to precipitate an adrenal crisis. Thyroxine increases the metabolism of cortisol, and an unmet demand for glucocorticoid secretion may result in an Addisonian crisis. LT4 circulates bound to thyroid-binding globulin and is converted in peripheral tissues by T4-50 -deiodonase to the more biologically potent thyroid hormone triiodothyronine (T3). Synthetic T3 is available as thyroid hormone replacement, but should not be used as monotherapy in the long-term treatment of hypothyroidism. Combined therapy with T4 and T3 analogues has been explored and there are conflicting reports in the literature. Despite early positive findings, combined therapy has been shown in multiple individual randomized, double-blind controlled trials and three separate metaanalyses to not be more advantageous in improving mood, quality of life, and neuropsychological function [165]. Combined therapy often is associated with multiple side effects of the T3 component including palpitations, anxiety, and diaphoresis. Proponents of combined therapy suggest that there is a benefit to those who remain symptomatic despite adequate T4 replacement, but the benefit is not clearly demonstrated by changes in biological endpoints, psychometric testing, or psychological assessments [166]. Despite equivalence of measurable outcomes in most studies, when preference of replacement method was assessed, more subjects preferred combined therapy in the treatment of primary hypothyroidism [165]. Oral L-thyroxine is available by multiple manufacturers and the mean replacement dose is 1.5 6 0.3 μg/kg per day [120]. There is considerable interindividual variation, however the dosing for hypothyroid replacement may vary widely. LT4 should be given consistently, and patients are instructed to take the medication on an empty stomach, separately from other medications to avoid effects on absorption. The mean bioavailability of LT4 is approximately 80% but reduced absorption has been demonstrated with concurrent intake of medication, inflammatory bowel disease, and short bowel syndrome. The half-life of LT4 is approximately 7 days, with daily administration providing a steady level of drug in the circulation. Hence, dose adjustments should be

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followed by a reassessment of the steady-state level 6 weeks after the dosing change. Additionally, holding a dose when required to do so for surgery or other reasons does not significantly affect drug levels and can be re-initiated as soon as possible without harm. If necessary, intravenous replacement can be done with LT4 (at a ratio of 1:2 for IV:PO dosing) during prolonged periods of time when a patient cannot take the medication by mouth. Concomitant medications may alter the metabolism of LT4 and higher doses may be required if medications such as rifampin, phenobarbital, carbamazepine, or phenytoin are being administered [165]. Monitoring replacement of levothyroxine for central hypothyroidism differs from that of primary hypothyroidism in that TSH measurement is not helpful in determining appropriate dosing. As in diagnosis, the TSH does not appropriately or reliably respond to changes in circulating T4 or T3 levels. Free thyroxine or free thyroxine index measurements are used to adjust dosing in central hypothyroidism. Free T3 levels have been advocated as well [85,120]. However, T3 levels are preserved in hypothyroid states and may not accurately guide replacement dosing [167]. Measurement of total T4 and T3 levels is not helpful in dosing changes [85,120]. The target for dosing LT4 replacement is to attain levels of free thyroxine in the upper half of the normal range for younger patients, and it may be more appropriate to target lower levels within the normal range for older patients. Retrospective data demonstrating a negative correlation between fT4 values and cardiovascular risk in patients with central hypothyroidism underscore the importance of dose optimization in reducing cardiovascular risk markers in these patients [168]. Dosing adjustments are necessary during pregnancy or after initiating estrogen replacement therapy due to the changes in levels of thyroid-binding globulin. Free thyroxine levels (preferably utilizing the free thyroxine index) are useful in reaching the appropriate dose. As with nonhypopituitary patients, adequate thyroxine levels are necessary for good fetal health, and increases of approximately 30% are expected during pregnancy [169]. After the initiation of estrogen therapy, dosing adjustments should be made every 6
8 weeks to ensure proper serum levels of thyroxine. The treatment of neonates and children requires higher doses of thyroxine compared to adults, and treatment should begin with the estimated full dose to prevent adverse effects of hypothyroidism on neurological development [170]. Treatment of neonatal central hypothyroidism should start with 12
17 μg/kg of LT4, with adjustment every 2
3 weeks as needed based on free T4 and T3 measurements. The goal of therapy is to achieve levels within the age-adjusted normal range. Progressively lower doses will be required as the child ages.

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Gonadal Steroid Replacement Deficiencies in LH and FSH manifest in various way depending upon the age of onset and the degree of deficiency. The method of treatment of hypogonadism also depends upon the desired outcome of therapy. Treatment of hypogonadism varies dramatically depending upon whether puberty has occurred, fertility is desired, or menopause has occurred in females. Similar considerations are made for males who have not undergone puberty or desire fertility. FEMALE HYPOGONADOTROPHIC HYPOGONADISM ESTROGEN REPLACEMENT IN ADULT WOMEN Large epidemiological studies evaluating the impact of hormone replacement therapy (HRT) for long-term preventative therapy during postmenopausal years have drastically altered the approach toward standard therapy during menopause [145]. The long-term use of HRT during menopause for cardiovascular risk reduction and prevention of osteoporosis is no longer recommended due to the risks associated with breast cancer and cardiovascular outcomes. In concordance with these findings, the treatment of women with HRT in the setting of hypopituitarism has been reevaluated. Women who have reached an age commensurate with the postmenopausal state are no longer treated with HRT for long-term preventative reasons. In younger women, it is recommended to take HRT until the average age of menopause (approximately 50 years of age). Studies in young women have demonstrated cardiovascular benefits to treatment of estrogen deficiency [171], and the risk for osteoporosis increases with early menopause from either secondary or primary causes [145,172]. Transdermal preparations of estradiol are preferred over oral preparations [173,174]. Oral estrogen replacement is subject to hepatic first-pass metabolism. Avoiding this first-pass metabolism has many beneficial effects including reduced synthesis of clotting factors and inflammatory proteins associated with increased cardiovascular risk [173], reduced synthesis of sex hormone-binding globulin, and absence of the growth hormone-resistant effect of estrogen on IGF-1 production in the liver [174,175]. As with other cases of estrogen replacement, women with an intact uterus taking estrogen due to pituitary insufficiency should take concomitant progesterone therapy. Multiple treatment options are available for progestin therapy, but daily or monthly administration is preferred. INDUCING PUBERTY IN FEMALES Secondary sexual characteristics can be attained in females with estrogen therapy alone, and treatment with gonadotrophins or GnRH analogues is not required for induction

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of puberty. Induction of fertility can be achieved later when fertility is desired. Secondary sexual characteristics result from administration of progressively increasing doses of estrogen, usually ethinyl estradiol or 17β-estradiol, over a 2-year period with dose increases every 6 months. Addition of progesterone and transition to oral contraceptives is then done for maintenance therapy [176]. INDUCING FERTILITY IN FEMALES Induction of fertility in females with hypogonadotrophic hypogonadism requires use of gonadotrophin analogues for successful ovulation. Women may require combinations of human menopausal gonadotrophin, hCG, and/or synthetic analogues of LH or FSH. Outcomes have been improved with the addition of GH during ovulation induction. Further gonadotrophin support is required during the luteal phase. Complications of multiple gestation and ovarian hyperstimulation syndrome may occur. MALE HYPOGONADOTROPHIC HYPOGONADISM TESTOSTERONE REPLACEMENT IN MEN Treatment of hypogonadism in males consists of replacement of deficiencies in testosterone and sperm production, if fertility is desired. Testosterone therapy is aimed at restoring serum testosterone levels to within the normal range. There are multiple testosterone preparations available to use for treatment of hypogonadism. Administration of testosterone is hampered by first-pass hepatic metabolism, rendering oral administration of testosterone ineffective. Testosterone enanthate and testosterone cypionate are injectable forms of testosterone that have been used for many years in the treatment of hypogonadism. Injection of these lipophilic preparations results in a slow release of testosterone into the circulation. Dosing averages approximately 100 mg per week, with less frequent dosing, result in higher excursions above the normal range immediately after injection. A standard approach toward treatment is initiating injections of 200 mg every 2 weeks with dose titration based on mid-cycle or nadir values. More frequent dosing may be more acceptable to some men who prefer not to have fluctuation in serum levels of testosterone seen with the less frequent injections. The advantage of testosterone injections is the ability to not apply medication on a daily basis. The disadvantage is that an intramuscular injection requires a large-bore needle and specialized training to administer. Transdermal preparations are supplied in patch and gel form. Each must be applied daily. The patch is applied to the arm or torso and delivers a stable amount of testosterone over the 24-hour period. One disadvantage of the patch delivery system is the frequent occurrence of rash. Gel preparations are welltolerated forms of administration that are applied

daily. As with the patch, serum levels of testosterone remain stable over a 24-hour period. Care must be given to limit the potential for transfer to another individual by contact, and instructions are given to patients for strict handwashing and placement of the gel in an area less likely to contact others. Successful replacement should lead to achieving normal serum testosterone levels and initiation of virilization or maintenance of virilization in adults. Libido and energy levels should improve with therapy, and, if persistent, may indicate another cause for these symptoms. Replacement therapy also leads to improved muscle strength and fat-free mass, as well as improvements in bone mineral density [124]. Improvements in bone health are most dramatic in those subjects with lower baseline evaluations and in previously untreated patients [177]. INDUCING PUBERTY IN MALES When hypogonadism precedes puberty and secondary sexual characteristics have not developed, the induction of puberty is required to achieve normal adulthood. Administration of testosterone is the treatment of choice for the induction of puberty in males when fertility is not immediately desired and development of secondary sex characteristics is the goal. Testosterone is administered at low doses and gradually increased in dose and frequency to achieve stimulation of a pubertal growth spurt and secondary sexual characteristics. In cases where puberty is constitutionally delayed, treatment is required only temporarily until spontaneous gonadotrophin secretion occurs. However, in cases of anterior pituitary failure as the underlying etiology, spontaneous gonadotrophin secretion is not expected and treatment modalities should not rely on normal pituitary function. Treatment of delayed puberty with GnRH treatment will result in release of both LH and FSH from the gonadotroph cells. Treatment will result in complete development with testicular growth, spermatogenesis, and virilization. For the majority of patients with hypogonadotrophic hypogonadism, GnRH therapy is ineffective in replacing the gonadal axis due to destruction of gonadotroph cells and an inability to secrete LH and FSH. LH, FSH, hCG, and TSH are glycoproteins with similar α-subunit structure and individualized β-subunits. hCG has been utilized as monotherapy to induce puberty in males and is necessary for testicular development and spermatogenesis [176]. INDUCING FERTILITY IN MALES Spermatogenesis can be achieved with treatment of secondary hypogonadism due to pituitary disease with use of gonadotrophins, or GnRH in men with hypothalamic disease. hCG is an LH analogue that has a longer half-life and

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stimulates Leydig cells in the testes to produce and secrete testosterone. Recombinant LH is available but is less effective due to its shorter half-life. Local production of testosterone produces testicular levels of testosterone much greater than replacement of testosterone alone. This higher local level of testosterone is necessary for spermatogenesis. In most cases, hCG treatment is sufficient to induce spermatogenesis [178]. Growth Hormone Replacement Treatment of GHD should be addressed after all other pituitary deficiencies have been addressed and sufficiently treated. The treatment of GHD is performed by daily subcutaneous injection of a recombinant form of human GH. Recombinant preparations of GH have replaced preparations made from cadaveric tissue since 1985. Cadaveric GH preparations were associated with cases of Creutzfeldt
Jakob disease, halting its use worldwide. ADULT-ONSET GHD

The FDA approved the treatment of GH-deficient adults with GH in 1996. The treatment of GH deficiency in adults is directed at improving symptoms of body composition (increased abdominal adiposity and loss of muscle mass and bone mineral density), increasing strength and exercise performance, improving the cardiovascular risk profile, correcting abnormalities in cardiac structure and intima media thickness or the carotid artery, and improving mood and sense of wellbeing [179]. Discontinuation of GH therapy results in reversal of beneficial effects. There are several preparations of GH commercially available and each is supplied at a potency of 3 IU/mg, using the World Health Organization reference preparation 98/574. Initiation of therapy should begin after careful exclusion of absolute contraindications for therapy, including active malignancy, benign intracranial hypertension, and preproliferative or proliferative diabetic retinopathy. Initiation of GH therapy in adults should be at low doses of 0.15
0.3 mg given subcutaneously prior to bedtime. A general principle to follow is to initiate dosing in younger subjects with a dose of 0.3 mg and titrate the dose every 1
2 months based on the IGF-1 response. For older adults, doses of 0.15 mg daily should be started with a monthly titration as tolerated. Initial trials of GH therapy used weight-based dosing regimens that resulted in an unacceptable level of adverse effects that were directly related to dose. Dosages have been reduced and the method of initiation and titration changed to reflect an individualized dose based on IGF-1 levels [180]. Trials comparing weight-based strategies to IGF-1-based titration demonstrated 50% fewer adverse events in

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the individualized, IGF-1-based regimens compared to weight-based dosing. Final doses were lower in the individualized titration groups [180]. In general, women require higher doses of GH to achieve normal IGF-1 levels compared to men. Additionally, women with matched IGF-1 responses may reap fewer expected benefits of GH therapy than men [181,182]. Changes in estrogen therapy, either discontinuation of estrogen or switching from oral to transdermal estrogen, may be accompanied by a lowering of GH dose. GH secretion diminishes with age, and dosing in older adults may need to be lower to avoid side effects and to maintain a serum IGF-1 level within the ageand gender-matched normative range. CLINICAL BENEFITS OF GH REPLACEMENT

Body Composition GH replacement imparts a reliable change in body composition through increased lipolysis. Subjects with GHD are found to have an increased amount of visceral adiposity, and GH treatment diminishes this visceral fat depot over months of therapy [183]. Effects are also seen in subcutaneous fat, with an overall treatment response of improvements in total body fat. Untreated subjects with GHD also have decreased lean muscle mass, and treatment of GHD has been shown to increase lean muscle mass [184
186]. Some studies have demonstrated an increase in strength, although this result has not been universally seen [186
189]. Some studies have shown an increase in exercise capacity and physical performance, but again, not all studies have demonstrated this improvement [190
192]. Bone Density Multiple studies have demonstrated decreased bone mineral density by DEXA or quantitative CT scan, independent of glucocorticoid replacement and hypogonadism. The prevalence of osteoporosis is increased in subjects with childhood-onset GHD compared to adult-onset GHD, with rates of 35% and 20%, respectively [130]. The severity of GHD also correlates with the extent of osteopenia [193]. Histological examination of bone marrow biopsies reveals an increase in cortical but not trabecular bone, increased reabsorption and bone turnover, and increased osteoid thickness in treated GHD individuals [194]. Fracture rates in GHD subjects are increased compared to control groups [195]. Replacement of GH in GH-deficient adults has an anabolic effect on bone, with effects on both bone formation and reabsorption. Long-term therapy exerts a beneficial effect on bone mineral density, as determined by DEXA, with subjects having the greatest incremental improvement when initial Z-score values were lowest [196]. Improved bone mineral density appears to be more dramatic in men compared with women [197]. Cardiovascular Markers GH treatment has multiple beneficial effects on cardiovascular health to improve

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risk factors associated with cardiovascular-related morbidity and mortality. Studies have demonstrated improved flow-mediated dilatation and a reduction in arterial stiffness [198]. Several studies have demonstrated a small reduction in blood pressure [199]. Creactive protein and homocysteine concentrations decrease with GH replacement [100,101]. Some studies have demonstrated increases in HDL and decreases in LDL and total cholesterol with replacement [184,185,199,200]. GH therapy may worsen insulin resistance and have a small increase in fasting plasma glucose levels. The effect of GH on insulin metabolism is variable; however, it is unclear what the long-term, individual effects on insulin resistance are in subjects treated with GH. Initial increases in free fatty acids may worsen insulin resistance. Longer-term effects on decreased adipose tissue and increased lean muscle mass may impart an improvement in glucose metabolism. Increased intima medial thickness has been shown to improve with GH replacement and cardiac function measured by echocardiography improves with GH therapy [185,201]. Notable improvements are seen in left ventricle (LV) mass, LV end diastolic volume, and stroke volume [202]. Despite the improvements in cardiovascular risk factors, replacement of GH has not been clearly shown to improve the increased cardiovascular and cerebrovascular mortality associated with hypopituitarism. Quality of Life The effects of GH replacement on quality of life have been varied in studies. Some studies have shown a benefit, and others have shown little or no change. Improvement in quality of life appears to be more likely when initial scores are low, however this improvement does not correlate to improvements in IGF-1 levels [203
205]. MONITORING THERAPY After dose titration is complete, monitoring should be implemented with a careful periodic review of side effects and bi-annual assessment of serum IGF-1 levels and clinical outcomes. A lipid panel and fasting blood glucose should be measured annually. Bone mineral density should be monitored with DEXA if the baseline assessment is found to be low at 1
2-year intervals, and consideration should be made for additional therapies directed at bone density loss. Measurement of thyroxine should also be performed, and adjustments made in replacement, if indicated [130]. CHILDREN AND ADOLESCENTS

Once the diagnosis of GH deficiency is made in children, treatment should begin to improve linear growth. There is wide variability in responses to GH therapy and dosing should be adjusted based on the growth response and the serum levels of IGF-1. Unlike adults,

dosing is weight-based, and initial dosing depends upon the preparation used but ranges from approximately 0.18
0.3 mg/kg per week subcutaneously divided into equal daily doses. The treatment goal is to achieve levels above the mid-normal range of IGF-1 adjusted for age and Tanner stage. GH therapy is effective at increasing final height and improving growth velocity. If administered at an early age, patients can reach a height within the mid-parental target range [206]. Predictors of response to GH therapy have been established based on large databases: gender, gap between target height and height at the beginning of puberty, dose of GH at the beginning of puberty, age at the onset of puberty, and age at the end of growth. Other factors such as diet, dosing schedule, response to testing stimuli, exercise, and psychological wellbeing may also contribute to the response to therapy. Special considerations should be made when treatment of adolescents with GH is continued into adulthood. While final height is achieved once the epiphyses are closed, GH replacement has ongoing benefits through the transition period into adulthood. Complete development of muscle mass and peak bone mass are GH-dependent and continue after final height is reached. It has been recommended that treatment be continued through the transition period, rather than interrupt GH therapy and resume treatment in adulthood [207]. Controlled clinical trials have demonstrated a detrimental effect on interruption of GH replacement on fat distribution, muscle mass, cardiac performance, and bone mass [208]. During the transition phase, dosing of GH should be closely monitored and reduced to conform to the normal decline in GH secretion that occurs in late puberty. During puberty, dosing of GH varies widely. But during the transition phase, the dosing should restart using a nonweight-based dosing method at 0.2
0.5 mg daily, and adjustments made based on the age- and gender-matched serum IGF1 level [209]. Serum IGF-1 levels should be monitored every 6 months and adjusted if necessary. Long-term follow up should include body composition measurements (BMI, hip and waist measurement, height, and weight), cardiovascular measurements (heart rate, blood pressure), and assessment of quality of life. Bone mineral density and lipid profiles should be assessed at baseline and proceed according to adult guidelines during the transition period. Thyroxine and glucocorticoid replacement should be reassessed with dose changes in GH therapy and adjusted if necessary [209].

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405. [196] Johannsson G, Rosen T, Bosaeus I, Sjostrom L, Bengtsson BA. Two years of growth hormone (GH) treatment increases bone mineral content and density in hypopituitary patients with adult-onset GH deficiency. J Clin Endocrinol Metabol 1996;81(8): 2865
73. [197] Drake WM, Rodriguez-Arnao J, Weaver JU, et al. The influence of gender on the short and long-term effects of growth hormone replacement on bone metabolism and bone mineral density in hypopituitary adults: a 5-year study. Clin Endocrinol 2001;54(4):525
32.

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[198] Smith JC, Evans LM, Wilkinson I, et al. Effects of GH replacement on endothelial function and large-artery stiffness in GHdeficient adults: a randomized, double-blind, placebocontrolled study. Clin Endocrinol 2002;56(4):493
501. [199] Maison P, Griffin S, Nicoue-Beglah M, Haddad N, Balkau B, Chanson P. Impact of growth hormone (GH) treatment on cardiovascular risk factors in GH-deficient adults: a metaanalysis of blinded, randomized, placebo-controlled trials. J Clin Endocrinol Metabol 2004;89(5):2192
9. [200] Newman CB, Carmichael JD, Kleinberg DL. Effects of low dose versus high dose human growth hormone on body composition and lipids in adults with GH deficiency: a metaanalysis of placebo-controlled randomized trials. Pituitary 2015;18(3):297
305. [201] Borson-Chazot F, Serusclat A, Kalfallah Y, et al. Decrease in carotid intima-media thickness after one year growth hormone (GH) treatment in adults with GH deficiency. J Clin Endocrinol Metabol 1999;84(4):1329
33. [202] Maison P, Chanson P. Cardiac effects of growth hormone in adults with growth hormone deficiency: a meta-analysis. Circulation 2003;108(21):2648
52. [203] Rosilio M, Blum WF, Edwards DJ, et al. Long-term improvement of quality of life during growth hormone (GH) replacement therapy in adults with GH deficiency, as measured by

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questions on life satisfaction-hypopituitarism (QLS-H). J Clin Endocrinol Metabol 2004;89(4):1684
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73. Murray RD, Shalet SM. Adult growth hormone replacement: lessons learned and future direction. J Clin Endocrinol Metabol 2002;87(10):4427
8. Reiter EO, Price DA, Wilton P, Albertsson-Wikland K, Ranke MB. Effect of growth hormone (GH) treatment on the nearfinal height of 1258 patients with idiopathic GH deficiency: analysis of a large international database. J Clin Endocrinol Metabol 2006;91(6):2047
54. Clayton PE, Cuneo RC, Juul A, et al. Consensus statement on the management of the GH-treated adolescent in the transition to adult care. Eur J Endocrinol Eur Fed Endocrine Soc 2005;152(2):165
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70.

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11 Pituitary Dysfunction in Systemic Disorders Maria Fleseriu

INTRODUCTION The pituitary gland may be affected by a wide variety of systemic disorders (Table 11.1). The pituitary could be directly involved by the same processes that affect other organs (e.g., inflammatory, autoimmune, or infectious disorders), or the primary disease process may elicit indirect, distant effects on pituitaryhypothalamic hormonal function (Table 11.2).

SYSTEMIC DISORDERS DIRECTLY AFFECTING THE PITUITARY GLAND

PITUITARY GRANULOMAS Pituitary granulomas are rare specific lesions encountered in sarcoidosis, granulomatosis with polyangiitis (GPA), giant-cell granulomatous hypophysitis, Langerhans histiocytosis, Erdheim Chester disease (ECD), and tuberculosis.

Sarcoidosis Sarcoidosis, a chronic multisystemic inflammatory disease of unknown origin, is characterized by noncaseating granulomatous inflammation of the organs involved. Neurosarcoidosis occurs in about 5% of patients; there is evidence of either hypothalamic or pituitary dysfunction in approximately one-third [1]. Isolated hypothalamic/pituitary disease is occasionally reported. Visual symptoms often result from direct involvement of the optic chiasm [1]. A diagnosis of neurosarcoidosis is challenging and requires the documentation of characteristic clinical symptoms and radiologic findings after the exclusion

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00011-8

of other diseases, including vasculitis, infection, and neoplasm. Noncaseating granulomatous inflammation should be confirmed by histology findings. The value of serum angiotensin-converting enzyme levels in the diagnosis of sarcoidosis remains controversial due to its inadequate sensitivity and specificity. Central nervous system (CNS) sarcoid lesions usually show contrast enhancement on computed tomography (CT) scanning, generally without surrounding edema or calcification [2]. Most lesions enhance with gadolinium on magnetic resonance imaging (MRI) [1 3]. Diffuse extensive leptomeningeal enhancement, with the involvement of the hypothalamus, pituitary stalk, and the optic chiasm has also been reported [4]. CNS sarcoid lesions can also be visualized on positron emission tomography using 18F-fluorodeoxyglucose [5]. Studies using provocative testing have demonstrated a high prevalence of hypothalamic dysfunction, with intact pituitary hormonal responses to releasing factors, but impaired responses to clomiphene, metyrapone, and insulin hypoglycemia [5]. Hyperprolactinemia has been reported, but is not a universal finding [1,5]. Disturbances of water metabolism are common and include diabetes insipidus (DI), primary polydipsia, and the syndrome of inappropriate antidiuretic hormone secretion (SIADH) [1,5]. Polyphagia with morbid obesity and temperature dysregulation has also been reported. Despite occasional beneficial responses, therapy with glucocorticoids (GCs) or other immunosuppressive agents does not usually reverse features of pituitary damage [1,5]. Transsphenoidal surgery is commonly used to resect advanced pituitary sarcoid lesions refractory to medical management. As histology and imaging are key to diagnosis, a multidisciplinary team approach is valuable for enabling diagnosis and subsequent treatment [6]. Hormone replacement therapy is prescribed as indicated.

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366 TABLE 11.1

11. PITUITARY DYSFUNCTION IN SYSTEMIC DISORDERS

Systemic Disorders Affecting the Pituitary

Direct

Indirect

Pituitary granulomas

Changes in pituitary morphology and function with aging

• • • • • •

Sarcoidosis Granulomatosis with polyangiitis Granulomatous hypophysitis Necrotizing hypophysitis Langerhans cell histiocytosis Erdheim Chester disease

Any severe systemic illness Pituitary alterations in specific systemic disorders • • • • • • •

Autoimmune • Lymphocytic hypophysitis • Immunoglobulin G4-related hypophysitis

Obesity Malnutrition Anorexia nervosa Diabetes mellitus Chronic kidney disease Liver disease Endocrine disorders: primary adrenal insufficiency, primary hypothyroidism, hyperthyroidism

Amyloidosis Infectious diseases • Acquired immune deficiency syndrome • Tuberculosis • Other infectious diseases: syphilis, Whipple and Chagas disease, and hemorrhagic fever • Pituitary abscess Iron overload Snakebite Metastatic cancer Genetic multiglandular syndrome • Multiple endocrine neoplasia • Carney complex • McCune Albright syndrome Other stalk and pituitary lesions • • • •

Stalk hemangioblastoma Choroid glioma Hemangiopericytoma Fabry disease

Granulomatosis With Polyangiitis (Formerly Wegener’s Granulomatosis) GPA is a systemic disease characterized by pauciimmune, necrotizing, small-vessel vasculitis [7]. Most organ systems can potentially be affected, with a predilection for ear, nose, and throat (ENT), lungs, and kidneys. Pituitary disease is usually accompanied by the involvement of other organs, most commonly ENT; however, on rare occasions, pituitary disease can be an isolated finding. The number of patients diagnosed with GPA involving the pituitary is steadily increasing [8 12]. An association with antineutrophil cytoplasmic antibodies (ANCAs) remains controversial. Despite results of a metaanalysis showing that .90% of active cases of GPA are positive for ANCA, this statistic may exaggerate the sensitivity of the ANCA test, because it applies only to well-established and mostly

biopsy-proven cases. Before diagnosis, the presence of ANCAs may be negative in 50 70% of cases [7]. In a large study by Kapoor et al. [11], secondary hypogonadism and DI were the predominant manifestations of pituitary disease (87.5% and 75%, respectively). Other anterior pituitary function abnormalities have been described. Radiologic studies usually show pituitary enlargement (80%), sellar mass with peripheral enhancement, areas of central necrosis, and stalk thickening [11]. The enhancement pattern on MRI can be either heterogeneous or homogeneous. Cystic changes in the pituitary, infundibular thickening with contrast enhancement, and the absence of a normal hyperintense signal on T1weighted pituitary images have been described [11]. Treatment of GPA is complex [13]. The disease appears to respond well to remission induction therapy with GCs and cyclophosphamide- or rituximabbased treatment. Rituximab is the preferred agent if

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TABLE 11.2 Specific Indirect Effects of Aging and Systemic Disorders on Pituitary/Hypothalamic Function Aging

Systemic illness

Obesity

Malnutrition

Anorexia nervosa

Adrenal

Generally intact (ACTH, cortisol can slightly increase with flattening of diurnal cortisol release)

m Cortisol m ACTH m Adrenal androgens

m Serum cortisol m UFC m ACTH

m Cortisol m ACTH

HPA axis activation m Cortisol m UFC m ACTH

Thyroid

m TSH k T3 Stable T4

k T3 m Reverse T3 T4 normal initially TSH normal initially

Normal TSH Normal free T4

“Euthyroid sick “Euthyroid sick syndrome” syndrome”

Growth k hormone

m GH k IGF-1

k GH secretion Normal IGF-1

m Basal GH k IGF-1

Prolactin Normal

m

Normal (response to pharmacologic agents may be blunted)

Normal basal

Gonadal

m LH (initially) then k LH k FSH k estrogen k testosterone

Premenopausal women Hyperandrogenism m LH k Estrogen Men k Total testosterone Normal free testosterone

Primary hypothalamic abnormality

Women m LH m FSH k Estrogen Men k Testosterone Normal LH Normal FSH

Diabetes m AVP insipidus Loss of circadian AVP rhythm Pituitary size

k

GH “resistance” (m GH k IGF-1)

Women Hypothalamic amenorrhea Men k Testosterone k LH k FSH m Baseline ADH Low NA Low osmolality

Diabetes mellitus

m basal GH k IGF-1 (poorly controlled DM)

Chronic kidney disease Liver disease HPA axis upregulation

Normal HPA axis (k CBG impacts cortisol measurements)

m TSH Normal/ low T4, T3

k T3 m Reverse T3 Normal serum T4 m/normal TSH

m/normal m Basal GH basal GH k IGF-1 normal/m IGF1 m

m

Central 1 primary hypogonadism

Women k Estradiol m Estrone Men k Testosterone m/normal LH m/kFSH

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pituitary disease is due to GPA relapse, or if disease occurs or progresses while a patient is receiving cyclophosphamide [11]. However, pituitary hormone deficits often persist despite an adequate systemic disease response and resolution of head MRI findings. DI might be potentially reversible, thus desmopressin acetate (dDAVP) therapy holidays are encouraged in all patients [11]. Follow-up should include both imaging and pituitary function assessments.

Granulomatous Hypophysitis Idiopathic granulomatous hypophysitis is rare (,100 cases have been reported) and most often it mimics symptoms associated with a pituitary adenoma at presentation [14]. Initially described by Simmonds, in 1917, the pathogenesis remains unknown. Males and females are affected equally. Diagnosis is confirmed by histopathology of a resected tissue [15]. Clinical presentation often includes headache, with a few patients presenting with isolated hypopituitarism. Visual field defects, ophthalmoplegia, nausea/ vomiting, DI, hyperprolactinemia, and fatigue are also common presentations. Prolactin (PRL) may be high secondary to stalk involvement or low, usually indicative of permanent panhypopituitarism. Adrenal insufficiency (AI) has rarely been reported in association with this type of hypophysitis. On imaging, the pituitary is frequently diffusely enlarged and a thickened stalk is infrequently observed.

Necrotizing Hypophysitis Necrotizing hypophysitis is extremely rare and may represent a separate disease entity or a variant of other types of hypophysitis. Histologically, diffuse necrosis is surrounded by dense infiltration with lymphocytes, plasma cells, and a few eosinophils with considerable fibrosis. Presentation can be insidious with a risk of unrecognized AI and a possible catastrophic outcome if not appropriately treated. The natural history of any inflammatory hypophysitis remains elusive and treatment is controversial. High-dose GCs therapy or antiinflammatory and immunosuppressive (methotrexate, cyclosporine A, azathioprine) treatments used as preoperative medical management and postoperative therapy have been reported, with variable outcomes [14]. Transsphenoidal surgery is, however, both diagnostic and therapeutic and, therefore, should be performed in cases with progressive optic chiasm compression, and/or when a definitive diagnosis is

required for an atypical presentation. Spontaneous remission has not been reported in granulomatous or necrotizing hypophysitis.

Langerhans Cell Histiocytosis Langerhans cell histiocytosis (LCH), also known as histiocytosis X or Hand Schuller Christian disease, is characterized by the involvement of more than one site or system by lesions composed of lipid-laden histiocytes, eosinophils, lymphocytes, and plasma cells. The hypothalamus and posterior pituitary are frequently involved. DI is the most common endocrine disturbance, occurring in approximately one-quarter of patients, followed by growth retardation in children, and other anterior pituitary hormone deficiencies in a minority of subjects [16]. Hyperprolactinemia, observed in some patients, points to a hypothalamic or stalk disturbance, and may contribute to the suppression of gonadotrophin secretion. These patients respond to growth hormone (GH)-releasing hormone (GHRH) and gonadotrophin-releasing hormone (GnRH), especially when given in repeated pulsatile doses [17], reflecting hypothalamic or stalk etiology for pituitary failure. Autoimmune factors may also play a role in the pathogenesis of LCH. Autoantibodies to vasopressinsecreting hypothalamic cells have been detected in 54% of LCH patients with DI. Since histiocytes may function as antigen-presenting cells, hypothalamic infiltration could lead to immunologically mediated destruction of vasopressin neurons. Such a mechanism could account for the occurrence of DI in patients who have not demonstrated hypothalamic or posterior pituitary lesions at autopsy. An enhancing mass lesion on CT or a bright, gadolinium-enhancing area on MR images represents the usual radiological appearance. Thickening of the pituitary stalk is common and the posterior pituitary bright spot is frequently absent in patients with DI [16]. GCs, cytotoxic chemotherapy and radiation therapy are rarely effective in restoring endocrine function in patients with hypothalamic involvement [16]. Hormone replacement is prescribed as indicated.

Erdheim Chester Disease ECD, also known as Erdheim Chester syndrome or polyostotic sclerosing histiocytosis, is a rare form of non-LCH with multiorgan involvement. There has been a dramatic rise in the incidence and now patients are diagnosed much earlier. Most patients are affected in the fifth decade of life, with a slight male preponderance [18].

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AUTOIMMUNE

ECD is a clonal, neoplastic disorder originating from monocytic myeloid blood components that exhibit prominent inflammatory characteristics. Most of these clones seem to be predominantly driven by molecular elements that involve the RAS/RAF/MEK/ ERK signal transduction pathway, such as BRAF and NRAS, among others [19]. A V600E BRAF mutation in approximately 50% of patients has been reported [19]. Clinical manifestations are protean and virtually every organ system can be affected. DI of unexplained etiology may precede the onset of other disease symptoms by as many as 12 years. Curiously, anatomical disruptions of the hypothalamic pituitary region may appear on MRI months to years following diagnosis. This time delay suggests that ECD indolently progresses from microscopic involvement (with the functional disturbance of the posterior pituitary) to a macroscopic process, evident on MRI as the absence of a high-signal intensity of the posterior pituitary on T1weighted images and/or as thickening of the pituitary stalk. Other common clinical features include skeletal involvement with typical bilateral osteosclerotic lesions of long bones of the lower limbs, and cardiovascular involvement with circumferential thickening of the aorta and retroperitoneal fibrosis. Cardiovascular and CNS involvement are associated with a poor prognosis. Biopsy is required to confirm a definite diagnosis, typically with the histological identification of CD68 1 /CD1a-/S100-low, Langerin (CD207)-foamy or lipid-laden histiocytes. Interferon treatment is the established ECD therapy and might improve pituitary enlargement/stalk thickening. Vinblastine has been administered to ECD patients as a single agent or in combination with interferon with anecdotal reports of objective response. The BRAF-inhibitor vemurafenib has also been used in small groups of ECD patients. Despite improvement seen on imaging, the complete recovery of pituitary function after treatment has not been documented [18 21].

AUTOIMMUNE Lymphocytic Hypophysitis Lymphocytic hypophysitis (LyH) is considered the most common hypophysitis, and was first described at autopsy of a deceased female, 14 months postpartum, with a diffusely infiltrated anterior pituitary, Hashimoto’s thyroiditis, and atrophic adrenals [22]. There is a large female predominance, with 81% of cases reported in women during late pregnancy or

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postpartum [23]. LyH is associated, in some cases, with other types of endocrine autoimmunity [23]. Antibodies to pituitary tissue have been found in some LyH patients and in a variety of other endocrine autoimmune disorders, as well as in some patients with the primary empty sella syndrome and Sheehan syndrome. However, the functional significance of these antibodies remains unclear [15,23,24]. Two other novel forms of hypophysitis have been described: drug-induced hypophysitis related to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody therapy (see chapter: Drugs and Pituitary Function) and hypophysitis due to immunoglobulin (Ig) G4-related disease (IgG4-RD) [15,25].

Immunoglobulin G4-related Hypophysitis Immunoglobulin G4-related hypophysitis is a novel disease, characterized by elevated serum IgG4 concentration and infiltration of IgG4-positive plasma cells in the pituitary. Immunoglobulin G4-related disease (G4RD) is an increasingly recognized immune-mediated condition comprised of a collection of disorders that share pathologic, serologic, and clinical features. Pathogenesis is poorly understood, with findings consistent with both an autoimmune disorder and an allergic disorder. Immunoglobulin G4-RD occurs more frequently in middle-aged and older men [25 27]. The role of serum IgG4 levels in IgG4-related hypophysitis remains controversial; typically, levels are elevated with a decrease upon the initiation of GC therapy and in later disease stages; however, IgG4 levels can also be normal throughout the course of the disease. Although some criteria for diagnosis of IgG4-related hypophysitis have been proposed, they are not fully established; however, on biopsy, the presence of a mononuclear cell infiltrate rich in plasma cells within the pituitary gland appears pathognomonic. The presence of at least 10 IgG4-producing plasma cells has been deemed as required for diagnosis. In other cases, pituitary imaging in addition to the presence of IgG4 lesions in other organs could be viewed as sufficient. It has been proposed that a combination of three factors, namely increased serum IgG4 levels, enlarged pituitary on MRI, and prompt response to GCs, establishes a diagnosis of IgG4-related hypophysitis [25]. IgG4-related hypophysitis is responsive to GC treatment; thus, pituitary surgery should be reserved only for extreme or resistant cases. Alternative therapy with azathioprine resulted in marked improvement in a patient with worsening pituitary enlargement while receiving high-dose GCs [28].

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Polyglandular Autoimmune States Clinical involvement of the pituitary is rare in polyglandular autoimmune states [29].

AMYLOIDOSIS The amyloidoses comprise a group of diseases characterized by progressive deposition of insoluble proteinaceous material in the extracellular spaces of organs and tissues [30]. Microscopic amyloid deposits are frequently observed in blood vessel walls and interstitial areas in normal human pituitaries derived from elderly subjects. However, amyloid may be deposited in pituitary blood vessels in patients with systemic amyloidosis, usually with intact pituitary function. Very few cases of hypopituitarism have been reported (e.g., in a postmortem examination panhypopituitarism and AI were found to be associated with systemic amyloidosis caused by tuberculosis). Deposition of endocrine amyloid has been demonstrated in approximately twothirds of PRL-secreting, and GH-secreting adenomas, and in half of nonfunctioning adenomas [31,32].

INFECTIOUS DISEASES Acquired Immune Deficiency Syndrome In acquired immune deficiency syndrome (AIDS), the hypothalamus and pituitary may be directly infected with the human immunodeficiency virus (HIV) or any other of a variety of opportunistic pathogens, such as cytomegalovirus, Pneumocystis carinii, Cryptococcus neoformans, and Toxoplasma gondii [33]; in some cases, pituitary function is also impaired [33]. CNS lymphomas may also involve the pituitary of AIDS patients. Even in the absence of direct pituitary infection, functional abnormalities of the pituitary are common in AIDS patients, reflecting changes that accompany severe systemic illness (see below) and include euthyroid sick syndrome and hypogonadotrophic hypogonadism. Occasionally, patients may have GH deficiency and enhanced secretion of thyroid stimulation hormone (TSH), luteinizing hormone (LH), GH, or PRL [34]. Hyponatremia due to SIADH is common and usually secondary to pulmonary or CNS pathology [35]. The hypothalamic pituitary adrenal (HPA) axis is frequently affected and both AI (frank or subclinical) and hypercortisolism (with either low or high adrenocorticotrophic hormone; ACTH) have been reported [36]. The cause of AI in HIV is multifactorial and

includes infection, hemorrhage, and necrosis at either the pituitary or adrenal level. High-dose GC treatment and megestrol acetate also play a role in HPA suppression. A large proportion of patients with HIV have increased basal cortisol and ACTH levels, but blunted responses to corticotrophin-releasing hormone (CRH). The mechanism proposed is a steroidogenesis shift from dehydroepiandrosterone (DHEA) and aldosterone to cortisol production and cytokine stimulation of the HPA axis; however, negative feedback appears intact [36]. Use of antiretroviral agents, while lifesaving, has led to the emergence of several comorbidities, including insulin resistance and lipodystrophy. The characteristic phenotype of treated AIDS patients is relatively similar to Cushing’s syndrome and the term pseudoCushing’s was initially used [36]. Partial GC resistance (with increased GC receptor and decreased GC affinity) has been observed in a subset of AIDS patients, possibly owing to HIV-induced altered cytokine secretion and action [36]. Gonadal function is also affected in patients with HIV, changes being variable over the disease course. In men, testosterone levels are elevated or normal earlier in the course of the disease with high basal LH and greater LH responses to GnRH compared with controls. Free testosterone is also elevated; data on sex-hormone-binding globulin (SHBG) are controversial, but have been normal in most studies. Later in the course of the disease, a pattern of central hypogonadism ensues [35]. In women, menstrual periods are normal in the early stages of HIV infection, however, amenorrhea is frequently seen in patients with AIDS; an effect of medications used in patients with multiple complications cannot be excluded [35]. The spectrum of pituitary abnormalities in AIDS is broad, and is reflective of direct viral infection as well as effects of opportunistic infections and consequences of immune suppression.

Other Infectious Diseases Other infectious diseases affecting the pituitary include tuberculosis, syphilitic infection, Whipple and Chagas disease, and hemorrhagic fever. Tuberculosis has a predilection for the involvement of the basilar meninges and may therefore occasionally involve tissues in the sellar region, sometimes producing anterior or posterior pituitary insufficiency. Tuberculous meningitis may also cause SIADH. Radiologically, pituitary tuberculomas may resemble other sellar mass lesions; thickening of the stalk is seen in some cases [37].

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SNAKEBITE

Syphilitic infections of the hypothalamus or pituitary are rare and usually take the form of a gumma, which may be asymptomatic or may produce local mass effects or pituitary hypofunction [38]. Whipple disease and Chagas disease may also occasionally involve the pituitary. Although Lyme disease may affect the CNS, there have been no reports of pituitary dysfunction in this condition. Hemorrhagic fever with renal syndrome is a viral illness characterized by fever, hypotension, capillary leak, and acute renal failure; several varieties of Asian and European hantaviruses cause the syndrome. Approximately 20% of infected patients in one series developed pituitary hormone deficiencies, including pituitary atrophy and empty sella. Autopsy studies have shown pituitary hemorrhage and necrosis [39,40]. A wide variety of viral infections causing encephalitis may occasionally result in hypothalamic pituitary dysfunction [39].

Pituitary Abscess Pituitary abscess is a rare but serious intrasellar infection. In one series [41], patients presented with complaints and physical findings consistent with a pituitary mass, but rarely with clinical evidence of a serious infection. Headache, endocrine abnormalities, and visual changes were the most common features. Fever, peripheral leukocytosis, and meningismus were also present in one-third of patients. A sellar mass with peripheral enhancement is suggestive of an abscess; however, this finding may be absent in some cases [42]. Interestingly, in most cases, a diagnosis is made when a surgeon discovers a pustular cystic mass during surgery; the rate at which a pituitary abscess is diagnosed before surgery seems however to be rising [43]. Antibiotic therapy is indicated for septic patients, or for patients in whom specific organisms are identified from cultures obtained during surgery. Mortality is overall higher (8.3%) than for other pituitary pathologies. With appropriate treatment, headache, and visual changes may improve; however, endocrine dysfunction likely persists and requires hormonal replacement therapy.

IRON OVERLOAD Patients with excessive tissue iron deposits from idiopathic hemochromatosis, multiple transfusions, or prolonged use of pharmaceutical iron supplementation develop hypogonadism, in more than half of cases [44,45]. Hypopituitarism, particularly corticotrophic insufficiency, seems to be prevalent in a considerable

number of middle-aged patients with hereditary hemochromatosis. Despite normal somatotrophic function, low insulin-like growth factor 1 (IGF-1) serum concentrations may be found in a subgroup of hemochromatosis patients [46]. In a study of patients with transfusion-associated hemochromatosis [47], the most common pituitary hormonal deficiency involved the pituitary gonadal axis; 54% of the total subjects had hypogonadotrophic hypogonadism. Two patients had blunted cortisol responses to CRH stimulation. No patient had deficient GH or thyrotrophin TSH. Female patients seem to be less affected than males, perhaps because of the protective effects of menstrual blood loss. Pituitary gonadotrophin insufficiency is responsible for hypogonadism in most cases [48], although occasional patients appear to have primary testicular failure or hypothalamic pathology, laboratory abnormalities, including low basal serum gonadotrophin levels, impaired GnRH, even with prolonged, pulsatile GnRH administration, and generally intact testosterone response to human chorionic gonadotrophin (hCG) stimulation. However, secretion of GH, TSH, and ACTH is normal in most patients [44,45]. A minority of patients have modestly impaired PRL secretion [44,45]. Histopathology studies have revealed that pituitary iron deposits are localized primarily to gonadotrophs and, less frequently, to lactotrophs. Pituitary iron deposition may also be visualized radiologically. In hemochromatosis, the anterior pituitary may exhibit abnormally low-signal intensity on T2-weighted MR images. Pituitary iron and volume might predict hypogonadism in transfusional iron overload [49]. However, many patients with moderate-to-severe pituitary iron overload retain normal gland volume and function, which represents a potential therapeutic window. There are conflicting results on changes in pituitary function after iron depletion. Gonadotrophin and thyrotrophin secretion and end-organ function may improve following iron-depletion therapy, but pituitary function may also remain unchanged. A subset of hypogonadal patients with preserved gland volumes could explain improvements in pituitary function observed following intensive chelation therapy [49]. In children who receive multiple transfusions for thalassemia major, early intensive iron chelation therapy may prevent pituitary damage and allow normal puberty. Liver transplant after neonatal hemochromatosis has been shown to reverse pituitary dysfunction [50].

SNAKEBITE In South Asia, bites and envenomation by Russell’s vipers (Daboia russelii and Daboia siamensis) are

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common [51]. Russell’s viper venom contains many toxins, including several biologically active procoagulant enzymes, such as activating factors V and X, and affects other steps in the blood-clotting cascade. Disseminated intravascular coagulation sometimes develops within 30 minutes [52]. Russell’s viper venom also contains a metalloproteinase “haemorrhagin”, which damages vascular endothelium and toxins that impair platelet function. Venom-induced disturbances lead to thrombosis and spontaneous hemorrhages, and subsequently to edema and shock. Acute renal failure is the usual cause of death. Focal hemorrhage and microvascular thrombin deposition in the pituitary may be responsible for the hemorrhagic infarction of the anterior pituitary [52] and functional consequences of acute and chronic pan-hypopituitarism in long-term survivors. Hypopituitarism may contribute to the morbidity and mortality of the acute stage of illness in the initial hours and days postenvenomation. Pituitary necrosis and hypopituitarism appear to be uncommon following the bites of other poisonous snake species, with one case being reported following a bite by the jararacucu of Brazil [53].

with stalk involvement, DI is the predominant clinical manifestation of hormonal deficiency. Occasionally, pituitary metastasis may be the presenting feature of an occult primary cancer. Pituitary gland metastases should be considered as part of the differential diagnosis for any patient presenting with a pituitary lesion, severe headache, extraocular palsies, and DI [9,58]. Stereotactic radiation therapy improves neurological symptoms and DI in some cases, but prognosis is dependent on the primary cancer source [60]. The degree of hypopituitarism can improve after successful treatment with chemotherapy, especially for lymphomas [57]. To avoid excess hormonal replacement, follow-up evaluation of endocrine function during and after chemotherapy is recommended.

GENETIC MULTIGLANDULAR TUMORAL SYNDROMES (SEE ALSO CHAPTER: GENETICS OF PITUITARY TUMOR SYNDROMES) Multiple Endocrine Neoplasia

METASTATIC CANCER (SEE ALSO CHAPTER: NONPITUITARY SELLAR MASSES) Cancer metastases have been found in the hypothalamus, pituitary, or sella area in approximately one-third of autopsied cancer cases [54]. Pituitary metastases are frequently located in the posterior lobe or pituitary stalk, which receives blood from the systemic circulation via the inferior hypophyseal artery. The anterior lobe, supplied principally by the hypothalamic pituitary portal system is less affected. The first case of metastasis to the pituitary gland was described in 1857 by Benjamin in an autopsy of a patient with disseminated melanoma [55]. Breast and lung cancers are the most common types associated with pituitary metastases followed by prostate, renal cell [56] gastrointestinal cancers, lymphoma, and melanoma [57 59]. However, in patients who present with a pituitary metastasis, the primary tumor might remain undiagnosed in 3% of cases, despite intensive investigation. In a retrospective study, the most common presenting signs and symptoms were headache (58%), followed by fatigue (50%), polyuria (50%), visual field defects (42%), and ophthalmoplegia (42%) [58]. Central hypothyroidism, DI, and/or AI, are reported in two-thirds of patients [9,58]. In patients

Pituitary tumors occur commonly in multiple endocrine neoplasia (MEN) syndrome type 1, along with pancreatic islet cell tumors and hyperparathyroidism. Patients with features of MEN type 2A (consisting of medullary carcinoma of the thyroid, pheochromocytoma, and hyperparathyroidism) also occasionally have pituitary adenomas, raising the concept of “overlap” syndromes. Many of these “overlap” cases involve the occurrence of both pheochromocytoma and acromegaly; however, Cushing’s and pheochromocytoma cases have been noted. Interestingly, in a few cases, the pheochromocytoma was noted to produce GHRH and the pituitary lesion was actually a somatotroph hyperplasia rather than an adenoma. Thus, at least some of these “overlap” cases may represent an isolated pheochromocytoma associated with a secondary pituitary hyperfunction. Similarly, other presumed cases of MEN type 1 represent an isolated pancreatic islet cell tumor that produced GHRH, resulting in somatotroph hyperplasia and acromegaly [29].

Carney Complex (see also chapter: Acromegaly) Carney complex consists of myxomas, spotty mucocutaneous pigmentation, and endocrine overactivity (testicular tumors, pigmented nodular adrenocortical hyperplasia, and acromegaly) [61,62]. First described

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OTHER STALK AND PITUITARY LESIONS (SEE ALSO CHAPTER: NONPITUITARY SELLAR MASSES)

by Dr J. Aidan Carney as “the complex of myxomas, spotting pigmentation and endocrine over-reactivity,” the syndrome is due to inactivating mutations in the gene coding for the 1a regulatory subunit of protein kinase A (PRKAR1A) [61,62]. In approximately 70% of Carney complex cases, there is an affected parent, and in the other patients, de novo germline mutations occur. In inherited cases, Carney complex is passed as an autosomal dominant trait with almost 100% penetrance. Up to 75% of patients have elevated GH, IGF-1, or PRL and/or an abnormal response of GH to oral glucose tolerance test, but no detectable tumor on imaging [62]. The incidence of acromegaly due to pituitary tumors is around 10 12%. Interestingly, in many patients who underwent surgery, pathology revealed somatomammotrophic hyperplasia, rather than adenoma per se. Biochemical resistance to somatostatin receptor ligands (SRLs) in these patients is variable. Prolactinomas are rarely described [61,62].

McCune Albright syndrome (see also chapter: Acromegaly) McCune Albright syndrome (MAS) is a distinctive form of both endocrine and nonendocrine neoplasia caused by a postzygotic mutation of the GNAS1 gene, with affected cells being distributed in a mosaic pattern. This unique molecular pathophysiology leads to variability in the clinical expression and severity of disease features [63]. MAS cyclic AMP-mediated stimulation of GH secretion appears to be responsible for occasional MAS acromegaly cases, due to activating mutations of the Gs alpha subunit of the G-protein, which activates adenylate cyclase [64]. Somatotroph hyperplasia constitutes the primary abnormality in acromegaly in MAS, and somatomammotroph hyperplasia has also been described. In addition to the presence of hyperplasia versus a defined tumor, surgery is complicated due to significant fibrous bone overgrowth and anatomical distortion. Most patients with MAS and acromegaly are treated with medical therapy. Current guidelines recommend strict GH/IGF-1 control to prevent further comorbidities, including worsening of craniofacial fibrous dysplasia. Some patients respond well to long-acting SRLs, but resistance has been reported. GH receptor antagonist treatment or combined medical therapy has been successful in some cases [65]. Radiotherapy is used with caution because of the concern for the increased risk of malignant transformation of fibrodysplastic bone.

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OTHER STALK AND PITUITARY LESIONS (SEE ALSO CHAPTER: NONPITUITARY SELLAR MASSES) Stalk Hemangioblastoma Hemangioblastomas (HBLs) of the sellar and suprasellar regions are rare. They constitute approximately 1.5 2.5% of all intracranial tumors [66], and 20 30% occur in association with von Hippel-Lindau (VHL) disease [67]. In general, a diagnosis of HBL in the sellar region is more common in younger patients than in those with cerebellar or spinal cases and most patients experience endocrinological dysfunction [66]. Radiologically, HBLs are often cystic, with a mural nodule within the wall of the cyst, and a lack of dural attachment. An accurate preoperative HBL diagnosis would be very challenging in patients with no other lesions, or VHL disease. A sporadic and isolated pituitary stalk HBL needs to be considered when a MRI study reveals the characteristic findings. Pituitary stalk HBLs often remain asymptomatic and can be managed with observation. Surgery should be reserved for patients with associated signs or symptoms. Complete surgical excision has been achieved in symptomatic pituitary stalk HBL; however, as expected, panhypopituitarism will ensue and life-long hormonal replacement is required.

Chordoid Glioma Chordoid glioma is classified by the World Health Organization as a neuroepithelial tumor of uncertain origin. Chordoid gliomas are located in the suprasellar region with the involvement of the hypothalamus and third ventricle. On MRI, primarily solid mass, and cystic components have been described. These gliomas homogeneously enhance with gadolinium administration, and as a result, the local involvement of the hypothalamic pituitary axis is expected. A predilection in middle- to older-aged women has been noted. Symptoms due to the mass effect of the optic apparatus and hypothalamic pituitary axis include headaches, weight gain, visual changes, and endocrine disturbances, particularly DI. Pathology consistently demonstrates chords and nests of epithelioid cells in a stromal matrix, along with “lymphoplasmocellular infiltrates with Russell bodies, as a rule.” Surgery is the treatment of choice, however, gross total resection is achieved in less than half of the patients treated. Mortality as a result of postoperative

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complications is high. The role of postoperative radiotherapy is unknown [68].

Hemangiopericytoma Intracranial hemangiopericytomas (HPCs) are rare aggressive tumors. Other than in the meninges, an HPC has rarely been reported in the periventricular and sellar region. While diagnosing an intracranial HPC, special attention should be given to identifying malignant features. Surgery and adjuvant radiation have been shown to be effective [69].

Fabry Disease Fabry disease (FD) is an X-linked lysosomal storage disease caused by the lack or incomplete activity of lysosomal enzyme α-galactosidase A (α-Gal A); rate of disease progression and specific organ damage is very variable. Cardiovascular and cerebrovascular disorders as well as renal insufficiency represent the major causes of morbidity and mortality, usually in patients between the ages of 40 and 50 years. Patients with FD have adrenal and thyroid abnormalities; however, the pituitary gland might theoretically represent an ideal target for FD because of the high vascularization and low proliferation rate. Maione et al. reported a high prevalence of empty sella and a significant reduction in pituitary gland size in patients with FD compared to a matched normal population [70]. α-Gal A enzyme activity was the strongest predictor of pituitary volume independent of age, regardless of the presence or absence of enzyme recombinant therapy. Interestingly, despite marked morphological changes, pituitary function seems to be normal in most cases; however, pituitary hormone appraisal should be periodically performed in FD patients who are already at risk of cardiovascular complications.

CHANGES IN PITUITARY MORPHOLOGY AND FUNCTION WITH AGING The overall size of the pituitary decreases slightly with age, although gland weight seems to be maintained, most likely due to a qualitative change in the pituitary; indeed, pituitary parenchymal cells decrease with age as fibrosis increases. Most of the decrease is related to fewer and smaller somatotrophs; lactotrophs change little and, conversely, gonadotrophs may be enlarged [71]. Functional pituitary changes are also evident with aging. In the absence of a specific disease state, the pituitary adrenal axis appears to be generally intact in the

elderly. However, both ACTH and cortisol levels may increase moderately with flattening of diurnal cortisol release and increased peripheral conversion from cortisone. GC negative feedback in the hypothalamus, hippocampus, and prefrontal cortex is reduced. Pregnenolone secretion, DHEA, and DHEAsulfate (both basal and stimulated) also decrease with aging. Aldosterone can either decrease with aging or increase in some patients as a result of higher ACTH stimulation [72 74]. Serum TSH increases slightly with advancing age, and TSH response to TRH is diminished in the elderly [75]; the TSH increase does not seem to be related to the prevalence of autoimmune thyroid failure, since it is observed even when subjects with positive antithyroid antibodies and any family history of thyroid disease are excluded [75]. With aging, serum T3 levels fall modestly, perhaps due to medical comorbidities and nonthyroidal illness syndrome, while serum T4 remains nearly stable [76]. The most dramatic change is loss of ovarian function at menopause, with a consequent elevation of serum LH and follicle-stimulating hormone (FSH) due to the activation of normal feedback mechanisms [77]. Similar changes, albeit more gradual and of lesser magnitude, occur in many normal elderly men, in whom some degree of testicular failure develops [78]. Total serum testosterone falls while serum SHBG increases, with more dramatic falls in free or bioavailable testosterone [72]. Serum LH and FSH are often normal, but may be elevated in some patients [72]. Although GnRH secretion appears to be diminished, testicular responsiveness to LH or hCG is also decreased. Concurrent obesity and medical comorbidities appear to further modulate the effects of aging [72]. Water metabolism disturbances represent an important risk in the elderly. Vasopressin is essential for circulatory and water homeostasis facilitated by vasopressin receptor type 1a (vascular), type 1b (pituitary), and type 2 (renal and vascular); and arginine vasopressin (AVP) secretion increases during aging. With aging, maximal urine concentration capacity decreases; up to 20% reduction in maximum urine osmolarity and almost half decrease in conserving solutes in the 60 80 years age group. This overall urine concentration decrease might be due to decreased renal responsiveness to AVP. Drugs affecting the regulation of AVP release, frequently used in the elderly, might also play a role. Interestingly, the AVP circadian rhythm seems to be lost in the elderly and has been deemed one of the causes for nocturia. Higher AVP levels might contribute to increased thrombotic risk. The thirst mechanism might also be affected with a higher osmotic set point than younger adults. Thus,

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both hyponatremia and hypernatremia risks increase with aging [79].

GENERAL EFFECTS OF SYSTEMIC ILLNESS ON PITUITARY FUNCTION Illness, injury, and stress, if sufficiently severe, produce a constellation of endocrine changes that are, in general, independent of the specific type of illness. ACTH and cortisol secretion are increased by acute stress and illness [80]. In addition to amplified release of CRH, other factors (e.g., vasopressin, interleukin-1) may also play a role in stimulating ACTH secretion [80,81]. Adrenal androgen secretion is acutely stimulated early, but falls below normal when an illness continues for a week or more [80]. Adrenal aldosterone secretion is impaired in critical illness despite stimulation with angiotensin II. The unified mechanism for these changes suggests an adaptive adrenocortical shift in steroid production away from androgens and mineralocorticoids in order to maximize cortisol secretion. The concept of “relative AI” has been proposed as a contributor to mortality in septic shock, but the existence, diagnosis, and treatment of this condition are controversial. There is no readily available laboratory test to diagnose sepsis-related AI, and, in addition, there has been no clear survival benefit shown for GC therapy in these patients [82,83]. Prolonged physical stress or illness (e.g., major surgery, severe infections) results in the “nonthyroidal illness syndrome” or “euthyroid sick syndrome” [84]. In this syndrome, production of T3 is decreased, while the production of reverse T3 is increased initially in the peripheral metabolism of T4; moreover, deiodination of reverse T3 is impaired, further contributing to a rise in its serum concentration. Serum T4 and TSH levels are normal, and T3 and reverse T3 concentrations return to normal following recovery. In advanced stages with severe, usually life-threatening, illness, a similar pattern of serum T3 and reverse T3 is observed, but serum T4 is decreased in these patients [84]. The fall in total T4 seems to be due to concentration or affinity changes of serum carrier proteins for thyroid hormones. Free T4 levels are generally normal, which would predict normal levels of TSH and, indeed, this seems to be the case in most patients. In the recovery phase, serum TSH may be transiently, mildly elevated, with a later return to normal as serum T4 normalizes. Patients with nonthyroidal illness syndrome do not achieve clinical benefit from treatment with either T3 or T4 [85]. Mortality risk seems also unaffected by replacement treatment in these patients.

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GH secretion is frequently stimulated by stress, injury, and illness, though serum IGF-1 is low. Both GH and IGF-1 return to normal after recovery [86]. Stress, injury, and illness often raise serum PRL levels, which return to normal with recovery. Mechanisms producing hypogonadotrophic hypogonadism during illness are unclear; the suppressive effect of circulating cytokines, increased aromatization of androgens to estrogens and overproduction of endogenous opioids have been postulated to play a role [87]. In the initial hours of stress or surgery, there is a transient rise in serum LH, which rapidly returns to normal [87,88]. When stress or illness continues for days to weeks, serum LH and FSH remain normal or fall to low levels, associated with a fall in serum total testosterone and free testosterone in men; similar decreases in serum gonadotrophins have been observed in women [88]. GnRH administration stimulates LH and FSH secretion during severe illness, although these responses are sometimes blunted [87]. These observations may relate, in part, to the duration and severity of illness and to the need for repetitive GnRH stimulation to elicit normal gonadotrophin responses in hypothalamic disorders. Several authors have drawn parallels between the hypogonadism of severe illness and nonthyroidal illness syndrome; indeed, the occurrence of hypogonadism seems to correlate with depressed serum thyroid hormone levels [87]. Although the mechanisms may be different, the changes in both cases may operate to conserve the body’s resources during periods of extreme stress.

PITUITARY ALTERATIONS ASSOCIATED WITH SPECIFIC SYSTEMIC DISORDERS Obesity Several aspects of pituitary function are altered in obesity. Serum cortisol, urinary free cortisol (UFC), and plasma ACTH are usually normal in obese subjects, though some patients with abdominal obesity may have modest increases in UFC, salivary cortisol, and cortisol responses to stress, ACTH, or administration of CRH-AVP. Serum concentrations of thyroid hormones and TSH are normal in obesity, both basally and following stimulation by TRH. The most dramatic hormonal change is decreased GH secretion [89,90]. Earlier studies noted that obesity was associated with GH suppression and decreased linear growth in animals. Further research showed a clear relationship between adipose tissue stores and regulation of the GH axis [90].

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Mechanisms responsible for decreased GH secretion in obesity are multifactorial, and a defect in ghrelin secretion or action seems to also play a role. Both spontaneous and stimulated GH secretion are blunted, and are improved following weight loss. Peak stimulated GH and IGF-1 are significantly discordant when used to identify subjects with reduced GH secretion in obesity [91]. Despite decreased GH secretion, total serum IGF-1 levels are generally within the normal range, although somewhat lower than age-matched subjects of normal weight. Serum free IGF-1 levels are increased, however, and may contribute to the suppression of GH secretion in obesity [92]. However, it is unclear if abnormalities of GH secretion in obesity (either spontaneous or stimulated) represent true GH deficiency; adipose tissue, metabolic and nutritional status are important confounding factors in the interpretation of biochemical testing. Consensus guidelines recommend using body mass index (BMI)-dependent cutoff for most GH stimulation tests (including GHRH arginine and glucagon) to avoid false-positive results [93]. As GH regulates nuclear factor NF-KB activity in adipocytes and macrophages, the hormone might modulate chronic inflammation and insulin resistance. Indeed, in most obese patients, reduced GH secretion is associated with a more abnormal metabolic phenotype in obesity, characterized by increased carotid media thickness, dyslipidemia, insulin resistance, and inflammation [94]. PRL secretion is minimally altered in obesity. Basal and 24-hour integrated PRL levels are normal in obese subjects, although PRL responses to a variety of pharmacologic agents (TRH, dopamine antagonists) may be blunted in some patients [95]. Although most of these responses improve after weight loss, the blunted PRL response to insulin-induced hypoglycemia may persist, suggesting an intrinsic hypothalamic abnormality [95]. In most obese men, testicular function is normal. Serum total testosterone may be low, however, due to reduced concentrations of carrier protein, SHBG; free testosterone concentrations are usually normal, as are serum LH and FSH [95]. In some extremely obese men, however, even free testosterone is depressed, likely due to LH suppression by excessive estrogens produced by androgen aromatization in adipose tissue [95]. Obese postmenopausal women also have increased androgen aromatization to estrogens in adipose tissue. Obese premenopausal women, especially those with abdominal obesity, may exhibit hyperandrogenism, increased serum LH, and polycystic ovary syndrome.

Malnutrition In malnutrition, ACTH and cortisol secretion are increased [96 98]. Starvation or severe carbohydrate restriction induces the “euthyroid sick” or “nonthyroidal illness” syndrome, often accompanied by a slight decrease in basal and TRH-stimulated TSH secretion [84,96,98]. Malnutrition impairs production of IGF-1 [99], and is often associated with increased basal GH secretion, perhaps due to decreased negative feedback [99]; additionally, paradoxical increases in GH may be seen following glucose loading or TRH administration [99]. Basal serum PRL concentrations are usually not altered by fasting or malnutrition, but the PRL response to TRH may be diminished, modestly increased, or unchanged [96]. Changes in gonadotrophin secretion induced by malnutrition are variable; hypogonadism is frequently observed in states of prolonged decreases in calorie intake with normal or slightly decreased serum gonadotrophins [96 99]. This variation reflects specific nutrient deficiencies, initial body weight, and concurrent illness. The gonadotrophin response to GnRH is, on balance, probably normal [99,100]. Taken together, these findings suggest a primary hypothalamic abnormality in malnutrition- or fasting-induced pituitary hormone changes.

Anorexia Nervosa Anorexia nervosa is a disorder characterized by an obsessive desire to lose weight by refusing to eat despite severe malnutrition; the disorder is prevalent in adolescent girls and women, but also occurs in boys and young men, in approximately 10% or more of cases. Many endocrine axes are affected in response to very low caloric intake, and marked fat reduction, as well as absolute protein and carbohydrate intake. The HPA axis is usually activated in these patients; besides direct CRH ACTH cortisol activation, ghrelin also seems to play a role. However, levels of UFC are lower than 2 3 ULN in most patients and they lack cushingoid features. Cortisol increase may represent an adaptive mechanism to preserve glucose levels in the absence of caloric intake. The cortisol increase has been linked to both favorable prognosis for weight regain after treatment, but also greater eating psychopathology, independent of BMI [101]. Changes in the thyroid axis mimic those seen in euthyroid sick syndrome; low total T3, normal, low normal or even low free T4 with normal or low normal TSH. The TSH response to TRH is blunted [101].

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Despite higher GH levels, IGF-1 levels are low, reflecting GH resistance. This has been further demonstrated by blunted or absent IGF-1 increase after supraphysiological doses of GH administration. Both GH receptor downregulation and low GH binding protein levels have been implicated as mechanisms of GH resistance, which is reversed with weight gain. GH increase is most likely due to decreased negative feedback from low circulating IGF-1 levels, and ghrelin increase. While bone metabolism is affected due to low IGF-1, GH effects not mediated by IGF-1, such as those on glucose and lipid metabolism, are maintained [102]. Women with anorexia nervosa have hypothalamic amenorrhea; LH pulses have very low amplitude, similar to those seen in prepubescent girls. Most women restore gonadal function after an increase in fat mass, however there is usually a lag between weight regain and menstrual cycle restoration. Men with anorexia nervosa have abnormal gonadal function with low testosterone and gonadotrophins, as well as low leptin levels [101]. The posterior pituitary seems to also be affected, as low plasma sodium and osmolarity are frequently observed. Antidiuretic hormone is high at baseline, but, interestingly, does not increase appropriately after water deprivation. Oxytocin secretion seems to also be impaired and has been linked to lower fat mass.

Diabetes Mellitus A variety of pituitary abnormalities have been reported in patients with DM. Infarction of the adenohypophysis may occur without previous hypotension, and may lead to hypopituitarism. A significant association between pituitary autoimmunity and type 1 DM has been reported [103]. Functional abnormalities may also occur in the absence of anatomic changes; many of these reports are contradictory, with normal, increased, or decreased responses to various stimulation tests recorded [104]. The most consistent abnormality is elevated basal serum GH in poorly controlled diabetics; investigators have inconsistently found that basal GH returns to normal when diabetes control improves. Despite chronic hyperglycemia, GH responses to provocative stimuli are generally intact [104]. Serum IGF-1 levels are decreased in poorly controlled diabetics, and increase with improved diabetic control [104].

Chronic Kidney Disease Hormonal abnormalities at the level of the hypothalamic pituitary axis are frequently noted in patients

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with worsening renal function and chronic kidney disease (CKD) [105]. Few studies have evaluated the HPA axis in CKD and data interpretation is limited by concomitant and multiple medications used by most patients. Overall, the HPA axis is likely upregulated, with increased serum concentrations of cortisol, with intact diurnal variation. Furthermore, cortisol levels after an overnight dexamethasone suppression test are often abnormal due to a prolonged halflife of serum cortisol, coupled with accelerated degradation and poor oral absorption of dexamethasone. Aldosterone is also high, especially in patients on hemodialysis (HD); 11-β HSD2 is reduced in patients receiving HD with consecutive increases in cortisol metabolites. High aldosterone, coupled with high cortisol levels, has been associated with sudden cardiac death and increased mortality [105]. The pituitary thyroid axis is mildly deranged in CKD, usually in parallel with renal function decline. Serum TSH is increased in about 15% of patients with a depressed response to TRH. In general, changes observed in uremia resemble those in “euthyroid sick” or “nonthyroidal illness” syndrome, with the exception of normal serum reverse T3 level. HD does not improve thyroid abnormalities. Iodine accumulation may also inhibit thyroid T4 production (Wolff Chaikoff effect). However, renal transplantation may largely correct the thyroid axis; and residual abnormalities that persist after transplantation may be due to effects of immunosuppressive therapy [105]. Basal serum GH is normal or elevated in CKD and GH regulation is disturbed, with diminished responses to hypoglycemia, exaggerated responses to L-dopa and GHRH, and paradoxical GH responses to TRH and hyperglycemia. Serum IGF-1 concentrations are normal or elevated in uremic plasma, but bioactivity is decreased due to the presence of circulating inhibitors and changes in IGF-binding proteins. Serum PRL is modestly increased in many CKD patients, due to both increased secretion (suppressed dopaminergic activity) and decreased clearance. Secondary hyperparathyroidism might also cause hyperprolactinemia. Clinical consequences are reflected in commonly observed reproductive abnormalities in women and men with CKD. PRL may also impact cardiovascular risks; studies in CKD patients have shown a strong and independent association between cardiovascular outcomes, endothelial dysfunction, and hyperprolactinemia in CKD patients with and without HD. Interestingly, bromocriptine therapy reduces blood pressure and regression of left ventricular hypertrophy, but it is unclear if the effects were mediated by decreased PRL. Hyperprolactinemia

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persists despite dialysis, but is reversed by successful renal transplantation. Hypogonadism is common in CKD and probably multifactorial in origin. There is evidence for both central and gonadal defects in the pathogenesis of uremic hypogonadism. In most patients, serum gonadotrophins are normal or elevated, with a normal or blunted response to GnRH. Administration of estrogen does not induce a midcycle LH surge, which suggests a central hypothalamic abnormality. Testicular responsiveness to hCG is also blunted. Hypogonadism in women with CKD has been associated with depression, sleep disturbances, osteoporosis, decreased cognition, and increased cardiovascular mortality. Men with hypogonadism associated with CKD have increased risk of cardiovascular events and mortality risk, and decreased quality of life. Cardiovascular risk may possibly also be induced by testosterone supplementation in these patients. In HD patients, 50% of patients report no improvement in gonadal function by dialysis, but the disorder is mostly reversed by renal transplantation. It has been suggested that pituitary hormonal disorders associated with CKD can have a further detrimental effect on progression of CKD per se, and its chronic consequences [105].

Liver Disease Chronic liver disease causes a wide variety of endocrine disturbances. The pituitary adrenal axis is altered minimally in cirrhosis. However, serum concentrations of corticosteroid-binding globulin are often decreased, and the serum halflives of both cortisol and dexamethasone are prolonged in cirrhotics, apparently as a consequence of hepatic parenchymal damage. Thyroid hormone measurements are altered in patients with most chronic liver diseases, primarily as a consequence of changes in peripheral thyroid hormone metabolism. Thus, serum T3 is usually reduced and serum reverse T3 elevated in cirrhosis, while serum T4 is generally normal and serum TSH is normal or mildly elevated. This “euthyroid sick” or “nonthyroidal illness” syndrome appears to be related to the degree of hepatocyte dysfunction and is not a feature of portosystemic shunting. In contrast to advanced alcoholic cirrhosis, patients with infectious hepatitis, chronic active hepatitis, and primary biliary cirrhosis may have elevated serum levels of thyroxinebinding globulin, thereby raising total serum T4 and T3, although free hormone levels and serum TSH are usually normal [106,107].

In patients with cirrhosis, basal serum GH is increased, and paradoxical increases in GH are observed following glucose ingestion or TRH administration [108,109]. These features appear to be independent of the etiology or structural changes of liver disease, and are observed with cirrhosis of any cause. Serum levels of IGF-1 are depressed in patients with chronic liver disease and reduced negative feedback effects of IGF-1 on GH secretion may contribute to disordered GH regulation, as may changes in brain neurotransmitters that occur as a result of altered amino acid metabolism. Abnormalities of GH secretion appear to return toward normal after successful liver transplantation, although effects of immunosuppressive drugs and residual encephalopathy complicate interpretation of the results. Serum PRL is sometimes mildly elevated in cirrhotic patients; this may reflect potentiating effects of estrogens on PRL release and a disordered hypothalamic neurotransmitter function. Gonadal function and gonadotrophin secretion are altered in patients with cirrhosis; many of the changes appear to correlate with the degree of liver dysfunction, although some may be specifically due to toxic effects of ethanol on the testis. Men with advanced cirrhosis usually have decreased serum concentrations of total and free testosterone; estradiol is normal or mildly increased, and estrone considerably increased. Increased estrogen concentrations are primarily a consequence of increased peripheral aromatization of androgens (especially androstenedione) rather than decreased hepatic removal. Serum LH is normal or moderately elevated. It has been argued that the failure of LH to rise substantially in the face of low free testosterone concentrations suggests concurrent hypothalamic pituitary dysfunction; however, these findings may be due to gonadotrophinsuppressing effects of elevated serum estradiol, estrone, and other estrogen metabolites (e.g., 16hydroxy-estrone). Seminiferous tubule damage may occur in cirrhotics and alcoholics, with a rise in serum FSH concentrations. Gonadotrophin responses to GnRH are intact or blunted. Premenopausal women with alcoholic liver disease have lower serum estradiol but higher serum estrone levels than healthy women, again presumably due to altered peripheral steroid metabolism. Gonadotrophin concentrations are normal or low, and respond normally to GnRH. In summary, gonadal function is depressed in cirrhotics and further worsened by chronic alcohol intake. There is partial reversal of these abnormalities following liver transplantation [106,107].

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Shirley McCartney, PhD, Oregon Health & Science University, Portland, Oregon for editorial assistance.

Primary Adrenal Insufficiency Patients with primary AI may have serum TSH and PRL elevations, in addition to the elevation of ACTH secretion (which is expected due to feedback from loss of cortisol). TSH and PRL increases appear to be related to GC deficiency and are reversed with GC replacement therapy; however, concomitant primary hypothyroidism needs to be excluded.

Primary Hypothyroidism In addition to serum TSH elevation, other pituitary abnormalities are associated with primary hypothyroidism. Thyrotroph hyperplasia (due to the activation of the thyrotrophs by loss of thyroid hormone negative feedback) can sometimes mimic a pituitary tumor. Serum cortisol and ACTH concentrations are generally normal, though cortisol turnover can be decreased [110], GH synthesis is decreased, and secretion is blunted, both basally and in response to provocative testing [110,111]. Moderate hyperprolactinemia (usually less than 100 ng/mL) is occasionally seen, generally in patients with severe hypothyroidism; the mechanism is unclear, but may be due to TRH increase. In hypothyroid men, total, free, and bioavailable serum testosterone levels are low, as is serum SHBG; however, serum gonadotrophin levels are in the normal range, suggesting a hypothalamic pituitary defect; these changes are normalized when euthyroidism is restored. Severely hypothyroid children may experience precocious puberty associated with mildly elevated serum FSH levels [112]; very high serum TSH concentrations may also stimulate gonadal FSH receptor.

Hyperthyroidism Thyroid-stimulating hormone secretion is suppressed by negative feedback in patients with thyroid hormone excess. GH secretion is normal or blunted [111]. In addition, PRL response to TRH or dopamine antagonists is usually blunted in hyperthyroidism, although the basal serum PRL level is normal [113]. Serum gonadotrophin concentrations are normal or mildly elevated in hyperthyroid patients.

Acknowledgments The author acknowledges Harold E. Carlson, MD, SUNY, Stony Brook, NY, the author of the corresponding chapter in the previous edition of this textbook, for use of material in this chapter, and

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[20] Cives M, et al. Erdheim Chester disease: a systematic review. Crit Rev Oncol/Hematol 2015;95(1):1 11. [21] Gianfreda D, et al. Sirolimus plus prednisone for ErdheimChester disease: an open-label trial. Blood 2015;126(10): 1163 71. [22] Goudie RB, Pinkerton PH. Anterior hypophysitis and Hashimoto’s disease in a young woman. J Pathol Bacteriol 1962;83:584 5. [23] Caturegli P, et al. Autoimmune hypophysitis. Endocr Rev 2005;26(5):599 614. [24] Ricciuti A, et al. Detection of pituitary antibodies by immunofluorescence: methodological approach and results in patients with pituitary diseases. J Clin Endocrinol Metab 2014;99 (5):1758 66. [25] Leporati P, et al. IgG4-related hypophysitis: a new addition to the hypophysitis spectrum. J Clin Endocrinol Metabol 2011;96 (7):1971 80. [26] Kamisawa T, et al. IgG4-related disease. Lancet 2015;385 (9976):1460 71. [27] Haraguchi A, et al. Putative IgG4-related pituitary disease with hypopituitarism and/or diabetes insipidus accompanied with elevated serum levels of IgG4. Endocr J 2010;57(8):719 25. [28] Caputo C, et al. Hypophysitis due to IgG4-related disease responding to treatment with azathioprine: an alternative to corticosteroid therapy. Pituitary 2014;17(3):251 6. [29] Owen CJ, Cheetham TD. Diagnosis and management of polyendocrinopathy syndromes. Endocrinol Metab Clin North Am 2009;38(2):419 36 x. [30] Ozdemir D, Dagdelen S, Erbas T. Endocrine involvement in systemic amyloidosis. Endocr Pract 2010;16(6):1056 63. [31] Ishihara T, et al. Amyloid protein of vessels in leptomeninges, cortices, choroid plexuses, and pituitary glands from patients with systemic amyloidosis. Hum Pathol 1989;20(9):891 5. [32] Las MS, Surks MI. Hypopituitarism associated with systemic amyloidosis. N Y State J Med 1983;83(11 12):1183 5. [33] Milligan SA, et al. Toxoplasmosis presenting as panhypopituitarism in a patient with the acquired immune deficiency syndrome. Am J Med 1984;77(4):760 4. [34] Dobs AS, et al. Endocrine disorders in men infected with human immunodeficiency virus. Am J Med 1988;84(3 Pt 2): 611 16. [35] Sellmeyer DE, Grunfeld C. Endocrine and metabolic disturbances in human immunodeficiency virus infection and the acquired immune deficiency syndrome. Endocr Rev 1996;17 (5):518 32. [36] Chrousos GP, Zapanti ED. Hypothalamic-pituitary-adrenal axis in HIV infection and disease. Endocrinol Metab Clin North Am 2014;43(3):791 806. [37] Beatrice AM, Selvan C, Mukhopadhyay S. Pituitary dysfunction in infective brain diseases. Indian J Endocrinol Metab 2013;17 (Suppl. 3):S608 11. [38] Bricaire L, et al. The great imitator in endocrinology: a painful hypophysitis mimicking a pituitary tumor. J Clin Endocrinol Metab 2015;100(8):2837 40. [39] Schaefer S, et al. Hypothalamic-pituitary insufficiency following infectious diseases of the central nervous system. Eur J Endocrinol 2008;158(1):3 9. [40] Stojanovic M, et al. High risk of hypopituitarism in patients who recovered from hemorrhagic fever with renal syndrome. J Clin Endocrinol Metab 2008;93(7):2722 8. [41] Vates GE, Berger MS, Wilson CB. Diagnosis and management of pituitary abscess: a review of twenty-four cases. J Neurosurg 2001;95(2):233 41. [42] Dalan R, Leow MKS. Pituitary abscess: our experience with a case and a review of the literature. Pituitary 2007;11(3):299 306.

[43] Zhang X, et al. Diagnosis and minimally invasive surgery for the pituitary abscess: a review of twenty nine cases. Clin Neurol Neurosurg 2012;114(7):957 61. [44] McDermott JH, Walsh CH. Hypogonadism in hereditary hemochromatosis. J Clin Endocrinol Metab 2005;90(4):2451 5. [45] Schafer AI, et al. Clinical consequences of acquired transfusional iron overload in adults. N Engl J Med 1981;304 (6):319 24. [46] Uitz PM, et al. Pituitary function in patients with hereditary haemochromatosis. Horm Metab Res 2013;45(1):54 61. [47] Kim MK, et al. Endocrinopathies in transfusion-associated iron overload. Clin Endocrinol (Oxf) 2013;78(2):271 7. [48] Young J. Approach to the male patient with congenital hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2012;97 (3):707 18. [49] Noetzli LJ, et al. Pituitary iron and volume predict hypogonadism in transfusional iron overload. Am J Hematol 2012;87 (2):167 71. [50] Indolfi G, et al. Neonatal haemochromatosis with reversible pituitary involvement. Transpl Int 2014;27(8):e76 9. [51] Schiermeier Q. Africa braced for snakebite crisis. Nature 2015;525(7569):299. [52] Antonypillai CN, et al. Hypopituitarism following envenoming by Russell’s Vipers (Daboia siamensis and D. russelii) resembling Sheehan’s syndrome: first case report from Sri Lanka, a review of the literature and recommendations for endocrine management. QJM 2010;104(2):97 108. [53] Milani Junior R, et al. Snake bites by the jararacucu (Bothrops jararacussu): clinicopathological studies of 29 proven cases in Sao Paulo State, Brazil. QJM 1997;90(5):323 34. [54] Al-Aridi R, et al. Clinical and biochemical characteristic features of metastatic cancer to the sella turcica: an analytical review. Pituitary 2014;17(6):575 87. [55] Komninos J, et al. Tumors metastatic to the pituitary gland: case report and literature review. J Clin Endocrinol Metab 2004;89(2):574 80. [56] Gopan T, et al. Symptomatic pituitary metastases from renal cell carcinoma. Pituitary 2007;10(3):251 9. [57] Nakashima Y, et al. Pituitary and adrenal involvement in diffuse large B-cell lymphoma, with recovery of their function after chemotherapy. BMC Endocr Disord 2013;13(1):45. [58] Ariel D, et al. Clinical characteristics and pituitary dysfunction in patients with metastatic cancer to the sella. Endocr Pract 2013;19(6):914 19. [59] Sidiropoulos M, et al. Melanoma of the sellar region mimicking pituitary adenoma. Neuropathology 2012;33(2):175 8. [60] Kano H, et al. Stereotactic radiosurgery for pituitary metastases. Surg Neurol 2009;72(3):248 55 discussion 255-6. [61] Bertherat J. Carney complex (CNC). Orphanet J Rare Dis 2006;1:21. [62] Correa R, Salpea P, Stratakis CA. Carney complex: an update. Eur J Endocrinol 2015;173(4):M85 97. [63] Salenave S, et al. Acromegaly and McCune-Albright syndrome. J Clin Endocrinol Metab 2014;99(6):1955 69. [64] Vasilev V, et al. McCune-Albright syndrome: a detailed pathological and genetic analysis of disease effects in an adult patient. J Clin Endocrinol Metab 2014;99(10):E2029 38. [65] Galland F, et al. McCune-Albright syndrome and acromegaly: effects of hypothalamopituitary radiotherapy and/or pegvisomant in somatostatin analog-resistant patients. J Clin Endocrinol Metab 2006;91(12):4957 61. [66] Lonser RR, et al. Pituitary stalk hemangioblastomas in von Hippel-Lindau disease. J Neurosurg 2009;110(2):350 3. [67] Neumann HP, et al. Von Hippel-Lindau syndrome. Brain Pathol 1995;5(2):181 93.

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[68] Dziurzynski K, et al. Diabetes insipidus, panhypopituitarism, and severe mental status deterioration in a patient with chordoid glioma: case report and literature review. Endocr Pract 2009;15(3):240 5. [69] Juco J, et al. Hemangiopericytoma of the sella mimicking pituitary adenoma: case report and review of the literature. Clin Neuropathol 2007;26(11):288 93. [70] Maione L, et al. Pituitary function and morphology in Fabry disease. Endocrine 2015;50(2):483 8. [71] Kovacs K, Horvath E, Ezrin C. Anatomy and histology of the normal and abnormal pituitary gland. In: De Groot LJ, et al., editors. Endocrinology. Philadelphia: WB Saunders Co; 1989. p. 264 83. [72] Kaufman JM, Vermeulen A. The decline of androgen levels in elderly men and its clinical and therapeutic implications. Endocr Rev 2005;26(6):833 76. [73] Seeman TE, Robbins RJ. Aging and hypothalamic-pituitaryadrenal response to challenge in humans. Endocr Rev 1994;15 (2):233 60. [74] Gupta D, Morley JE. Hypothalamic-pituitary-adrenal (HPA) axis and aging. Compr Physiol 2014;4(4):1495 510. [75] Stan M, Morris JC. Thyrotropin-axis adaptation in aging and chronic disease. Endocrinol Metab Clin North Am 2005;34 (4):973 92 x. [76] Visser WE, Visser TJ, Peeters RP. Thyroid disorders in older adults. Endocrinol Metab Clin North Am 2013;42(2):287 303. [77] Hall JE. Neuroendocrine changes with reproductive aging in women. Semin Reprod Med 2007;25(5):344 51. [78] Golan R, Scovell JM, Ramasamy R. Age-related testosterone decline is due to waning of both testicular and hypothalamicpituitary function. Aging Male 2015;18(3):201 4. [79] Tamma G, et al. Aquaporins, vasopressin, and aging: current perspectives. Endocrinology 2015;156(3):777 88. [80] Schuetz P, Muller B. The hypothalamic-pituitary-adrenal axis in critical illness. Endocrinol Metab Clin North Am 2006;35 (4):823 38 x. [81] Arafah BM. Hypothalamic pituitary adrenal function during critical illness: limitations of current assessment methods. J Clin Endocrinol Metab 2006;91(10):3725 45. [82] Loriaux DL, Fleseriu M. Relative adrenal insufficiency. Curr Opin Endocrinol Diabetes Obes 2009;16(5):392 400. [83] Sprung CL, et al. Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008;358(2):111 24. [84] Mebis L, et al. Changes within the thyroid axis during the course of critical illness. Endocrinol Metab Clin North Am 2006;35(4):807 21 x. [85] Kaptein EM, Beale E, Chan LS. Thyroid hormone therapy for obesity and nonthyroidal illnesses: a systematic review. J Clin Endocrinol Metab 2009;94(10):3663 75. [86] Mesotten D, Van den Berghe G. Changes within the growth hormone/insulin-like growth factor I/IGF binding protein axis during critical illness. Endocrinol Metab Clin North Am 2006;35(4):793 805 ix-x. [87] Langouche L, Van den Berghe G. The dynamic neuroendocrine response to critical illness. Endocrinol Metab Clin North Am 2006;35(4):777 91 ix. [88] Spratt DI, et al. Both hyper- and hypogonadotropic hypogonadism occur transiently in acute illness: bio- and immunoactive gonadotropins. J Clin Endocrinol Metab 1992;75 (6):1562 70. [89] Savastano S, et al. The complex relationship between obesity and the somatropic axis: the long and winding road. Growth Horm IGF Res 2014;24(6):221 6. [90] Sperling MA. Traditional and novel aspects of the metabolic actions of growth hormone. Growth Horm IGF Res 2015.

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C H A P T E R

12 Drugs and Pituitary Function Maria Fleseriu

INTRODUCTION Many medications can alter pituitary function, acting either directly on the pituitary or central nervous system, or, in some cases, by disrupting normal feedback relationships by acting on downstream end-organ systems (e.g., thyroid, gonad) or altering peripheral hormone metabolism or action. In some instances, persistent drug-induced pituitary dysfunction results in signs and symptoms of hypopituitarism or pituitary hormonal excess. Many therapeutic and recreational drugs alter pituitary gland function, usually as a side effect unrelated to the primary indication for which the drug was given. A summary of the most important therapeutic drug-related changes in pituitary hormone secretion is depicted in Tables 12.1
12.6.

OPIATES AND OPIATE ANTAGONISTS Opiates Opioid prescription use and opioid abuse have risen significantly in recent years, across all age groups, but short- and long-term endocrine opioid effects are not well described [1
3]. Opiates (including morphine, heroin, codeine, fentanyl, β-endorphin, enkephalins) have profound and generally consistent effects on human pituitary function. Three major classes of opioid peptides include endorphins, enkephalins, and dynorphins, each derived from three distinctive precursors. Synthetic, semisynthetic opioids, and opioid antagonists act with varying potency at three types of receptors (mu, gamma, and kappa), and this may explain the different effects observed (Table 12.7). Most research has focused on

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00012-X

opioid-induced hypogonadism, but an evolving topic of concern is central adrenal insufficiency (AI). Opiates suppress adrenocorticotrophic hormone (ACTH) and cortisol secretion [2] by acting mainly at the pituitary level (opiates blunt ACTH responses to lysine, vasopressin, and corticotrophin-releasing hormone (CRH)). The suppressive effect may involve other hypothalamic factors, since opiates do not directly alter ACTH secretion in vitro [4]. Morphine infusion blunts ACTH and cortisol response up to 90 minutes after CRH administration [4]. Fentanyl was shown to attenuate the response of ACTH and cortisol early on, in postsurgery patients [5]. Chronic administration of opiates and narcotic addiction was initially thought to result in partial tolerance to endocrine effects. Serum cortisol is normal or slightly decreased with chronic opioid use, but it may show an exaggerated response to stimulation (e.g., hypoglycemia during insulin tolerance test). Nevertheless, occasional cases of AI have been reported in patients receiving chronic opiate therapy [1]. The mode of administration and type of opiate can result in different hypothalamic
pituitary
adrenal (HPA) axis consequences. Transdermal fentanyl may suppress the HPA axis more than oral morphine [1,6], while transdermal buprenorphine increases cortisol levels after 6 months of treatment [7]. It is important to determine which patients are at a higher risk of developing AI. One small study suggested that a mureceptor polymorphism might induce more opioid suppression of hypothalamic CRH [8]. Reversibility of AI after opioid discontinuation is unknown. All patients with AI should be treated with glucocorticoid (GC) replacement until the normalization of their HPA axis is achieved. Small but significant increases in thyroidstimulating hormone (TSH) in response to opiates [2]

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384 TABLE 12.1

12. DRUGS AND PITUITARY FUNCTION

Drugs That Alter Adrenocorticotrophin Secretion

TABLE 12.3

Drugs That Alter Prolactin Secretion

Increase secretion

Decrease secretion

Increase secretion

Decrease secretion

Nicotine

Opiates

Risperidone

Apomorphine

Opiate antagonists

Glucocorticoids

Most Antipsychotics (typical and atypical)

Opioid withdrawal

Megestrol

Beer (?)

Dopamine agonists

Methylphenidate

Ipilimumab

Nicotine

Bromocriptine

Imipramine

Nivolumab

Opiates

Cabergoline

Desipramine

Imatinib

Cocaine (?)

Aripiprazole

Chlorimipramine

Amphetamines (IV)

HER2/ErbB2 antagonists

Physostigmine

Imipramine ( 6 )

Ketoconazole

Desipramine ( 6 )

Mifepristone

Chlorimipramine

Mitotane

Amoxapine

Amphetamines

Monoamine oxidase inhibitors Buspirone

TABLE 12.2

Drugs That Alter Thyrotrophin Secretion

Increase secretion

Decrease secretion

Lithium

Dopamine

Metoclopramide

L-dopa

Sulpiride

Bromocriptine

Domperidone

Verapamil

Estrogens ( 6 )

Androgens ( 6 )

Sunitinib

Glucocorticoids

Nivolumab

Metformin

Mifepristone

Ipilimumab

( 6 ), in some but not in others.

have been reported; however, TSH might be decreased in chronic opium smokers. Morphine and other opiates acutely stimulate growth hormone (GH) secretion [2,9] through central action on mu-receptors. In animals, morphine sulfate markedly induces plasma GH levels, while morphine6 glucuronide did not produce significant changes in GH or insulin-like growth factor 1 (IGF-1). There is some evidence that the stimulation of GH release by enkephalin analogues may involve a reduction in somatostatin secretion, or reset a hypothalamic GH pulse generator and stimulate GH-releasing hormone (GHRH) release. Effects of opioids on GH seem to be influenced by sex steroids. In humans, GH stimulation occurs at a morphine dose of approximately 15 mg. Chronic effects are however less well defined. Interestingly, some patients administered intrathecal opioids for 3 years had low

Metoclopramide Sulpiride Domperidone Physostigmine Reserpine ( 6 ) Methyldopa ( 6 ) Labetalol Verapamil ( 6 ) Cimetidine (IV) Ranitidine (IV) Estrogens ( 6 ) IV, intravenous administration. (?), uncertain. ( 6 ), in some but not in others.

IGF-1 and 15% met criteria for GH deficiency [10]. In patients with acromegaly, morphine (albeit at a higher dose) increases GH, most likely through modulatory effects on GH secretion [2,9]. Prolactin (PRL) secretion is stimulated by all opiates in both sexes [2], likely due to decreased tuberoinfundibular dopamine release [2]. Naloxone inhibits PRL release. In males, opiates decrease serum luteinizing hormone (LH), followed by a fall in serum testosterone levels [2,11]. In females, opiates suppress serum LH in premenopausal subjects, often resulting in irregular menses or amenorrhea [2]. Both morphine and an enkephalin analogue suppress LH in postmenopausal women. Serum follicle-stimulation hormone (FSH) is also acutely decreased by opiates, but to a lesser

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OPIATES AND OPIATE ANTAGONISTS

TABLE 12.4

Drugs That Alter Growth Hormone Secretion

Increase secretion

TABLE 12.6

Drugs That Alter Vasopressin Secretion

Decrease secretion

Decrease secretion

Nicotine

Atropine

Increase secretion/potentiate ADH effect

Opiates

Pirenzepine

Nicotine

Opiates

Amphetamines

Yohimbine

Lithium

Glucocorticoids

Methylphenidate

Phentolamine

Serotonin reuptake inhibitors

Temozolomide

Benzodiazepines ( 6 )

Cimetidine (?)

Thiazide and thiazide-like diuretics

Ipilimumab

Imipramine

Glucocorticoids

Carbamazepine

Desipramine

Tyrosine kinase inhibitors

Antidepressants

Chlorimipramine

Opiates

Antipsychotics

Apomorphine

Nonsteroidal antiinflammatory drugs

Dopamine

Amlodipine Angiotensin-converting enzyme inhibitors

L-dopa

Bromocriptine

Chlorpropamide

Physostigmine

Anticancer drugs: vinca alkaloids, platinum compounds, alkylating agents

Propranolol

Opiates

Clonidine

Tyrosine kinase inhibitors

Estrogens

TABLE 12.7

Androgens Interferon (?), uncertain. ( 6 ), in some but not in others.

TABLE 12.5

Opioid Effects on Pituitary/Hypothalamic Function

Acute effects

Chronic effects

Adrenal

HPA axis suppression

Significant HPA axis suppression (extent depends on type of opioid, dose, type of administration): k CRH k ACTH k cortisol k DHEA

Thyroid

m TSH

Not well described ? k TSH Unclear effects on T3/T4

Growth hormone

m GH

k GH k IGF-1

Prolactin

Not well described

k Dopamine m PRL

Gonadal

k GnRH k LH (LH . FSH) k FSH k Estradiol and progesterone in women k Testosterone in men

k GnRH k LH (LH . FSH) k FSH k Estradiol and progesterone in women k Testosterone in men

Diabetes insipidus

m AVP

k AVP

Drugs That Alter Gonadotrophin Secretion

Increase secretion

Decrease secretion

Opiate antagonists

Opiates

Endocrine disruptors

Dopamine

Cancer chemotherapy

Bromocriptine

Estrogen receptor antagonists

Verapamil

Aromatase inhibitors (in men)

Estrogens

Flutamide (in men)

Androgens

Ketoconazole

Glucocorticoids

Mitotane

Megestrol

degree [2]. In animals, opiates suppress gonadotrophin secretion by inhibiting gonadotrophin-releasing hormone (GnRH release), [2] but opiates do not impair the gonadotrophin response to GnRH in humans [12]. Opiates, on balance, appear to inhibit vasopressin secretion, although conflicting results have been reported [2]. Plasma oxytocin levels were decreased by opiate administration during labor, but not in late pregnancy before the onset of labor [2]; oxytocin secretion was also shown to be suppressed during breastfeeding [2].

Chronic heroin or methadone administration decreases serum LH, FSH, and testosterone levels [2]. Thyroid function and serum TSH are normal [13], as is GH secretion [13]. Serum PRL is mildly elevated.

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Chronic methadone maintenance therapy produces a mild impairment in renal concentrating ability that responds to vasopressin administration, suggesting decreased endogenous vasopressin secretion. Men with chronic heroin and cocaine dependence have been reported to have mild pituitary enlargement, perhaps due to lactotroph hyperplasia.

Opiate Antagonists Naloxone, naltrexone, and nalmefene are opiate antagonists, almost devoid of agonist activity. They have proved invaluable in exploring the role of endogenous opiates in human pituitary function. Opiate antagonists acutely stimulate both cortisol and ACTH secretion [1,14,15]. Naloxone decreases GH in a dose-dependent matter in neonatal rat pups, probably due to different affinities and/or selectivity to opioid receptors. All three opiate antagonists cause an acute increase in serum LH in men and luteal-phase women, with little or no effect in the early follicular phase of the menstrual cycle or in postmenopausal women; smaller changes are generally seen in serum FSH levels. Naloxone and nalmefene have no major effects on basal serum TSH and PRL; however, small rises in serum PRL and decreased nocturnal TSH secretion [16] have been reported with naloxone. Chronic naltrexone administration, in contrast to the acute response, does not alter serum testosterone or gonadotrophins. Naloxone has little or no effect on vasopressin secretion; it may affect the oxytocin response to nicotine and inhibit the oxytocin response to orgasm. Naloxone had no effect on plasma oxytocin concentrations in males, in females during late pregnancy or labor, or during breastfeeding [2].

AMPHETAMINES AND METHYLPHENIDATE These stimulant drugs act by promoting the release and/or inhibiting neuronal reuptake of dopamine, norepinephrine and, to a lesser extent, serotonin. Acute oral administration of dextroamphetamine and methylphenidate consistently stimulates GH and cortisol release, but has either no effect or a suppressive action on serum PRL. Intravenous amphetamine stimulates GH and cortisol secretion but, in contrast, also appears to slightly stimulate PRL release, suggesting that high serum amphetamine concentrations achieved by bolus intravenous administration may activate additional neurotransmitter systems (e.g., serotonergic mechanisms) [17]. Intravenous methylphenidate raises

serum GH, ACTH, and cortisol, with minimal PRL effects [18]. Chronic treatment of children with attention deficit disorder with stimulant drugs commonly produces slowing of linear growth [19]. The mechanism for this growth retardation is unclear, but is likely related to anorexia and decreased food intake; GH secretion, serum IGF-1, and serum GH-binding protein are all normal [20].

CAFFEINE Caffeine, a widely consumed stimulant, exerts its major actions through antagonism at adenosine receptors; in vivo metabolites, such as theophylline and theobromine, may also contribute to its effects. Acute administration of large doses (500 mg, equivalent to five cups of coffee) to naı¨ve normal subjects has minimal effect on pituitary function, producing only slight increases in plasma ACTH, cortisol, β-endorphin/β-lipotrophin, and GH; TSH and PRL are unchanged [21]. Despite caffeine activating the HPA axis in the short term, moderate caffeine doses do not modulate HPA axis responses to stressful stimuli [22]. There are no data regarding caffeine effects on gonadotrophin secretion in humans, although theophylline, a related methylxanthine has no effect on serum LH concentrations. Theophylline has been reported to cause the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Chronic consumption of caffeine produces tolerance to most of the drug’s actions, suggesting that longterm endocrine consequences are unlikely [21].

BENZODIAZEPINES Alprazolam and temazepam appear to modestly suppress serum cortisol levels, while diazepam does not appear to affect the secretion of basal cortisol, β-endorphin, and β-lipotrophin [24]. However, diazepam blocks responses to hypoglycemia of cortisol, β-endorphin, and β-lipotrophin, and alprazolam blunts ACTH and cortisol responses to social stress. It is not clear whether these effects on cortisol are due to specific actions at hormone regulatory centers or reflect a more general relief of anxiety and stress by the drug itself. The fact that diazepam suppresses nocturnal release of cortisol and GH, and that alprazolam suppresses the release of ACTH and cortisol induced by naloxone or metyrapone administration, would favor a specific action on hormone regulation [23]. In contrast with healthy volunteers, benzodiazepines did not attenuate pituitary
adrenal responses to CRH in

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ANTIPSYCHOTIC DRUGS

patients with Cushing’s syndrome [24]. Furthermore, acute alprazolam administration inhibited cortisol secretion in patients with subclinical but not overt Cushing’s syndrome [25]. Several studies have reported that acute administration of benzodiazepines (diazepam, bromazepam, metaclazepam, and alprazolam) stimulate GH secretion in normal males [26]. Serum PRL, TSH, LH, and FSH are not affected. With chronic benzodiazepine administration, basal serum GH and PRL levels are normal, and the GH response to diazepam appears blunted [27]. Thus, signs and symptoms of GH excess do not develop in patients receiving long-term benzodiazepine therapy. Flumazenil, a benzodiazepine receptor antagonist had no effect on basal or naloxone-stimulated release of ACTH or cortisol, suggesting that endogenous benzodiazepine-like ligands do not tonically regulate ACTH secretion.

ANTIDEPRESSANTS Acute oral or intravenous administrations of imipramine, desipramine, and clomipramine have been reported to have different effects, possibly due to the use of different drug doses. Effects vary from no effect or a stimulatory effect on GH, PRL, cortisol, and ACTH in normal subjects [28]. Oral nefazodone and intravenous citalopram modestly stimulate PRL release [29
31]. Chronic administration of chlorimipramine modestly raised serum PRL to about twice baseline [32,33], while chronic treatment with desipramine, imipramine, nortriptyline, and amitriptyline had little or no effect on serum PRL [15]. Interestingly, escitalopram has been linked to galactorrhea despite normal PRL. Chronic treatment with fluvoxamine or fluoxetine modestly raised serum PRL in a minority of depressed patients [34,35]. There is probably little or no change in serum cortisol, TSH, or GH levels during chronic treatment with the tricyclic antidepressants, selective serotonin reuptake inhibitors, or monoamine oxidase inhibitors [36]. Amoxapine, an antidepressant structurally related to the antipsychotic agent, loxapine, has dopamine antagonist activity and is a marked stimulator (three to four times basal) of PRL secretion [37]. Bupropion, a nontricyclic antidepressant, has no effect on serum PRL or GH [38]. Monoamine oxidase inhibitors such as chlorgyline and pargyline also double basal PRL concentrations when given chronically [39]. Thus, most antidepressants have either no effect on anterior pituitary hormone secretion or effects limited to PRL. In particular, these drugs only occasionally cause significant hyperprolactinemia and rarely produce galactorrhea [40].

Hyponatremia, apparently due to inappropriate secretion of antidiuretic hormone (ADH), has been reported with many antidepressants, including tricyclics, monoamine oxidase inhibitors, and bupropion [41].

LITHIUM Used primarily in the treatment of bipolar affective disorder, lithium has two major effects on pituitary function, both indirect. Lithium acts on the thyroid gland to inhibit hormone release, resulting in the activation of feedback mechanisms and increased pituitary TSH secretion [42]. In most normal subjects, the TSH rise is minor and transient, but in susceptible individuals (often those with preexisting thyroid damage due to autoimmune thyroiditis or radiation) frank hypothyroidism may be produced and goiter may develop [42]. To detect such cases of emerging hypothyroidism, serum T4 and TSH should be monitored every 3
4 months during the first year of lithium therapy, and yearly thereafter. Lithium also impairs vasopressin action on the kidney [43]. Mild nephrogenic diabetes insipidus results in enhanced vasopressin release with no change in the osmotic threshold for vasopressin secretion [44]. Lithium may also mildly stimulate thirst [44], an additional action that could also contribute to polyuria and polydipsia seen in some patients receiving lithium therapy. Treatment with amiloride improves renal concentrating ability in patients taking lithium [44].

ANTIPSYCHOTIC DRUGS The major antipsychotic drugs (also known as neuroleptics) act as antagonists at the D-2 dopamine receptor; first generation phenothiazines, butyrophenones, and thioxanthenes share this property, which is probably the basis of antipsychotic actions [45]. Since D-2 dopamine receptors participate in regulation of pituitary hormone secretion, it is not surprising that these drugs have important effects on pituitary function. Neuroleptic drugs have little or no effect on ACTH [46]. Phenothiazines decrease TSH response to thyroidreleasing hormone (TRH). Nonphenothiazines can elevate TSH levels. Atypical antipsychotics may decrease TRH-stimulated TSH [47]. Neuroleptics may have an inhibitory effect on GH secretion [48], but clinically, there is no evidence of GH deficiency in patients receiving neuroleptics. The most consistent endocrine effect of the classic neuroleptic drugs is elevation of serum PRL levels. This effect is due to antagonism of the PRL-inhibitory effects of endogenous dopamine at the pituitary

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lactotroph D-2 dopamine receptor. There is a reasonably good correlation between antipsychotic potency, (e.g., risperidone, haloperidol) D-2 dopamine receptor antagonism, and PRL stimulation [49]. The major exceptions are clozapine and quetiapine, atypical neuroleptics with weak D-2 binding affinity, which produce only minimal elevations in serum PRL and aripiprazole, which has partial agonist activity at D-2 dopamine receptors [45]. Olanzapine, another atypical neuroleptic, has a higher affinity than clozapine for D2 receptors, and has a slightly higher incidence of mild hyperprolactinemia [49]. Chronic administration of neuroleptics may result in tolerance to PRL-elevating drug effects, with some patients demonstrating normal or near-normal serum PRL concentrations after longterm therapy. However, patients can benefit from switching to aripiprazole or other newer atypical antipsychotics in some cases of symptomatic hyperprolactinemia [50]. Reports of PRL-secreting pituitary tumors developing during chronic neuroleptic therapy [51] are viewed as coincidental rather than causal, particularly in the context of a high frequency of incidental pituitary tumors in the general population. Hyperprolactinemia induced by neuroleptic drugs can suppress GnRH and gonadotrophin secretion with consequent hypogonadism and amenorrhea [45,49], although this effect is highly variable, and many patients have normal gonadal function despite mild hyperprolactinemia [35,45,49,52]. SIADH has also been reported in patients treated with neuroleptics.

OTHER DOPAMINE ANTAGONISTS Metoclopramide and domperidone are potent D-2 dopamine antagonists which, like the antipsychotics, induce PRL secretion. Since TSH and LH, like PRL, are also under mild tonic inhibition by dopamine, acute administration of these drugs produces a small, transient rise in serum TSH and LH levels. Chronic endocrine effects are similar to those observed with antipsychotics [53]. Acutely, metoclopramide, but not sulpiride, domperidone, or haloperidol, stimulates vasopressin secretion [54]. Buspirone, an antianxiety drug, is a D-2 dopamine receptor antagonist as well as a serotonin receptor agonist, and acutely elevates human serum PRL, cortisol, and GH levels, with no effects on oxytocin or vasopressin [55].

DOPAMINE AGONISTS Drugs with dopamine agonist activity suppress PRL secretion; these include apomorphine, dopamine,

L-dopa, L-dopa/carbidopa

combinations, and the dopaminergic ergot alkaloids, bromocriptine, and cabergoline [56]. Dopaminergic drugs acutely stimulate GH secretion, probably through hypothalamic effects. TSH and LH are acutely suppressed, but not with chronic administration. Dopamine infusions, given for hypotension, may contribute to the hypothyroxinemia seen in severe systemic illness by suppressing TSH [57].

CHOLINERGIC AGONISTS AND ANTAGONISTS Muscarinic blockers such as atropine and pirenzepine appear to enhance somatostatin release from the median eminence of the hypothalamus and thereby decrease GH secretion [58]. The GH response to insulin-induced hypoglycemia is relatively less affected by these drugs than other GH-provocative tests [58]. Cholinergic agonists such as physostigmine and pyridostigmine have an opposite effect, inhibiting somatostatin and stimulating GH [58]. In addition, physostigmine has been reported to stimulate PRL, ACTH, cortisol, and β-endorphin.

ANTIHYPERTENSIVES Reserpine, which depletes catecholamines, and methyldopa, which both depletes catecholamines and serves as a precursor for false neurotransmitters, modestly stimulate PRL secretion, although in many patients serum PRL concentrations remain within the normal range. Methyldopa has been reported to also cause SIADH. Most β-blockers have little or no effect on other pituitary hormones. β-Adrenergic blockade with propranolol enhances the GH response to various stimuli [58]; selective β1-antagonists do not share this action. Intravenous labetalol, a drug with both α- and β-adrenergic blocking effects as well as β-agonist actions, stimulates PRL secretion via unknown mechanisms [59]. Clonidine and related α2-adrenergic agonists stimulate GH secretion, particularly in children, with little or no change in other anterior pituitary hormones [60,61]. Clonidine has been used as a provocative test to assess GH reserve and diagnose deficiency. Yohimbine, an α2-adrenergic antagonist, and phentolamine, a nonspecific α-blocker, blunt GH responses to a variety of stimuli, but prazosin, an α1-antagonist, does not [58]. Clonidine has been reported to cause SIADH. Calcium channel blockers have divergent effects on pituitary hormone secretion. Diltiazem and nifedipine appear to have little or no effect, but verapamil has

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been reported to decrease gonadotrophin and TSH secretion and enhance PRL release [62]. Mild hyperprolactinemia and galactorrhea have occurred in patients receiving verapamil therapy [62].

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Cancer chemotherapy commonly produces gonadal damage, with a consequent rise in serum FSH and LH due to activation of normal feedback mechanisms. Recovery may occur spontaneously years later in some male patients, while females may show progressive ovarian failure [69].

ANTIHISTAMINES H1-Antihistamines

Immunotherapies

H1 histamine receptors appear to play little role in the regulation of human pituitary function. Intravenous diphenhydramine, an H1-antagonist, had no effect on serum GH, PRL, or TSH, [63] and did not alter the TSH and PRL response to TRH or the GH response to L-dopa [64]. However, other H1-antihistamines (meclastine and chlorpheniramine) blunt the GH response to arginine infusion but not the response to hypoglycemia [65]. It is possible that this effect is due to the anticholinergic properties of these drugs.

Novel immune checkpoint blockade with ipilimumab, an antibody blocking the cytotoxic T-lymphocyteassociated protein 4 (CTLA4), is revolutionizing cancer therapy. However, ipilimumab induces symptomatic, sometimes severe, endocrine immune-related adverse events. The pituitary expresses CTLA-4 at both RNA and protein levels, particularly in a subset of PRL- and thyrotrophin-secreting cells. Notably, these cells are sites of complement activation, featuring deposition of C3d and C4d components and an inflammatory cascade akin to that seen in type II hypersensitivity [76]. The clinical presentation of CTLA-4 hypophysitis is now recognized and occurs in about 4
9% of patients treated with ipilimumab, though most of the data are derived from patients with metastatic melanoma [77
79]. Hypothyroidism/thyroiditis develops in about 6% of patients and primary adrenal dysfunction is rarely encountered. Hypophysitis usually emerges between the second and fourth cycles of ipilimumab administration and seems to be dose independent. The degree of the endocrine adverse events of this therapy correlates with improved efficacy. Pituitary imaging shows a moderately enlarged gland in a few patients. The HPA axis is most frequently affected, followed by thyroid and/or gonadotroph axes; diabetes insipidus is more rare than reported in other types of hypophysitis. Glucocorticoid treatment of CTLA-4 hypophysitis results in markedly improved symptoms, and induces resolution of focal symptoms, but pituitary deficiencies rarely recover. Long-term follow-up to monitor development of other hormonal deficits and appropriate hormonal replacement is required in all these patients [77
79]. There is still a debate surrounding the need for high-dose GC treatment (instead of the replacement dose for AI) even in the absence of visual changes. Similarly, it is also not clear whether ipilimumab therapy cessation affects the natural progression of hypophysitis. Therapy with a combination of ipilimumab and nivolumab, an antiprogrammed cell death 1 receptor antibody, is also associated with a 9% incidence of hypophysitis and a 22% incidence of either thyroiditis or hypothyroidism. The mechanism of endocrine dysfunction induced by either of these drugs has not been fully elucidated and studies identifying the most susceptible patients are needed [77
79].

H2-Antihistamines Cimetidine and ranitidine stimulate PRL secretion when given as an intravenous bolus that achieves high serum drug levels; other pituitary hormones are not affected [66]. Animal studies suggest that the PRLreleasing effects of cimetidine and ranitidine are not mediated by H2-histamine antagonism [67]. Endocrine effects of long-term cimetidine therapy are controversial, although no change in serum PRL or other pituitary hormones has been consistently observed.

CANCER THERAPIES Antineoplastic Chemotherapy Cytotoxic drugs may directly alter anterior pituitary function [68,69]. The TKIs sunitinib and sorafenib can alter thyroid function, resulting in suppressed or, more commonly, elevated serum TSH [70]. Imatinib induces HPA axis dysfunction and partial glucocorticoid deficiency. It is therefore important to start immediate GC replacement with an intercurrent illness; [71] GH deficiency and hypoglycemia may also occur in patients treated with imatinib [72]. Bexarotene, a retinoid X-receptor agonist used in treatment of cutaneous T-cell lymphoma, suppresses TSH secretion and has resulted in central hypothyroidism, with no effect on serum PRL or cortisol levels [73]. Several cancer chemotherapeutic agents have been reported to cause SIADH [15,74,75]. These include temozolomide, cyclophosphamide, melphalan, vincristine, vinblastine, vinorelbine, cisplatin, and imatinib.

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ESTROGENS Estrogens have several effects on the human pituitary gland. Serum TSH may be slightly increased by exogenous estrogens, although some studies have failed to show this effect. GH secretion is potentiated, both basally and in response to a variety of provocative tests [58,80]. PRL secretion is also mildly to moderately increased, in a dose-related fashion [49,81]. Estrogens modulate apoptosis of lactotrophs and somatotrophs in female rats. In male rats, dihydrotestosterone (DHT) and estradiol (E2) have opposite effects on apoptosis in the anterior pituitary gland and it has been suggested that expression and/or activity of 5α-reductase and aromatase changes may play a role in the development of anterior pituitary tumors [82]. However, there is no direct evidence that exogenous estrogen is involved in the genesis of pituitary prolactinomas in humans. Gonadotrophin secretion is suppressed by estrogen, especially at high doses [80,83]. Estrogen also exerts a positive feedback effect on gonadotrophins, as exemplified by LH and FSH surges at the midpoint of the normal menstrual cycle. Males may also exhibit positive feedback of estrogens on LH release.

ANDROGENS Androgens suppress gonadotrophin secretion through actions on both the hypothalamus and pituitary. While part of this effect may be attributable to estrogens produced by biotransformation in vivo, nonaromatizable androgens also suppress gonadotrophins [84]. Fluoxymesterone, a nonaromatizable androgen, modestly suppresses TSH secretion, but does not alter PRL. Studies examining effects of testosterone on these two hormones have been confounded by concurrent increases in serum estrogens produced by aromatization in vivo. Testosterone administration enhances spontaneous GH secretion with a subsequent rise in serum IGF-1; however, effects of a synthetic nonaromatizable androgen (oxandrolone) on the GH axis, especially in children, are controversial.

ANTIANDROGENS Finasteride, a 5α-reductase inhibitor, has no consistent observed effect on serum LH, FSH, cortisol, TSH, or T4 in men or women. Flutamide, an androgen receptor blocker, may increase serum LH and testosterone in men, with no change in FSH or PRL. In normal and

hirsute premenopausal women, no change was observed in serum LH, FSH, or testosterone. In postmenopausal women, serum testosterone fell, while LH and FSH were unchanged.

GLUCOCORTICOIDS In addition to feedback suppression of ACTH and β-lipotrophin secretion, GCs in large doses also exert suppressive effects on GH, TSH, gonadotrophins, PRL, and vasopressin. By suppressing TSH, lowering serum thyroxine-binding globulin and blocking extrathyroidal conversion of T4 to T3, large doses of GCs may contribute to the derangements of pituitary
thyroid function seen in the “euthyroid sick” or “nonthyroidal illness” syndrome. In the absence of concomitant illness, thyroid axis changes produced by GCs are usually modest.

ENDOCRINE-DISRUPTING CHEMICALS Endocrine-disrupting chemicals (EDCs) are abundant “exogenous chemical(s), or mixtures of chemicals that interfere with aspects of hormone action” [85]. Fetal development and early childhood are specifically susceptible stages for exposure to chemicals (e.g., bisphenol A (BPA), phthalates, pesticides, industrial chemicals, etc.). In humans, a growing number of epidemiological studies report an association with altered pubertal timing and progression. Although the data are not always consistent between experimental and epidemiological studies, they suggest that some EDCs may adversely affect the anterior pituitary in women in addition to direct effects on the uterus and vagina. Some EDCs are also associated with abnormal puberty, irregular cyclicity, reduced fertility, infertility, polycystic ovarian syndrome, endometriosis, fibroids, preterm birth, and adverse birth outcomes. Antiandrogens, xenoestrogens, and dioxins are the best-characterized endocrine disruptors of the male reproductive system. Antiandrogenic chemicals act additively, irrespective of their mechanism of action (i.e., whether they are receptor antagonists or inhibitors of hormone synthesis) [86]. Cadmium affects the hypothalamo
pituitary gonadal axis by acting at the hypothalamic level and affecting gonadotrophin secretion, but also by affecting testicular or ovarian structure and activity. Cadmium may induce chronotoxicity and induce oxidative stress of this axis [87]. A limitation to understanding the effects of EDCs is the potential for confounding due to the long temporal

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ALCOHOL

lag from early-life exposures to adult outcomes. Contemporaneous exposures can also disrupt the hypothalamic
pituitary
gonadal axis. Mixed exposures of compounds might individually have opposing actions on the reproductive axis [88,89].

MISCELLANEOUS DRUGS Anticonvulsants have been shown to possibly affect posterior pituitary hormone secretion. Phenytoin may inhibit ADH secretion, while carbamazepine may cause SIADH. Sodium valproate inhibits the vasopressin response to hypernatremia and upright posture and the oxytocin response to angiotensin II. As shown in animal models, chronic administration of nonsteroidal antiinflammatory drugs might disturb, in a sex-dependent manner, the hypothalamic
pituitary
gonadal axis [90]. Blockade of cortisol biosynthesis by ketoconazole or blockade of glucocorticoid receptor by mifepristone has been used as adjunctive therapy in the treatment of Cushing’s syndrome. Ketoconazole, an antifungal agent, interferes with biosynthesis of both testosterone and cortisol and can lead to elevations in serum gonadotrophins and, less often, ACTH, by activating feedback mechanisms. Mifepristone increases cortisol and ACTH dose dependency. Mean peak increases in ACTH are about twofold (with a wide range) and usually observed in two-thirds of patients. In a long-term study of patients with Cushing disease [91], ACTH declined to near baseline levels after mifepristone discontinuation. Mifepristone may also increase TSH [92]. It is likely that altered serum TSH levels are due to anti-GC properties of mifepristone, but the clinical significance of these biochemical alterations in thyroid homeostasis remains to be determined. Titration of thyroid replacement is necessary in some cases. Mitotane is an antineoplastic medication used in treating adrenocortical carcinoma; it is also used as an adrenal steroidogenesis inhibitor in Cushing disease. Mitotane also has direct effects on the pituitary, and both ACTH secretion and cell viability are affected [93]. Mitotane also impairs TRH-induced TSH secretion, inhibits cell viability, and promotes apoptosis, mimicking central hypothyroidism [94]. Secondary hypogonadism is frequent in male patients undergoing adjuvant mitotane therapy. In vitro studies showed that mitotane impedes cell viability, stimulates apoptosis, alters the cell cycle progression, and impairs gonadotrophin secretion [95]. Testing of pituitary functions and hormonal replacement as needed is recommended. Heparin therapy may precipitate pituitary apoplexy; thus, awareness is needed in patients at risk.

Several proton-pump inhibitors cause SIADH, especially in older patients [15]. Megestrol, a synthetic progestin, is used clinically to stimulate appetite and promote weight gain. The drug also has GC agonist activity and suppresses serum ACTH, cortisol, LH, and testosterone levels, and may result in AI and hypogonadism; with high doses of megestrol, Cushing’s syndrome has also been reported. Metformin, used in the treatment of diabetes mellitus, modestly decreases serum TSH, occasionally to subnormal levels, in diabetic patients with primary hypothyroidism, independent of thyroid hormone treatment. Interferons may have multiple effects on the pituitary. Interferon-α, used in the treatment of chronic viral hepatitis, causes hypophysitis and hypopituitarism in a small number of patients [96]. Interferon-β, used to treat multiple sclerosis, acutely stimulates the secretion of ACTH, cortisol, PRL, and GH after each injection for the first several months of therapy; hormonal responses are blunted by indometacin and eventually partially abate with long-term therapy [97]. A summary of the most important recreational and illicit drug-related changes in pituitary hormone secretion is detailed in Table 12.8.

ALCOHOL Consumption of beverages containing ethanol may alter pituitary function in several ways: (1) by direct effects on the brain or pituitary gland; (2) by altering the function of end organs (e.g., testis) and inducing feedback-mediated changes in pituitary hormone secretion; and (3) by modifying the peripheral metabolism or action of hormones with resulting effects on pituitary function. Acute and chronic alcohol use has been reported to upregulate HPA axis function and induce elevated levels of basal cortisol and ACTH [98]. The ethanol dose, presence of nausea, alcohol dehydrogenase genotype, or family history of alcoholism have all been suggested as possible predictors of HPA activation. Chronic consumption of ethanol may lead to persistent hypercortisolemia, and some alcoholics may develop physical stigmata of Cushing’s syndrome along with nonsuppression of plasma ACTH and cortisol by dexamethasone, the so-called “alcohol-induced pseudoCushing’s syndrome” [99]. The syndrome is usually reversible after several months of abstinence [99]. Paradoxically, ACTH and cortisol responses to insulininduced hypoglycemia and other stimuli are frequently blunted in alcoholics [99]. Furthermore, animal studies showed that selective GC receptor

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392 TABLE 12.8

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Recreational Drugs That Alter Pituitary Function

Alcoholic beverages Cigarettes and nicotine Marijuana Opiates Cocaine Amphetamines and methylphenidate Caffeine Benzodiazepines

antagonists reduce the severity of ethanol withdrawal and related HPA activation, and thus might be considered as putative pharmacotherapies for the treatment of ethanol dependence [100]. Findings from both preclinical and clinical studies suggest profound sex differences regarding neuroadaptation of amino acid signaling systems and the HPA axis to chronic alcohol intake. Acute ethanol consumption does not affect daytime serum concentrations of TSH and thyroid hormones, although the nocturnal TSH surge is suppressed. Chronic alcoholics may have a blunted TSH response to TRH, but basal TSH levels are normal. Alcoholinduced liver damage can result in the “euthyroid sick” or “nonthyroidal illness” syndrome, generally with low serum T3, elevated reverse T3, normal or low T4, and normal or slightly elevated TSH [101]. GH secretion is minimally affected by ethanol per se in the absence of associated liver disease or malnutrition. Serum GH levels remain unchanged after acute administration of alcohol [102]. The effects of alcohol on PRL are controversial, from no acute effects to an acute decrease or even a small, but statistically significant dose-related rise in serum PRL following ethanol administration. In ovariectomized rats, ethanol causes hyperprolactinemia by elevating PRL release and by increasing the number of anterior pituitary lactotrophs [103]. Interestingly, beer exerts a fairly potent stimulus effect on PRL secretion, independent of its ethanol content. However, serum PRL is mildly elevated in a minority of patients with alcoholic liver disease [104]. Reports of ethanol on gonadal function have yielded conflicting data. These inconsistencies may result from the use of different study populations, varying doses of alcohol, performing studies at different times of the day with blood sampling at varying intervals, the development of tolerance to the effects of alcohol, and the presence of variable degrees of hepatic dysfunction in chronic studies [105]. Acute ingestion of ethanol in males has been reported to increase, decrease, or have

no effect on plasma testosterone and LH levels [105]. Chronic alcohol consumption, in the apparent absence of liver disease, it may lower serum testosterone concentrations by inhibiting testicular synthesis and increasing hepatic metabolism of testosterone; serum LH may rise, at least transiently, as a consequence. A later fall in LH may occur as a response to rising serum estrogen levels produced by increased hepatic aromatase activity. Plant-derived phytoestrogens are also present in alcoholic beverages and could also contribute to gonadotrophin suppression. Ethanol is toxic to the seminiferous tubules, and it may produce testicular atrophy and subsequent increased serum FSH levels [105]. In premenopausal women, acute administration of ethanol has minimal effects on serum LH or estradiol concentrations. Chronic administration, however, has been associated with plasma luteal estrogen concentrations, but not with androgen levels, nor estrone or E2 measured in the follicular phase [106]. In postmenopausal women, serum gonadotrophins are altered minimally by ethanol. Effects on oxytocin and vasopressin secretion are also controversial. A decrease in the number of vasopressin-containing neurons in the supraoptic and paraventricular nucleus of the hypothalamus has been described in chronic alcoholics [107].

SMOKING Cigarettes and Nicotine The acute release of several pituitary hormones after cigarette smoking might be due to nicotine. Several studies have reported increases in plasma cortisol, dehydroepiandrosterone (DHEA), ACTH, and β-endorphin/β-lipotrophin levels in response to smoking one or more medium- or high-nicotine cigarettes compared with sham smoking or smoking lownicotine cigarettes [108]. The crosstalk between the endogenous opioid system and nicotinic pathways does not extend to cooperative actions in nicotineinduced HPA-axis activation [109]. Intravenous or nasal spray nicotine stimulates ACTH and cortisol secretion. Interestingly, chronic habitual smoking induces small, but significant increases in morning serum cortisol and DHEA as well as salivary cortisol over the entire day [110]. Neither serum TSH nor free T4 are acutely altered by smoking in humans [111]. Chronic smokers and individuals exposed to second-hand smoke may have slightly lower serum levels of TSH than nonsmokers, a situation that may reflect direct or indirect nicotine effects on thyroid hormone secretion, metabolism, or action [112].

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Similarly, a rise in serum GH following smoking has been fairly consistently observed [108], but serum IGF-1 does not differ in chronic smokers versus nonsmokers. Serum PRL levels are increased by smoking; however, some reports have ascribed these hormonal changes to the nausea induced by rapid smoking [113]. Serum FSH levels are slightly higher in chronic smokers, but no consistent changes in serum LH have been observed [113]. While release of vasopressin and its precursor protein, neurophysin I, is stimulated by smoking and nicotine infusion, oxytocin is not affected.

Marijuana Although animal studies have shown a wide variety of effects of marijuana on hormonal systems, it has been difficult to show consistent changes in human studies, in which lower doses of marijuana or tetrahydrocannabinol (THC) are given [15]. Additional experimental difficulties in some studies involve possible inaccuracies of reported intake, uncertainty regarding concurrent use of alcohol or other drugs, and development of tolerance to the effects of marijuana. In a small study, Δ-9-THC raised plasma cortisol levels in a dose-dependent manner, but frequent users showed blunted increases relative to healthy controls. Frequent users also had lower baseline plasma PRL levels relative to healthy controls. It is unclear whether these group differences may be related to development of tolerance to neuroendocrine effects of cannabinoids [114]. In a single study, serum T4 and TSH levels were not altered by chronic marijuana smoking, though serum T3 was slightly decreased [115]. Serum PRL is slightly decreased or unchanged in chronic marijuana users [114,116]. Though blunted, both GH and cortisol responses to insulin-induced hypoglycemia are intact following oral THC administration [117]. In both men and women, no consistent changes have been seen in serum gonadotrophins or sex steroids [116].

COCAINE Cocaine is a potent stimulant of the sympathetic nervous system and causes structural changes on the brain, heart, lung, liver, and kidney. Use of cocaine may produce alterations to the endocrine system. Recent research on behavioral and neuroendocrine effects of cocaine dates has increasingly focused on alterations of the HPA axis, which appears to be the chief target of

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cocaine effects. Such activation is likely involved, though via a still undefined mechanism, in behavioral and cardiovascular changes of drug abusers as well as in the reinforcement/relapse phenomena [118]. In newly abstinent cocaine abusers, some individuals have mild or moderate hyperprolactinemia, which lasts for weeks after withdrawal. Hyperprolactinemia, if present, may result from disordered dopaminergic or opioidergic neurotransmission induced by the drug [119]. Several studies have reported that cocaine acutely increases serum LH but does not change serum testosterone or GH levels [15,118]. Plasma oxytocin levels are suppressed in postpartum women who abuse cocaine [119]. The cocaine and thyroid function interaction is not well understood. TSH response to TRH is reduced in cocaine abusers, but response recovers after a month of abstinence. The exact mechanism is not known, but chronic cocaine might induce dopamine- and norepinephrine-mediated TRH increase which downregulates TRH receptors [120]. Cocaine abuse effects have also been shown to influence thyroid hormone receptor and retinoid X receptor signaling, retinoic acid metabolism, and transcriptional regulation of neurogranin, a gene essential for adult neuroplasticity [121]. However, in another study, thyroid function in heavy cocaine users was not significantly different from normal controls [122]. Intranasal cocaine abuse can result in destructive lesions in the nasal septum and sinuses, often associated with antineutrophil cytoplasmic antibodies; it has been reported to also induce hypopituitarism in these patients.

Acknowledgments The author acknowledges Harold E. Carlson, MD, SUNY, Stony Brook, NY, the author of the corresponding chapter in the previous edition of this textbook, for use of material in this chapter, and Shirley McCartney, PhD, Oregon Health & Science University, Portland, Oregon for editorial assistance.

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13 The Pituitary Gland in Pregnancy Andrea Glezer and Marcello D. Bronstein

INTRODUCTION

NORMAL PITUITARY DURING PREGNANCY

During pregnancy, pituitary morphology and function are modified by placental hormonal secretion. Pituitary volume is increased due to lactotroph hypertrophy and hyperplasia. Placental sex steroids inhibit gonadotrophinreleasing hormone (GnRH) and gonatrophin secretion. The placenta secretes a variant isoform of growth hormone (GH), increasing growth factor 1 (IGF-1) levels. TSH secretion decreases in the first trimester, secondary to thyroid stimulation by human chorionic gonadotrophin. Hepatic production of thyroid-binding globulin is stimulated by estrogens increasing total T4 and T3. Placental CRH stimulates pituitary and placental ACTH, increasing cortisol levels throughout pregnancy. Infertility is common in patients harboring pituitary tumors. Pathophysiology includes hormonal hypersecretion and tumoral mass effect. Hypopituitarism can also result from surgery and/or radiotherapy for pituitary adenoma treatment. Hyperprolactinemia, occurring in prolactinomas, the most common subtype of pituitary adenomas, and other pituitary tumors, inhibits GnRH pulsatility. Different mechanisms seem to play a role on hypogonadism of patients harboring acromegaly: tumoral gonadotroph compression, hyperprolactinemia, and GH/IGF-1 excess disrupting gonadal axis. Regarding Cushing disease, hypogonadism is mainly due to the negative impact of hypercortisolism and hyperandrogenism in the gonadotrophic axis. Clinically nonfunctioning pituitary tumors seldom are associated with pregnancy. In pituitary adenomas, treatment is often necessary to control hormonal hypersecretion and tumor mass effect related to infertility. Other causes of infertility include lymphocytic hypophysitis and Sheehan syndrome. This chapter reviews the pathophysiology, diagnosis, and therapeutic approaches for women with pituitary diseases, before and during pregnancy, in order to induce fertility and avoid the deleterious effects of hormonal hyper- or hyposecretion.

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00013-1

During pregnancy, pituitary size and function are modified mainly by placental hormonal secretion. These changes play a pivotal role both in mother and fetus during pregnancy, labor, and puerperium.

Prolactin Pituitary size and volume are increased due to lactotroph hypertrophy and hyperplasia [1], reaching 12 mm of height immediately postpartum [2]. Thyrotrophin-releasing-hormone (TRH) also stimulates prolactin (PRL) secretion [3]. As a consequence, serum PRL levels gradually increase during the course of gestation, reaching up to 10 times nonpregnant upper limit values at the end of the third trimester [4,5]. Maternal decidua, not controlled by TRH or dopamine, is responsible for increased amniotic fluid PRL levels with little contribution to serum PRL concentrations [6] (Fig. 13.1).

Gonadotrophins Elevated sex steroids and regulatory peptides (e.g., inhibin) inhibit gonadotrophin-releasing hormone (GnRH) and gonadotrophin secretion, which start to decrease at 6 7 weeks, reaching undetectable levels from the second trimester to the end of pregnancy [3].

Growth Hormone The human growth hormone (GH) and chorionic somatomammotrophin hormone (CSH) gene cluster includes five related genes located at 17q22 24: GH1 encodes pituitary GH (GH-N) and GH2 encodes

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FIGURE 13.1 PRL axis during pregnancy: estrogen stimulates lactotroph hyperplasia with consequent increased serum PRL levels. Maternal decidua produces amniotic PRL with a small contribution to serum PRL. Source: From Karaca Z, Tanriverdi F, Unluhizarci K, Kelestimur F. Pregnancy and pituitary disorders. Eur J Endocrinol 2010;162(3):453 75, [7].

placental GH, also known as GH variant (GH-V) [8]. CSH is synthesized by placental syncytiotrophoblasts and secreted into the maternal and fetal circulation, with levels progressively rising from the third week of pregnancy to a peak at 35 36 weeks of gestation. As CSH increases IGF-1 secretion from cultured human fetal pancreas, an inhibitory action of GH-V on GH-N secretion via insulin-like growth factor 1 (IGF-1), has also been suggested [9]. As a result, GH-V stimulates production of maternal IGF-1, which subsequently inhibits GH-N secretion through a negative feedback [10 13]. Therefore, in normal pregnancy, increased GHV levels replace GH-N as the predominant circulating GH in maternal blood by the second trimester [14,15].

Thyrotrophin Thyroid-stimulating hormone (TSH) secretion is relatively stable during pregnancy, except for the first trimester, when serum TSH levels are decreased secondary to thyroid stimulation by human chorionic gonadotrophin (hCG), which has structural similarity to TSH. Both TSH and hCG contribute to TSH bioactivity (Fig. 13.2).

FIGURE 13.2 Changes in thyrotrophic axis during pregnancy. Mean ( 6 SE) serum concentrations of hCG, TSH, and bioassayable thyroid-stimulating activity (Bio-TSH) during normal pregnancy. Source: From Harada K, Hershman JM, Reed AW, et al. Comparison of thyroid stimulators and thyroid hormone concentrations in the sera of pregnant women. J Clin Endocrinol Metab 1979;48:793, [16].

Hepatic production of thyroid-binding globulin (TBG) is stimulated by estrogens with the consequent increase of total T4 and T3, reaching a plateau at 12 14 weeks [17]. Free T4 levels remain relatively constant throughout gestation, with a small increase during the first trimester, and minimal decrease thereafter [18]. The increased T4 requirement during pregnancy is mainly due to elevated serum TBG. Nevertheless, T4 metabolism by the fetal placental unit could also contribute to an increased need for this hormone in late pregnancy and to decreased demand after delivery [19].

Corticotrophin Normal human pregnancy is associated with increased maternal Hypothalamic pituitary adrenal axis function (Fig. 13.3). Due to high concentrations of corticosteroid-binding globulin (CBG) induced by elevated estrogen levels, cortisol clearance is reduced with a subsequent increase in total plasma cortisol levels. Additionally, as a result of placental corticotrophinreleasing hormone (CRH) stimulation of adrenocorticotrophic hormone (ACTH), production of cortisol

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FIGURE 13.3 Hypothalamic pituitary adrenal (HPA) axis during pregnancy. 16α-OH-A4, 16α-hydroxyandrostenedione; CRH, corticotrophin-releasing hormone; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate. Source: From Bronstein MD, Paraiba DB, Jallad RS. Management of pituitary tumors in pregnancy. Nat Rev Endocrinol 2011;7(5):301 10, [21].

increases, along with the free fraction of plasma cortisol, leading to very high cortisol levels, mainly in the third trimester. Despite high levels of free cortisol, pregnant women do not develop hypercortisolism owing to the antiglucocorticoid activity of high concentrations of progesterone. Additionally, effects of hypercortisolism are partially buffered by CRH-binding protein (CRHBP), thereby avoiding pituitary adrenal axis hyperstimulation. In the last weeks of pregnancy, however, a progressive decrease in CRHBP levels leads to greater quantities of free CRH, which stimulate the pituitary adrenal axis for labor [20]. In the fetus, glucocorticoid levels are much lower than maternal levels owing to high activity of placental corticosteroid 11β-dehydrogenase isozyme 2 (11β-HSD2), which catalyzes conversion of physiologically active glucocorticoids cortisol and corticosterone into inert forms such as cortisone. Thus, this barrier protects the fetus from exposure to maternal glucocorticoids, although still allowing passage of about 10 20%.

Posterior Pituitary During pregnancy, the plasma osmolality set point for vasopressin release is lowered by about 10 mOsm/

kg, as compared to nonpregnant women; consequently, serum sodium levels decrease by about 4 5 mEq/mL [22]. To maintain normal plasma vasopressin levels, vasopressin secretion rises to counteract the action of placental vasopressinase. However, vasopressinase activity during pregnancy may unmask mild diabetes insipidus (DI), or may exacerbate established DI [23]. Oxytocin plasma levels remain stable until the late stages of labor, rising as a result of vaginal wall distension. As a consequence, oxytocin stimulates myometrial contractions for delivery and also plays a role in breastfeeding [24].

PITUITARY TUMORS AND PREGNANCY Pituitary adenomas account for about 15% of all intracranial neoplasms. The induction and management of pregnancy in women harboring pituitary tumors is usually challenging. Infertility is common in these patients, as the gonadotroph axis is frequently compromised, either by tumor mass effect of macroadenomas or by functional suppression secondary to

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hyperprolactinemia or hypercortisolism, irrespective of tumor size. As a result, fertility is often impaired in women with microadenomas or macroadenomas [25]. Progress in successful hormone therapy for ovulation induction, as well as surgical and medical therapy for pituitary adenomas, has made pregnancy possible for many affected women. Nevertheless, this achievement has highlighted risks of complications for both mother and fetus. Fertility restoration and management before, during, and after pregnancy of patients bearing pituitary adenomas remain challenging for improved efficacy and safety of the desired pregnancy. Hyperprolactinemia, occurring with prolactinomas and other pituitary tumors, secondary to PRL secretion or pituitary stalk disconnection, respectively, suppresses GnRH pulsatility, probably via kisspeptin inhibition, leading to infertility [26]. Prolactinoma is the most common pituitary adenoma subtype, with a prevalence of 500 cases/million persons and an incidence of 27 cases/million/year [27]. Growth hormone-secreting pituitary adenomas leading to acromegaly is the second most common functioning pituitary adenoma, with a prevalence of 40 [28] to 130 cases [29] per million persons. Different mechanisms contribute to hypogonadism in acromegaly: gonadotroph compression by the tumor, hyperprolactinemia, lactotrophic effect of GH, and GH/IGF-1 excess inhibiting gonadotrophic axis, and directly or indirectly altering ovarian sex steroid (via hyperinsulinism), similar to polycystic ovarian syndrome [30 32]. Cushing disease (CD) has an estimated prevalence of 40 cases per million and an incidence of 1.2 2.4 cases per million per year [33]. Hypogonadism in CD is mainly due to the negative impact of hypercortisolism and hyperandrogenism on the gonadotroph axis and, less commonly, to tumor mass effect of macrocorticotrophinomas. Additionally, frequent and important CD-associated metabolic disorders may also impact fertility [25]. Clinically nonfunctioning pituitary tumors rarely affect young females. Infertility is encountered with macroadenomas, due to tumor compression and/or hyperprolactinemia secondary to pituitary stalk disruption. Thyrotrophinomas are very rare tumors and seldom associated with pregnancy [25]. In pituitary adenomas, treatment should include control of hormonal hypersecretion and tumor mass effect related to infertility.

Prolactinomas Prolactinomas are the most common pituitary adenomas and a frequent cause of infertility among young

women, usually associated with oligomenorrhea or amenorrhea [34,35]. Medical treatment with dopamine agonists (DAs), including cabergoline (CAB), and bromocriptine (BRC), is the gold standard for both microprolactinomas and macroprolactinomas. They effectively normalize serum PRL levels, leading to restoration of eugonadism and tumor shrinkage in most cases. CAB is currently the drug of choice due to its higher affinity to dopamine receptor type 2 (D2R) and better tolerance as compared to BRC [36]. A head-to-head study evaluating 459 women with hyperprolactinemia treated with CAB or BRC pointed to serum PRL normalization in 83% of patients on CAB and 59% of those on BRC. Ovulatory cycles were restored in 72% of patients on CAB and 52% on BRC [37]. Nevertheless, use of CAB for induction of pregnancy is still a matter of debate, and only BRC is currently approved for this purpose [38]. It is important to consider the patient’s desire for pregnancy, especially for those with macroprolactinoma, and to discuss the potential risks involved. The first concern is related to tumor mass effect. Hyperestrogenism secondary to pregnancy can cause hypertrophy and hyperplasia of both normal and tumoral lactotrophs, leading to tumor increase associated with mass effect symptoms in nontreated patients. The second point to be considered is fetal exposure to DA during embryogenesis that potentially could lead to malformations. Also, the question of breastfeeding should be considered. Regarding the risk of tumor growth during pregnancy, a systematic review [39] included 764 microprolactinomas, 238 macroprolactinomas without previous surgery or radiotherapy, and 148 macroprolactinomas in which these treatments had been already performed. Symptomatic tumor growth occurred in 2.4%, 21%, and 4.7% of cases, respectively. In the vast majority of cases, DA was withdrawn when pregnancy was confirmed, usually at 6 weeks of gestation. Interestingly enough, in one study, the authors showed a reduced risk of tumor growth in pregnant patients treated with BRC for at least 12 months [40]. For optimal pregnancy outcome and fetal development, in women harboring prolactinomas who desire pregnancy, BRC is the DA of choice [38], as more than 6000 pregnancies induced by this drug are reported. Moreover, the drug is usually withdrawn after pregnancy is confirmed and BRC has a shorter half-life as compared to CAB. In fact, as CAB can be detected in the circulation up to 30 days after drug withdrawal [41], early fetal exposure is unavoidable. Therefore, although the number of reported CAB-induced pregnancies is increasing (938 cases), CAB safety in pregnancy is still a concern. Current data do not show different risk of premature labor and fetal malformations, compared to BRC and the general population,

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but the number of pregnancies induced by CAB is still significantly lower than that with BRC [39]. Data regarding DA use throughout pregnancy are scant. BRC use during entire pregnancy was reported in 100 cases, with two cases of malformations: one undescended testicle and another with a talipes deformity [42 44]. For CAB, one fetal death was reported in 15 pregnancies [45]. There are few data about the development of children conceived while mothers received DA. Bronstein [46] reported on 70 children born after BRC exposure and followed for 12 to 240 months, and noted one case of idiopathic hydrocephalus, one with tuberous sclerosis, and another with precocious puberty. In two studies including 64 children [47] between the ages of 6 months and 9 years, and 988 children [42] 4 months to 9 years, respectively, no impaired physical development was observed. For CAB, Bronstein [46] and Ono et al. [48] did not find abnormalities in 5 and 83 children, followed by 41 months and 12 years, respectively. Lebbe et al. following 88 children described two cases of slight delay in verbal fluency and one case of difficulty in

FIGURE 13.4 Management of prolactinoma during pregnancy. Source: From Bronstein, MD. PRLomas and pregnancy. Pituitary 2005;8:31 8, [46].

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achieving complete continence [49]. Stalldecker et al. followed 61 children and found 2 cases of seizures and 2 cases of pervasive developmental disorder [50]. Fig. 13.4 depicts a suggested algorithm for management of prolactinoma during pregnancy. Discontinuation of DA is recommended in patients with microadenomas or enclosed macroadenomas as soon as pregnancy is confirmed. In macroadenomas, tumor shrinkage within sellar boundaries is required before allowing pregnancy. For patients with invasive tumors, the decision of DA maintenance throughout pregnancy or reintroduction of the drug, if needed, depends on clinical circumstances. Serum PRL assessment is not recommended routinely, since median values of pregnant women with and without prolactinomas seem to be similar [46]. For microprolactinomas, clinical evaluation in each trimester of pregnancy should be done, and sellar magnetic resonance imaging (MRI) without contrast and neuroophthalmologic evaluation is performed if there is evidence of tumor size increase. For expanding macroprolactinomas, ophthalmologic evaluation should be performed in each trimester, and pituitary MRI without contrast is indicated if there is evidence of tumor growth. In the presence of mass effect symptoms, with no rapid improvement after DA introduction, decompressive neurosurgery, especially in the second trimester, should be considered [46]. Fig. 13.5 depicts the image evolution of the pregnancy of a 19-year-old patient harboring a prolactinoma, secondary amenorrhea, no visual impairment, and serum PRL levels of 520 ng/mL. After 12 months on CAB 1.5 mg/week, she resumed regular menses, serum PRL levels dropped to 19 ng/mL, and MRI depicted tumor reduction within sellar boundaries.

FIGURE 13.5 Sellar noncontrasted MRI follow-up of a patient with a macroprolactinoma. At diagnosis coronal (A) and sagittal (B) views and after 12 months on CAB (C and D) showing tumor shrinkage. In the 7th month of pregnancy, 3 months without CAB, the patient presented severe headaches; with tumor increasing dimensions (E and F). After BRC reintroduction, tumor regression and improvement of mass effect symptoms can be seen (G and H).

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Despite being advised to maintain barrier contraception, she returned in the 3rd month of pregnancy, and CAB was withdrawn at this time. After 1 month, however, she presented with severe headaches without visual complaints. Her serum PRL levels were 110 ng/ mL and sellar imaging showed tumor progression. BRC was then administered, headache subsided and serum PRL dropped to 46 ng/mL. During the 7th month of gestation, a sellar MRI showed tumor reduction. Cesarean delivery was uneventfully performed at term. Interestingly, women harboring prolactinomas in the second and third trimesters of pregnancy exhibit levels of serum PRL comparable to normal (Fig. 13.6). As decreased or even normalization of PRL levels after pregnancy are well documented in the literature, PRL levels and tumor size should be reassessed after delivery. Peillon et al. [51] observed hemorrhagic zones in the tumor, leading to the hypothesis that hyperestrogenism during pregnancy can induce areas of tumor necrosis and microinfarction, leading to reduction, or complete tumor involution after pregnancy. In our series [52], 60% of patients with microprolactinoma and 72% of those with macroprolactinoma showed decreased serum PRL levels after delivery when compared with levels before pregnancy. In 62 patients (with both microprolactinomas and macroprolactinomas), serum PRL levels dropped from 336 6 105 ng/mL (mean 6 SD) to 133 6 20 ng/mL (P , 0.05), assessed before and after pregnancy. Moreover, 11% of our patients maintained normal PRL levels, with ovulatory menses, and no further drug reinstitution. Eight became pregnant again, with no complications and no hyperprolactinemia recurrence. Remission of hyperprolactinemia

FIGURE 13.6 Mean serum PRL levels throughout the three trimesters of pregnancy in patients with prolactinomas compared to PRL levels in normal pregnancy. MIC, microprolactinomas; MAC, macroprolactinomas; dotted lines denote mean serum PRL during normal pregnancy. Source: From Bronstein, MD. PRLomas and pregnancy. Pituitary 2005;8:31 8, [46].

after pregnancy in eight studies was 27%, ranging from 10 to 68% [46,49,50 59]. Domingue et al. [54] found no correlation with nadir PRL, serum PRL both pre- and postpregnancy, CAB dose, and length of use as predictors of hyperprolactinemia remission after pregnancy. Small adenoma size and a normal sellar MRI after pregnancy were related to hyperprolactinemia remission. Breastfeeding is not associated with risk of pituitary tumor growth risk [53 59], and should be allowed in patients not requiring DA during pregnancy.

Acromegaly The placenta secretes a variant GH isoform, different from pituitary GH in 13 amino acids, and increases IGF-1 levels leading to reduced pituitary GH secretion during the second-half of pregnancy [14]. In healthy pregnant women, pituitary GH secretion is suppressed, while in acromegaly, this secretion is autonomously sustained (Fig. 13.7). Placental GH secretion increases throughout pregnancy and induces rising IGF-1 levels. Current GH immunoassays do not differentiate placental [60] and pituitary isoforms and suppression of GH during an oral glucose tolerance test has not been properly assessed in pregnant

FIGURE 13.7 Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) axis in nonpregnant women compared with healthy pregnancy and in pregnant women with acromegaly. GH variant (GH-V) is the major hormone responsible for stimulating the production of maternal IGF-1, which inhibits maternal pituitary secretion of normal GH (GH-N) through negative feedback. GH-V replaces GHN as the predominant form of GH in maternal blood by the second trimester. In pregnant woman with acromegaly, GH-N secretion is autonomous. Both GH-N and GH-V levels are persistently elevated in the blood throughout pregnancy. Similar to normal pregnancy, IGF-1 levels rise in patients with active acromegaly owing to stimulation by GH-V. Source: From Bronstein MD, Paraiba DB, Jallad RS. Management of pituitary tumors in pregnancy. Nat Rev Endocrinol 2011;7 (5):301 10, [21].

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patients, rendering the diagnosis of acromegaly during pregnancy difficult. Documentation of pulsatile GH secretion and/or paradoxical GH release after TRH stimulation, which induces release of GH-N but not GH-V, may help diagnose the presence of a true increase in GH-N levels [14,15,61,62]. If a patient becomes pregnant before the diagnosis of acromegaly has been established, the confirmation of the disease is often only possible after delivery. Nevertheless, if clinical findings and the laboratory assessment suggest acromegaly, pituitary MRI without gadolinium is warranted to visualize the tumor. Pregnancy in women with acromegaly is rare due to the presence of central hypogonadism, hyperprolactinemia, insulin resistance, and polycystic ovary syndrome [32,63 65]. Moreover, IGF could be involved in activation of ovarian steroidogenesis [66]. Nevertheless, reports of pregnancies in women with acromegaly have increased, even in patients in whom the disease is not controlled [67]. During pregnancy in acromegaly, there is a potential risk of tumor mass effect secondary to physiological pituitary size increase. Nonetheless, there are few cases reported with this complication. Interestingly, improvement of acromegaly, even with IGF-1 level normalization, has been described [68 70]. Although excess maternal GH/IGF-1 seems to be harmful during pregnancy because it could predispose to gestational diabetes, hypertension, and heart disease, these complications have rarely been described [71,72]. Data regarding the fetus are also scant, with few reports of low birth weight, related to treatment with somatostatin receptor ligands (SRLs) [31,67,73]. Concerning the therapeutic approach to acromegaly in pregnancy, two scenarios should be considered. The first concerns patients already diagnosed who decide to become pregnant. In this case, they require assistance to improve fertility and prevent maternal and fetal complications. The second scenario concerns patients with acromegaly who become pregnant without planning. In this case, the decision should be made as to whether the patient requires active acromegaly treatment or whether expectant management can be adopted. With a planned pregnancy in patients harboring microadenomas and noninvasive macroadenomas, SRLs, which cross the placenta, ideally should be discontinued at least 2 months before attempts to conceive [70,74]. For unplanned pregnancies, SRLs should be discontinued as soon as pregnancy is detected [77]. Nevertheless, despite expression of somatostatin receptors in the placenta and the fetal pituitary gland, neonates born to women with acromegaly treated with SRLs are usually of normal size, without malformations [70,74 76]. Also, reversible and mild changes in maternal fetal hemodynamics in pregnant acromegaly

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FIGURE 13.8 Management of women with acromegaly before and during pregnancy. Source: From Bronstein MD, Paraiba DB, Jallad RS. Management of pituitary tumors in pregnancy. Nat Rev Endocrinol 2011;7(5):301 10, [21].

women exposed to SRLs have been described [77]. Araujo et al. [78] compiled 89 reported cases of pregnancy in acromegaly women receiving SRLs or DA with no serious adverse events during pregnancy, delivery, and newborn development. Medical treatment was withdrawn in the first trimester of pregnancy in most cases [30,31,67,73,78]. Nonetheless, due to lack of well-controlled studies, these drugs should only be used under special circumstances, such as with recurrence of GH hypersecretion with return of clinical symptoms of acromegaly and/or reexpansion of the pituitary adenoma. Close visual field monitoring is essential, and pituitary gland MRI should be performed if evidence of tumor growth is present. In cases not responsive to drug therapy, surgery should be considered, especially during the second trimester [21] (Fig. 13.8). Outcomes of 27 pregnant women receiving the GH receptor antagonist pegvisomant were compiled [79]. Three patients received the drug throughout pregnancy, and no adverse effects were observed. One study reported use of pegvisomant throughout pregnancy, with normal fetal GH and IGF-1 levels, minimal fetal and breast milk levels of pegvisomant, normal fetal growth parameters, and a healthy baby [80].

Cushing Disease The diagnosis of Cushing’s syndrome (CS), including CD, during pregnancy is often a challenge. Three scenarios should be distinguished: (1) patients with diagnosis of CS who become pregnant; (2) patients with CS developed during pregnancy, and (3) normal pregnant women with clinical features of CS, including hypertension, glucose intolerance, and/or striae, all

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FIGURE 13.9 Nonpregnant woman with Cushing disease exhibiting striae in the breast and thighs (author’s collection).

prevalent in normal pregnancy. Muscle weakness, large purple striae (mainly in regions outside the abdomen) (Fig. 13.9), and osteoporosis are clues highly suggestive of CS. Nonetheless, differential diagnosis solely on clinical features can be confusing, usually requiring laboratory and imaging support. During healthy pregnancy, a physiological status of hypercortisolism is expected due to pituitary adrenal axis activation by both placental ACTH and CRH and also due to estrogen-induced increased CBG levels, leading to urinary cortisol levels 2 3 times those seen in nonpregnant women. As a consequence, high urinary-free cortisol, especially if lower than three times the upper limit of normal range, usually cannot distinguish normal pregnancy from CS, especially during the second and third trimesters. Additionally, cortisol levels during a dexamethasone test are usually nonsuppressed during normal pregnancy [20,81,82]. Therefore, due to overlapping results with normal pregnancy, laboratory diagnosis of CS during pregnancy could be difficult. The best laboratory clue to differentiate CS with normal pregnancy should be based on the demonstrated absence of circadian cortisol rhythm in CS, which is preserved during normal pregnancy. Therefore, measuring late-night salivary cortisol could be an important tool. Recently, cutoff values for nighttime salivary cortisol were established, with levels increasing throughout subsequent trimesters [83]. However, more studies are necessary to validate nighttime salivary cortisol to differentiate CS from healthy pregnancy. Pituitary MRI may not be informative for CD microadenomas [84,85], particularly during pregnancy, when physiological pituitary hyperplasia may mask a tiny tumor. Noncontrast imaging should be performed only if surgery is indicated prior to birth. Adrenal CT scans are contraindicated, and ultrasonography is the initial approach. Nongadolinium MRI could be indicated for challenging cases. The presence of adrenal incidentalomas should also be considered in the differential diagnosis.

Corticotrophinoma growth during pregnancy is controversial [86 90]. Jornayvaz et al. [91] followed 20 pregnancies in patients with CD previously treated by bilateral adrenalectomy and no radiotherapy, with uneventful deliveries. Pregnancies did not induce corticotroph tumor progression to Nelson syndrome. Pregnancy is rare in CS, and is more commonly associated with adrenal rather than pituitary disease. Adrenal adenomas (purely cortisol-secreting) interfere less with ovulation, as compared to pituitarydependent CS. About 50 pregnancies in women with CD have been reported [20,92,93]. In CD, infertility occurs mainly due to hypogonadism secondary to hypercortisolism and hyperandrogenism, and even hyperprolactinemia. Moreover, pregnancy in these patients is associated with maternal and fetal morbidities, including hypertension, preeclampsia, gestational diabetes, cardiac failure, poor wound healing after cesarean section, abortion, premature labor, intrauterine growth retardation, and perinatal death [94,95]. The high incidence of fetal morbidities is probably secondary to maternal complications, as the presence of 11β-dehydrogenase type 2 in placenta degrades cortisol and protects the fetus from hormonal excess [97]. In a review of 69 pregnancies in patients with CS, two-thirds of women presented with hypertension and one-fourth with diabetes [97]. In six pregnancies in five patients with CD followed by Chico et al. [94], four underwent transsphenoidal surgery and developed mild hypercortisolism before pregnancy. Lindsay et al. reviewed 136 pregnancies in women with CS: in 42.5%, no specific treatment was indicated and others underwent transsphenoidal surgery, medical treatment, or bilateral adrenalectomy. Both medical and surgical treatment of hypercortisolism improve fetal outcome [20]. Metyrapone has been used as therapy, but may lead to systemic hypertension and preeclampsia, and one case of fetal hypoadrenalism was described [98]. Ketoconazole (not approved during pregnancy) has been successfully used in some reports with careful attention to potential feminization of male fetus and teratogenic effects [20,99,100]. The use of CAB throughout pregnancy was described in one case of CD, without complications [101]. Transsphenoidal surgery for CD should be reserved for severe cases without improvement with medical treatment, or in the unlikely case of symptomatic tumor growth, preferably during the second trimester. Radiotherapy and mitotane are contraindicated due to teratogenic effects [21]. In conclusion, the correct diagnosis and treatment of CD during pregnancy is often challenging and should be carefully monitored, especially due to potential maternal and fetal morbidities [102].

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Thyrotrophinomas Pregnancy in patients with thyrotrophinomas is extremely rare given that this kind of tumor type comprises less than 3% of all pituitary adenomas [103]; they are usually macroadenomas, associated with central hyperthyroidism. Symptoms related to tumor mass effect should be carefully investigated throughout pregnancy. There are only four [70,104 106] such cases reported in the literature to date: octreotide was used during pregnancy in two cases, neurosurgery was performed in another, and in one case the diagnosis was made during pregnancy. Maternal fetal adverse effects were not encountered. Our experience is limited to one patient treated with octreotide LAR throughout pregnancy, also without complications (unpublished data).

Clinically Nonfunctioning Pituitary Adenomas These tumors are associated with central mass effect with no hormonal hypersecretion except for hyperprolactinemia due to pituitary stalk disruption. Although frequent, representing one-third of all pituitary adenomas, they more commonly affect older individuals. Moreover, they are usually macroadenomas at the time of diagnosis, usually associated with hypopituitarism, making pregnancy a very rare occurrence in women harboring those tumors, who usually require ovulation induction by GnRH or gonadotrophins [107]. Physiological pituitary enlargement due to hyperestrogenism can lead to symptomatic tumor mass effect, requiring first-line dopaminergic therapy. Surgery should be reserved for cases with no improvement with medical treatment, preferably during the second trimester [21,98].

NONTUMORAL PITUITARY DISTURBANCES RELATED TO PREGNANCY Sheehan syndrome (ShS), first described by Simmonds in 1913 [108], is a consequence of hypopitutarism secondary to pituitary necrosis due to hypovolemic shock related to uterine bleeding during or after delivery. Although rare nowadays due to improved perinatal care, this condition should be borne in mind in puerperal patients, especially for those with severe postpartum bleeding and agalactia with no history of hypopituitarism and/or pituitary tumor [7]. During pregnancy, pituitary hyperplasia renders the gland more susceptible to ischemia, associated with small sella size, severe arterial hypotension, and even autoimmunity [109,110].

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As symptoms of hypopituitarism are frequently indolent, ShS is rarely diagnosed in the acute-phase [111] and the time between delivery and diagnosis ranges from a few years up to 30 years, with a mean of 14 years [112 114]. Acute-phase ShS should be distinguished from pituitary apoplexy, as the latter might be treated surgically [115]. The degree and severity of hypopituitarism is variable, and although failure of lactation is the hallmark of ShS, GH deficiency is the most prevalent hormone disorder [114,116,117]. Panhypopituitarism was reported in 55 86% of patients [114,116,117]. DI is rare, occurring in less than 5% of cases [118,119], and is a diagnostic clue that could be used to differentiate ShS from hypophysitis. Sellar imaging depicts early nonhemorrhagic enlargement, later resulting in an empty sella [7]. Anemia and other cytopenias are described in patients with ShS, and improving after hormonal replacement [120]. Heavy vaginal bleeding after delivery, significant hypotension or shock requiring urgent medical assistance, failure to lactate in the postpartum period, amenorrhea after delivery, hypopituitarism, and imaging of empty sella are diagnostic clues for ShS [121]. Treatment of ShS is based on hormonal replacement of the deficient hormone(s) and, as hypogonadism is implicated in infertility, ovulation induction can be considered. Interestingly, spontaneous pregnancies have been reported in patients with ShS, and pregnancy was also associated with recovery of some pituitary function [122 126].

Lymphocytic Hypophysitis Lymphocytic hypophysitis (LH), an autoimmune disease characterized by predominant lymphocytic infiltration, may lead to pituitary tissue destruction and fibrotic reaction, with subsequent hypopituitarism [127]. It is rarely encountered: a surgical series estimated the annual incidence of 1 case per 9 million [128], although some believe that this number is an underestimate [127]. LH can be classified according to anatomical disturbances, with adenohypophysitis (LAH), lymphocytic infundibuloneurohypophysitis (LINH), and panhypophysitis lipotropic hormone (LPH). Among the different types of primary hypophysitis, LH is the one clearly related to pregnancy. In 1962, Goudie and Pinkerton described a 22-yearold woman who died due to adrenal insufficiency 14 months after delivery of her second child; at autopsy, her pituitary was small, with lymphocytic infiltration [129]. Since then, 379 cases of LH have been reported [127]. LAH is about six times more common in women, with mean age at presentation of 35 years, and 57% presented during pregnancy or postpartum.

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LINH and LPH have a mean age at presentation of 42 years and are not associated with pregnancy. This association with LH is not clearly explained: increased pituitary mass may lead to release of pituitary antigens and hyperestrogenemia could modify circulating pituitary patterns, increasing the systemic circulation, and rendering the pituitary more accessible to the immune system during pregnancy [127]. In about 30% of cases, LH is associated with other autoimmune diseases, especially Hashimoto’s thyroiditis [127,130]. Although considered autoimmune, clearcut antigens in LH have not been identified to date. Antipituitary antibodies (APAs) were described in research studies and cannot yet be used as a diagnostic tool [131]. Antibodies to Tpit were found in 10% of patients with LH, but without specificity in diagnosis [132]. High titers of APA were also found in patients previously diagnosed with idiopathic hypopituitarism: 15% with ACTH deficiency, 26% with GH deficiency, and 21% with central hypogonadism [133]. Other authors pointed to positive APA titers in diseases other than pituitary autoimmune diseases [134]. Symptoms are related to mass effects, including headaches (53%) and visual impairment (43%), hypopituitarism (preferentially ACTH deficiency (42%) in contrast to pituitary tumors), and hyperprolactinemia (23%). In other series, DI occurred in 15% of cases [135,136]. LH is not related to maternal or fetal morbidities during pregnancy, except one report of a mother dying during labor [137]. Previous pregnancies and a history of LH are not associated with subsequent episodes of LH [127]. Typically, MRI depicts a sellar mass with homogeneous enhancement by gadolinium, pituitary stalk thickening and loss of spontaneous high signal intensity on T1-weighted imaging, corresponding to the posterior pituitary. An empty sella may be found after years of continued disease [134,138]. Nevertheless, pituitary imaging can be misleading, as a pituitary adenoma may be inaccurately diagnosed. Definitive LH diagnosis is made by pathological analysis of pituitary tissue from a pituitary biopsy. Clues for diagnosis include hypopituitarism in young women, particularly with ACTH deficiency, relationship to pregnancy and puerperium, symmetrical sellar mass, and no history of peripartum hypovolemia [136,139]. Hormonal replacement of deficient hormones is required, especially for the adrenal axis. Spontaneous regression has been reported in some cases. Nevertheless, in cases with a central mass effect, immunosuppression with corticosteroids is recommended; if there is no improvement, neurosurgery is indicated [134,140].

Hypopituitarism Management of patients with hypopituitarism should aim to mimic as closely as possible changes in the hormonal milieu observed during normal pregnancy. Also, knowledge of hormonal production by the placenta and fetus is important to avoid overtreatment. In a hypopituitary woman with hypogonadism, sex steroid replacement is indicated to prepare the uterine environment for pregnancy, stimulating proliferation of endometrium and vaginal epithelium [141]. Highdose estrogen and GH, if deficient, could improve the prognosis of hypopituitarism [7]. Pregnancy and birth rates are reported to be 47% and 42%, respectively [142]. In isolated hypogonadotrophic hypogonadism, ovulation and conception rates are higher and spontaneous miscarriage rates lower [143]. As uterine size is similar in both conditions, other hormone deficiencies, if not replaced, could contribute to an adverse outcome. Although the GH IGF-1 axis is not essential for fertility, some studies showed that GH replacement, in those with GH deficiency, could improve fertility rates [144 147]. Proposed mechanisms for fertility improvement by the GH/IGF-1 axis include stimulation of follicular growth and maturation, improved ovulation by ovarian sensitivity to gonadotrophins, and reduced apoptosis rate in preovulatory ovarian follicles [7]. GH treatment in pregnant women with normal pituitary function does not seem to be beneficial [147], although some authors suggest rhGH replacement prior to ovulation induction [148]. Although GH deficiency is reported to result in uneventful pregnancies [148 150], its use is controversial during pregnancy [151]. Gestational GH therapy should be performed only during the first and second trimesters, as after the 20th week of pregnancy, GH secretion by the placenta increases, providing an adequate replacement to the lack of pituitary GH secretion [14]. For adrenal insufficiency, the glucocorticoid dose may be increased (by 50% for hydrocortisone) in the third trimester. As in other stress conditions, the hydrocortisone dose should also be increased during labor until 48 hours postpartum [152]. Breastfeeding is allowed as glucocorticoid transfer to breast milk is insufficient to cause hypoadrenalism in the newborn [153]. In central hypothyroidism, which lacks TSH as a marker, replacement with levothyroxine (LT4) is recommended to maintain free T4 in the upper half of the normal range [154]. Additionally, due to increased serum T4 binding to TBG during pregnancy, the dose of LT4 should be progressively increased up to 50% of pregestational doses, after pregnancy confirmation [19]. Wang and Yang [155] studied perinatal treatment and pregnancy outcomes in 31 hypopituitary women and showed that there was no significant difference in LT4

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REFERENCES

dosage between early pregnancy (51 6 36 μg/d) or postpartum (38 6 34 μg/d) and pregestation (33 6 35 μg/d) (P . 0.05). However, the required dose of LT4 during the second (68 6 42 μg/d) and third trimesters (76 6 42 μg/d) increased by about 35% on average as compared to the pregestational period. The diagnosis of DI during pregnancy is challenging because water metabolism changes during pregnancy. As the water deprivation test is normally not recommended [156,157], the diagnosis is usually limited to assessment of urine and plasma osmolalities. Mild DI may be exacerbated in pregnant patients, and asymptomatic disease could become symptomatic during pregnancy. During pregnancy, desmopressin use is safe, although it may be associated with increased uterine contractility, especially after intravenous administration [158,159]. As placental vasopressinase does not degrade desmopressin, patients on this drug before pregnancy may maintain the same doses during this period [160,161]. Breastfeeding is safe in these patients as only small amounts of desmopressin are present in breast milk [160,161]. Gestational DI may occur during the late phases of pregnancy and usually remits 4 6 weeks after delivery. It is a rare condition, occurring in 2 4 of 100,000 pregnancies [156]. As liver dysfunction (mainly the HELLP syndrome) attenuates vasopressinase degradation, leading to worsening DI [156,157,162 164], assessment of liver function in patients with gestational DI is mandatory [156,165,166].

CONCLUSIONS Pregnancy is associated with changes in the hypothalamic pituitary axis and the respective target glands and tissues. It is also associated with development of clinical disorders including ShS and LH. Due to advances in medical and surgical treatments of pituitary adenomas, pregnancy has become a reality for women harboring such tumors. Moreover, ovulation induction and proper hormonal replacement mimicking the altered hormonal milieu during normal gestation have enabled safe pregnancies for patients with hypopituitarism.

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[150] Verhaeghe J, Bougoussa M, Van Herck E, de Zegher F, Hennen G, Igout A. Placental growth hormone and IGF-I in a pregnant woman with Pit-1 deficiency. Clin Endocrinol 2000;53:645 7. [151] Vila G, Akerblad AC, Mattsson AF, Riedl M, Webb SM, Ha´na V, et al. Pregnancy outcomes in women with growth hormone deficiency. Fertil Steril 2015;104(5):1210 17. [152] Arlt W, Allolio B. Adrenal insufficiency. Lancet 2003;361: 1881 9. [153] McKenzie SA, Selley JA, Agnew JE. Secretion of prednisolone into breast milk. Arch Dis Childhood 1975;50:894 6. [154] LaFranchi S. Thyroid hormone in hypopituitarism, Graves’ disease, congenital hypothyroidism, and maternal thyroid disease during pregnancy. Growth Horm IGF Res 2006;16 (Suppl. A):S20 4 1620 242. [155] Wang YF, Yang HX. Clinical analysis of hypothyroidism during pregnancy. Zhonghua Fu Chan Ke Za Zhi 2007;42:157 60. [156] Ananthakrishnan S. Diabetes insipidus in pregnancy: etiology, evaluation, and management. Endocr Pract 2009;15:377 82. [157] Kalelioglu I, Uzum AK, Yildirim A, Ozkan T, Gungor F, Has R. Transient gestational diabetes insipidus diagnosed in successive pregnancies: review of pathophysiology, diagnosis, treatment, and management of delivery. Pituitary 2007;10:87 93. [158] Ray JG. DDAVP use during pregnancy: an analysis of its safety for mother and child. Obstet Gynecol Surv 1998;53:450 5.

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[159] Hague WM. Pre-existing endocrine disease in relation to pregnancy. Curr Obstet Gynaecol 1999;9:62 8. [160] Durr JA. Diabetes insipidus in pregnancy. Am J Kidney Dis 1987;9:276 83. [161] Kallen BA, Carlsson SS, Bengtsson BK. Diabetes insipidus and use of desmopressin (Minirin) during pregnancy. Eur J Endocrinol 1995;132:144 6. [162] Hamai Y, Fujii T, Nishina H, Kozuma S, Yoshikawa H, Taketani Y. Differential clinical courses of pregnancies complicated by diabetes insipidus which does, or does not, pre-date the pregnancy. Hum Reprod 1997;12:1816 18. [163] Kennedy S, Hall PM, Seymour AE, Hague WM. Transient diabetes insipidus and acute fatty liver of pregnancy. Br J Obstet Gynaecol 1994;101:387 91. [164] Yamanaka Y, Takeuchi K, Konda E, Samoto T, Satou A, Mizudori M, et al. Transient postpartum diabetes insipidus in twin pregnancy associated with HELLP syndrome. J Perinat Med 2002;30:273 5. [165] Bellastella A, Bizzarro A, Colella C, Bellastella G, Sinisi A, De Bellis A. Subclinical diabetes insipidus. Best Pract Res Clin Endocrinol Metabol 2012;26:471 83. [166] Marques P, Gunawardana K, Grossman A. Transient diabetes insipidus in pregnancy. Endocrinol Diabetes Metab Case Rep 2015;2015:150078.

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C H A P T E R

14 Psychiatric Disease in Hypothalamic Pituitary Disorders Caroline Sievers and Gunter K. Stalla

INTRODUCTION Psychiatric disorders have a great impact on mortality and quality of life and are a major burden for society and individuals worldwide. According to the Global Burden of Disease Study, major depressive disorder as the main psychiatric disorder is the second leading cause of disability-adjusted life years worldwide [1,2]. Psychiatric comorbidity occurs frequently in chronic somatic disease, especially in neuroendocrine disorders, where structural changes of the brain and metabolic changes occur (see [3]). Somatic disease, as a stressor, puts patients at risk for depression through direct and indirect effects on morphological brain alterations, as well as changes of the hormonal and adipokine milieu, and vascular pathologies, that disrupt the somatic “hardware” responding to the stressor. Detailed knowledge of factors, diagnostic procedures, and therapeutic options for treating psychiatric diseases associated with endocrine disorders are relevant for patients and physicians, and also from a perspective of health economics. The clinical aim is to identify neuroendocrine patients at risk for psychiatric comorbidity, to treat them effectively, and to develop new patient management strategies.

Historical Perspective Medical care for patients with endocrine disease was primarily the responsibility of psychiatrists at the beginning of the 20th century. Psychophysiological functions seen in conjugation with hormonal diseases

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00014-3

were summarized as “endocrine psychosyndrome” by Bleuler and colleagues [4,5]. Although nowadays endocrine disorders usually present in more subtle ways and are detected earlier by specialists, some patients are only diagnosed with an endocrine disease such as Cushing disease after they were hospitalized for a psychiatric disorder such as depression. A new research field, psychoneuroendocrinology, has emerged with extensive study of the interaction between hormones and the brain. Outcomes of hormonal excess syndromes as well as hormonal substitution can serve as a model to study physiological and pathophysiological roles of hormones in cognitive functions, mood, behavior, sleep, and cognition (see [3]).

PSYCHIATRIC DISEASES IN HYPOTHALAMIC PITUITARY DISORDERS Hypopituitarism Hypopituitarism can be found in about 45 patients per 100 000 inhabitants who often present with very nonspecific symptoms [6]. Patients may suffer from symptoms such as headache, loss or reduction of vision, lack of motivation, sleep disturbances, reduced libido, or reduced attention and memory (Table 14.1) [7]. These symptoms may predispose patients to depressive symptoms, and may also make it difficult to clearly distinguish between depressive symptoms as a separate entity versus symptoms of the underlying endocrine disturbance.

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14. PSYCHIATRIC DISEASE IN HYPOTHALAMIC PITUITARY DISORDERS

TABLE 14.1 Clinical Consequences of Hormone Deficiencies Overlapping Psychiatric Symptoms Hormone deficiencies

Symptoms

Growth hormone

Anergia, poor quality of life

LH/FSH (sex steroids)

Sexual dysfunction, mood disorders, reduced vigor

Corticosteroids

Weakness, poor energy, weight loss

Thyroid hormones

Poor energy, neuropsychiatric problems, weight gain

LH, luteinizing hormone; FSH, follicle-stimulating hormone. Adapted and modified from Schneider HJ, Kreitschmann-Andermahr I, Ghigo E, Stalla GK, Agha A. Hypothalamopituitary dysfunction following traumatic brain injury and aneurysmal subarachnoid hemorrhage: a systematic review. JAMA 2007;298(12):1429 38.

It seems to be a general finding that the quality of life is often permanently reduced in hypopituitarism despite hormonal substitution therapy and psychiatric symptoms are frequent [8,9]. However, elevated rates of clinical psychiatric diagnoses in patients with hypopituitarism could not be established [10].

Corticotroph Insufficiency The CRH-ACTH-cortisol system as the stress hormone system plays a role in the development of psychiatric symptomatology in patients with pituitary disease [11]. Any kind of disturbances of the stress hormone system can lead to psychiatric symptomatology. While acute corticotrophic deficiency can be a life-threatening condition, with symptoms such as weakness, dizziness, nausea, vomiting, fever, and shock, patients with chronic deficiency present with fatigue, anorexia, weight loss, and depression [6,12]. Primary or secondary adrenal insufficiency represents a major challenge for patient management in terms of quality of life and morbidity and mortality. Patients seem to hate their medication and the “right regimen of glucocorticoid therapy for the individual patient” is a major challenge, which might be due to suboptimal mimicking of normal circadian profiles of plasma cortisol of the classical glucocorticoid regimens or to insufficient outcome parameters for an adequate substitution therapy [67,68]. Substitution treatment divided into three doses per day, mimics the normal secretion pattern. Study of a new long-acting hydrocortisone in patients with primary adrenal deficiency shows enhanced improvement of quality of life scores, which may eventually improve psychological outcomes as well [6,13,14,15]. Dehydroepiandrosterone therapy in some women with substituted corticotroph deficiency and psychological complaints may be beneficial [16].

Thyrotrophin Insufficiency In addition to the HPA axis, there is also a clear and established link between the thyroid axis and psychiatric symptomatology [17,18]. Thyroid hormones are used in augmentation strategies to combat treatmentresistant depression. And on the other hand, low thyroid hormonal levels, independent of their cause, and central T3 deficiency increase the risk for psychiatric disorders such as depression, fear, or emotional instability [19 21]. Combination therapy with T4 and T3, instead of T4 only, may have positive effects on psychiatric symptoms in select patients with hypothalamic/ pituitary disease and thyrotrophin deficiency; however, study results are not consistent [22 24].

Gonadotrophin Insufficiency Across all studies, the prevalence of depressive disorders is significantly higher in women than in men [19]. This also applies to female patients with pituitary or hypothalamic lesions. Women with “hypoestrogenism” frequently report symptoms, including depression, fear, sleep disturbances, and cognitive restrictions [25]. A metaanalysis of 26 studies showed that estrogen substitution had moderate to significant effects on depressive mood in women, while added progesterone had no effect. Addition of androgens, such as testosterone, was of positive added value with regard to mood [26]. For men, positive correlations are found for testosterone and mood parameters, such as the quality of life and wellbeing, in various studies [27]. Men with untreated hypogonadism reported significantly higher scores on scales for depression, fear, and fatigue, while testosterone substitution by gel or intramuscular injection improved these parameters [28,29]. Hence, also in men, hormonal testosterone substitution may be advisable in select patients from a psychiatric perspective, since mood, energy, and motivation under optimal substitution are strongly improved [30].

Somatotrophin Insufficiency Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) receptors are present in many brain regions, may pass the blood brain barrier, and both are also produced in the brain (Fig. 14.1) [31,32]. Disturbances within this axis as in the case of acromegaly or growth hormone deficiency are associated with a wide range of psychiatric disorders, and in epidemiological studies, IGF-1 may be associated with the incidence of depressive diseases [33]. Various studies support the claim that the associated GH deficiency starting during childhood and/or adulthood may reduce the quality of life, depressive

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HORMONE EXCESS SYNDROME AND PSYCHIATRIC DISORDERS

415 FIGURE 14.1 GH/ IGF-1 transport system in plasma and brain. PI3K, phosphoinositide-3-kinase; MAPK, mitogen-activated protein kinase; IGFBP, insulin-like growth factor binding protein; BBB, blood brain barrier. Source: Reprinted with permission from Sievers C, Schneider HJ, Stalla GK. Insulin-like growth factor-1 in plasma and brain: regulation in health and disease. Front Biosci 2008;13:85 99.

symptoms, tiredness, and social isolation [19,31,32]. GH substitution may improve psychological wellbeing and mood; however, this effect is dose dependent and more noticeable in adult patients with acquired GH deficiency. Patients with childhood-onset GH deficiency seem to respond less profoundly to GH substitution therapy [34]. Traumatic brain injury or subarachnoid hemorrhage may be a common cause of hypopituitarism and GH deficiency in 10 20% of all affected patients [7]. In these cases, however, it is difficult to clinically distinguish whether brain injury, GH deficiency, or both cause the frequently observed brain dysfunctions, including a negative impact on mental health and patient quality of life. Few clinical intervention, studies in this field have been reported.

HORMONE EXCESS SYNDROME AND PSYCHIATRIC DISORDERS Acromegaly Excessive GH secretion as seen in acromegaly may result in psychiatric malfunctions [10,35 39]. A crosssectional study on 81 acromegaly patients showed elevated risk for major depression and dysthymia compared to two control groups of subjects with and without other chronic somatic disorders (Table 14.2). About 35% of patients with acromegaly have an affective disorder; this is twice as high as in patients with

other chronic illnesses [39]. Biochemical control achieved by either surgery, radiotherapy, radiosurgery, or medical therapy usually improves most somatic comorbidities, but often fails to restore full quality of life and mental health [37 42]. These long-term impairments might be due to irreversible neuronal changes in the presence of chronic GH-IGF-1 excess. In 44 acromegaly patients, larger gray matter and white matter volumes were exhibited, while cerebrospinal fluid was reduced [43]. There is also evidence that acromegaly patients have lower activity in the right dorsolateral prefrontal cortex and left parahippocampal cortex, areas of the brain related strongly to several cognitive functions. In another study, treatment-naı¨ve acromegaly patients were found to have decreased oscillation activity, specifically in electroencephalography (EEG) bands (alpha, beta, and gamma) of the prefrontal and middle temporal lobes of the brain. These areas are involved in working memory and learning and recall processes, in which alterations could lead to possible cognitive deficits [44 46]. It is not yet clear whether acromegaly-specific therapies or psychotrophic drugs influence these psychiatric symptoms.

Cushing’s Syndrome Psychiatric abnormalities are well documented in patients with Cushing’s syndrome [19]. An estimated

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416 TABLE 14.2

14. PSYCHIATRIC DISEASE IN HYPOTHALAMIC PITUITARY DISORDERS

Prevalence of Psychiatric Disorders in Acromegaly Patients and Controls A

Lifetime

DSM IV disorder

Acromegalic patients (n 5 81) (%)

B Controls with chronic somatic disorders (n 5 3281) (%)

C Controls without somatic disorders (n 5 430) (%)

A versus B

A versus C

P value

P value

Any affective

34.6

21.4

11.1

0.006

, 0.001

Any MDE

28.4

19.0

9.7

0.043

, 0.001

MDE

18.5

16.7

1.7

0.659

0.053

MDE GMC

9.9

2.3

0.5

0.001

, 0.001

Dysthymia

16.1

2.3

0.5

, 0.001

, 0.001

Any affective

21.0

13.3

4.9

0.056

, 0.001

Any MDE

14.8

10.7

3.7

0.260

0.005

MDE

7.4

9.5

3.6

0.563

0.345

MDE GMC

7.4

1.3

0.1

, 0.001

0.001

Dysthymia

16.1

5.2

1.3

, 0.001

, 0.001

1 YEAR

MDE, major depression episode; GMC, general medical condition. From Sievers C, Dimopoulou C, Pfister H, et al. Prevalence of mental disorders in acromegaly: a cross-sectional study in 81 acromegalic patients. Clin Endocrinol (Oxf) 2009;71 (5):691 701.

50 70% of patients develop depressive symptoms, while practically all patients with the disorder present with cognitive disturbances associated with the extent of the hypercortisolemia and hippocampal shrinking [47 50]. Psychotic symptoms and suicidal tendencies occur in approximately 10% of these patients. Frequently, normalization of hormone levels is not sufficient to normalize symptomatology in patients with Cushing’s syndrome [51]. Often, these patients require additional psychiatric hospitalization and therapy with psychotropic drugs, such as neuroleptics, antidepressants, and, in some cases, benzodiazepines.

Prolactinoma Even though patients with hyperprolactinemia report emotional problems, it is not clear whether this translates into higher prevalences of psychiatric illnesses [19,52]. This is all the more remarkable since treatment with dopamine agonists in higher dosages may in fact be a risk factor for psychiatric symptoms such as delusions and addictions [53,54]. A study in which patients with prolactinomas and patients with nonfunctioning pituitary adenomas were compared for psychiatric symptoms, men with prolactinoma showed an overall increased presence of impulse control disorders as compared to men with nonfunctioning pituitary adenomas (27.7% vs 3.7%). As yet, no relationship has been found between the presence of impulse control disorders and type of dopamine agonist used, or the treatment dose or duration [55].

RARE DISEASES OF THE HYPOTHALAMUS/PITUITARY GLAND AND PSYCHIATRIC DISORDERS Rare diseases of the hypothalamus/pituitary gland, such as thyroid-stimulating hormone-producing pituitary adenoma or gonadotrophinoma, may be associated with psychiatric symptoms [19]. Patients with craniopharyngiomas exhibit a hypothalamic syndrome with sleep disturbances, vegetative syndromes, severe hunger, disturbed body temperature regulation, increased fatigue, and reduced quality of life [56,57]. However, no large clinical studies on psychopathology and/or psychiatric symptoms in these patients are reported.

DIAGNOSING PSYCHIATRIC DISORDERS IN HYPOTHALAMLIC PITUITARY DISEASES Diagnosing and treating mood disturbances, such as depression, is a major challenge in patients with hypothalamic pituitary diseases. There are no guidelines on this topic. However, guidance can be drawn from more general recommendations such as guidelines by the National Institute of Health and Care Excellence (NICE), UK, on “Depression in adults with a chronic physical health problem” (Depression in adults with chronic physical health problem: recognition and management, NICE guidelines, 2009; Screening for Depression in Older Adults with

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THERAPY OF PSYCHIATRIC DISORDERS IN HYPOTHALAMIC PITUITARY DISEASE

Chronic Physical Health Problems, 2012). The authors particularly stress the need for further research in this field and recommend designs for clinical studies in patients with somatic disease and depression. For now, we generally recommend screening for and assessing psychiatric symptomatology by interviewing the patient and their environment/family. Short screening instruments that can be filled in during waiting times (such as the WHO-5-Well-BeingQuestionnaire, WHO, 1998) may be of assistance. To exclude other causes of depressive symptoms, EEG, MR imaging, cereborospinal puncture, and specialized hormonal analyses may be necessary. In patients with normal pituitary function, the DEX-Test and the DEX/CRH stimulation test are suitable for assessing outcome measures of depression and its treatment [58]. However, this can be challenging in patients with hypothalamic/pituitary disorders with pituitary hormone deficiency. Patients with hypothalamus pituitary disorders frequently present with sleep disorders and pain [59,69], but the causes differ depending on the underlying disorder, e.g., hypothalamic tumors with suprasellar and transfrontal surgery or peripheral obstruction due to acromegaly [60,61]. The diagnosis is clinically based, and sleep disturbances, like inadequate sleep hygiene or an obstructive sleep apnea syndrome, often appear together with insomnia. The specific diagnosis of sleep disturbance is performed in the sleep laboratory. For suspected cognitive disturbances, additional cognitive testing is advised.

THERAPY OF PSYCHIATRIC DISORDERS IN HYPOTHALAMIC PITUITARY DISEASE Since the problem of psychiatric comorbidities in hypothalamic pituitary disease is underdiagnosed and underestimated, treatment is usually suboptimal and no specific recommendations are published. General therapy guidance can be drawn from the NICE guidelines. In a stepped care approach, the importance of lowintensity psychosocial interventions is important, including group-based peer support groups, structured group physical activity programs, and individual guided or computerized self-help based on cognitive behavioral therapy. The important role of structured patient management programs has been emphasized lately by research groups and health insurance companies [62 65,67,68]. Patient management programs are usually composed of different elements, including provision of information, cognitive behavioural techniques such as self management/empowerment, stress

417

management, reframing, group elements, and media. Programs as they are currently designed, carried out, and evaluated for patients with adrenal insufficiency and other pituitary diseases may have great value in additionally improving quality of life and reducing of morbidity and/or mortality in the future. Antidepressants should not be routinely prescribed for subthreshold depression in somatic disease, but should be considered for patients with a past history of moderate to severe depression, in patients who present with symptoms over longer periods (i.e., 2 years), or cases where the depressive symptoms (even when subthreshold) may complicate treatment of the somatic disease. No studies have specifically looked at the effectiveness of antidepressants and/or psychotherapy in patients with hypothalamic/pituitary gland disorders. Therefore, the entire selection of antidepressants is theoretically available for these patients. Although they commonly act on different monoamine systems, the agents typically differ in side effect profiles [19]. Normalization of the HPA system appears to be the common effect, and is possibly the mechanism of action of all antidepressants. A significant initial inhibition of HPA hormones is documented for trimipramine and mirtazapine [66]. For chronic major depression, a combination of antidepressant administration and psychotherapy techniques is preferred over sole application of one of these forms of therapy. Suitable adjunctive therapy in treatment-resistant patients includes administration of lithium and thyroid hormones as augmentation therapy. Complementary therapies, such as electroconvulsive therapy, may be considered in treatment-resistant depression. For sleep disturbances, nonpharmacological methods and/or herbal substances can first be used (sleep hygiene, stimulus control, sleep restriction, relaxation therapies) [19]. Guidance for subsequent treatment with benzodiazepine-receptor agonists is to use (1) a low dosage, (2) the shortest possible prescription period, and (3) caution when stopping the medication. Antidepressants with sedative properties (amitriptyline, doxepin, mirtazapine, trazodone, trimipramine) lend themselves for the treatment of initiating and maintaining sleep, independent of the presence of depression. Nasal continuous positive-airway pressure is typically used in the treatment of sleep apnea syndromes that occur in acromegaly.

Acknowledgments The authors thank Natascha Williams for an English language review of the chapter.

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[39] Sievers C, Dimopoulou C, Pfister H, et al. Prevalence of mental disorders in acromegaly: a cross-sectional study in 81 acromegalic patients. Clin Endocrinol (Oxf) 2009;71(5):691 701. [40] Biermasz NR, van Thiel SW, Pereira AM, et al. Decreased quality of life in patients with acromegaly despite long-term cure of growth hormone excess. J Clin Endocrinol Metab 2004;89(11): 5369 76. [41] Bonapart IE, van Domburg R, ten Have SM, et al. The ‘bioassay’ quality of life might be a better marker of disease activity in acromegalic patients than serum total IGF-I concentrations. Eur J Endocrinol 2005;152(2):217 24. [42] Geraedts VJ, Dimopoulou C, Auer M, Schopohl J, Stalla GK, Sievers C. Health outcomes in acromegaly: depression and anxiety are promising targets for improving reduced quality of life. Front Endocrinol (Lausanne) 2015;5:229. [43] Sievers C, Samann PG, Dose T, et al. Macroscopic brain architecture changes and white matter pathology in acromegaly: a clinicoradiological study. Pituitary 2009;12(3):177 85. [44] Martin-Rodriguez JF, Madrazo-Atutxa A, Venegas-Moreno E, et al. Neurocognitive function in acromegaly after surgical resection of GH-secreting adenoma versus naive acromegaly. PLoS One 2013;8(4):e60041. [45] Psaras T, Milian M, Hattermann V, Will BE, Tatagiba M, Honegger J. Predictive factors for neurocognitive function and Quality of Life after surgical treatment for Cushing’s disease and acromegaly. J Endocrinol Invest 2011;34(7):e168 77. [46] Sievers C, Samann PG, Pfister H, et al. Cognitive function in acromegaly: description and brain volumetric correlates. Pituitary 2012;15(3):350 7. [47] Dorn LD, Burgess ES, Friedman TC, Dubbert B, Gold PW, Chrousos GP. The longitudinal course of psychopathology in Cushing’s syndrome after correction of hypercortisolism. J Clin Endocrinol Metab 1997;82(3):912 19. [48] Forget H, Lacroix A, Somma M, Cohen H. Cognitive decline in patients with Cushing’s syndrome. J Int Neuropsychol Soc 2000;6(1):20 9. [49] Tiemensma J, Biermasz NR, Middelkoop HA, van der Mast RC, Romijn JA, Pereira AM. Increased prevalence of psychopathology and maladaptive personality traits after long-term cure of Cushing’s disease. J Clin Endocrinol Metab 2010;95(10):E129 41. [50] Dimopoulou C, Ising M, Pfister H, Schopohl J, Stalla GK, Sievers C. Increased prevalence of anxiety-associated personality traits in patients with Cushing’s disease: a cross-sectional study. Neuroendocrinology 2013;97(2):139 45. [51] Osswald A, Plomer E, Dimopoulou C, et al. Favorable longterm outcomes of bilateral adrenalectomy in Cushing’s disease. Eur J Endocrinol 2014;171(2):209 15. [52] Sievers C. Neurological and psychiatric symptoms in patients with hypothalamo pituitary diseases. Aust J Clin Endocrinol Metab 2012;5(2):12 14. [53] Athanasoulia AP, Ising M, Pfister H, Mantzoros CS, Stalla GK, Sievers C. Distinct dopaminergic personality patterns in patients with prolactinomas: a comparison with nonfunctioning pituitary adenoma patients and age- and gender-matched controls. Neuroendocrinology 2012;96(3):204 11. [54] Athanasoulia AP, Sievers C, Ising M, et al. Polymorphisms of the drug transporter gene ABCB1 predict side effects of treatment with cabergoline in patients with PRL adenomas. Eur J Endocrinol 2012;167(3):327 35. [55] Bancos I, Nannenga MR, Bostwick JM, Silber MH, Erickson D, Nippoldt TB. Impulse control disorders in patients with

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dopamine agonist-treated prolactinomas and nonfunctioning pituitary adenomas: a case-control study. Clin Endocrinol (Oxf) 2014;80(6):863 8. Roemmler-Zehrer J, Geigenberger V, Stormann S, et al. Specific behaviour, mood and personality traits may contribute to obesity in patients with craniopharyngioma. Clin Endocrinol (Oxf) 2015;82(1):106 14. Roemmler-Zehrer J, Geigenberger V, Stormann S, et al. Food intake regulating hormones in adult craniopharyngioma patients. Eur J Endocrinol 2014;170(4):627 35. Ising M, Horstmann S, Kloiber S, et al. Combined dexamethasone/corticotropin releasing hormone test predicts treatment response in major depression - a potential biomarker? Biol Psychiatry 2007;62(1):47 54. Dimopoulou C, Athanasoulia AP, Hanisch E, Held S, Sprenger T, Toelle TR, et al. Clinical characteristics of pain in patients with pituitary adenomas. Eur J Endocrinol 2014;171 (5):581 91. Schneider HJ, Oertel H, Murck H, Pollmacher T, Stalla GK, Steiger A. Night sleep EEG and daytime sleep propensity in adult hypopituitary patients with growth hormone deficiency before and after six months of growth hormone replacement. Psychoneuroendocrinology 2005;30(1):29 37. Colao A, Ferone D, Marzullo P, Lombardi G. Systemic complications of acromegaly: epidemiology, pathogenesis, and management. Endocr Rev 2004;25(1):102 52. Bancos I, Hahner S, Tomlinson J, Arlt W. Diagnosis and management of adrenal insufficiency. Lancet 2015;3(3):216 26. Quinkler M, Hahner S. What is the best long-term management strategy for patients with primary adrenal insufficiency? Clin Endocrinol (Oxf) 2012;76:21 5. Repping-Wuts HJ, Stikkelbroeck NM, Noordzij A, Kerstens M, Hermus AR. A glucocorticoid education group meeting: an effective strategy for improving self-management to prevent adrenal crisis. Eur J Endocrinol 2013;169:17 22. Quinkler M, Beuschlein F, Hahner S, Meyer G, Scho¨fl C, Stalla GK. Adrenal cortical insufficiency—a life threatening illness with multiple etiologies. Dtsch Arztebl Int 2013;110 (51 52):882 8. Frieboes RM, Sonntag A, Yassouridis A, Eap CB, Baumann P, Steiger A. Clinical outcome after trimipramine in patients with delusional depression - a pilot study. Pharmacopsychiatry 2003;36(1):12 17. Tiemensma J, Andela CD, Pereira AM, Romijn JA, Biermasz NR, Kaptein AA. Patients with adrenal insufficiency hate their medication: concerns and stronger beliefs about the necessity of hydrocortisone intake are associated with more negative illness perceptions. J Clin Endocrinol Metab 2014;99 (10):3668 76 http://dx.doi.org/10.1210/jc.2014-1527. Epub 2014 Sep 16. Andela CD, Staufenbiel SM, Joustra SD, Pereira AM, van Rossum EF, Biermasz NR. Quality of life in patients with adrenal insufficiency correlates stronger with hydrocortisone dosage, than with long-term systemic cortisol levels. Psychoneuroendocrinology 2016;72:80 6. Leistner SM, Klotsche J, Dimopoulou C, Athanasoulia AP, Roemmler-Zehrer J, Pieper L, et al. Reduced sleep quality and depression associate with decreased quality of life in patients with pituitary adenomas. Eur J Endocrinol 2015;172 (6):733 43.

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15 Acromegaly Shlomo Melmed

INTRODUCTION Acromegaly, a spectacular clinical syndrome of disordered somatic growth and proportion, has intrigued physicians since earliest recorded history. It was, however, only in 1886 that Pierre Marie published the first clinical description of the disorder based on observing two of his patients, and his recognition of five other cases previously described by others [1]. He described features distinguishing the disorder from myxedema and osteodystrophy and proposed the name “acromegaly.” Marie did not recognize the relation of a pituitary tumor to this syndrome until 5 years later, when an adenohypophyseal tumor was observed in a patient with acromegaly. In 1900, Benda recognized that pituitary adenomas in patients with acromegaly comprised mainly adenohypophyseal eosinophilic cells, which he proposed to be hyperfunctioning [2]. Subsequent careful clinicopathologic studies by Cushing, Davidoff, and Bailey were supplemented by demonstrating clinical remission of soft tissue signs of acromegaly after surgical resection of eosinophilic pituitary adenomas [3 5]. The experiments of Evans and Long demonstrating features of gigantism in rats injected with anterior pituitary extracts confirmed the association of a pituitary factor with somatic growth [6]. Establishment of the unequivocal link between hyperfunctioning adenoma and acromegaly was the earliest example of a pituitary disorder to be clinically and pathologically recognized and appropriately managed.

Epidemiology Acromegaly is caused by unrestrained secretion of growth hormone (GH) and Insulin-like growth factor-1 (IGF-1) (Fig. 15.1). Several studies have undertaken a comprehensive ascertainment of acromegaly in the community. In a retrospective survey, the annual

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00015-5

incidence of pituitary tumors in the United States is about 45 cases per million population per year [8]. Assuming that acromegaly accounts for about 25% of these cases, the incidence of the disorder is calculated to be 10 cases per million population. Similarly, in Belgium, the annual acromegaly incidence rate is B10 cases per million population, with a prevalence of more than 75 cases per million [9]. In Iceland, the nationwide prevalence of acromegaly is 134 per million population and the annual incidence is 7.7 per million per year [10].

Animal Models of Hypersomatotrophism Transgenic mouse strains bearing either growth hormone-releasing hormone (GHRH), GH, or IGF-1 genes have enabled elucidation of the respective roles for these three hormones in the development of hypersomatotrophism [11 13]. Expression of GHRH in mice results in increased somatic growth [13], mammosomatotroph hyperplasia [14], and GH-cell adenomas [15]. In mice bearing a GH trans gene, heterologous GH mRNA is detectable from day 13 of gestation, and marked growth acceleration is evident at about 3 weeks of age [11]. All body organs, except the brain, exhibit increased growth but the liver and spleen undergo disproportionate allometric growth [11]. Therefore, although both GH and IGF-1 levels are elevated in these animals, overall body growth and individual organ growth respond in different relative proportions. These observations imply that the pattern of hypersomatotrophic body and organ growth is dependent on at least two variables: external growth factors (e.g., GH and IGF-1) that stimulate cell division and organ and body weight, as well as organ-specific intrinsic growth potentials that respond to the hormonal environment in achieving final size outcome. In contrast, transgenic mice expressing the human IGF-1

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hypophysectomized rats, does in fact mimic most of the somatic effects of GH by restoring growth [16]. These observations suggest that both GH and IGF-1 independently contribute to the pathologic findings of hypersomatotrophism [17]. Nevertheless, IGF-1 is required for GH to achieve a maximally robust bone and tissue response [18].

PATHOGENESIS Acromegaly may be caused by pituitary tumors or by extrapituitary disorders [19,20] (Figs. 15.1 and 15.2). Regardless of the etiology of the disorder, the disease is characterized by elevated levels of both GH and IGF-1, with resultant signs and symptoms of hypersomatotrophism [21].

Pituitary Acromegaly

FIGURE 15.1 Normal and disrupted GHRH GH IGF-1 axis. Pituitary somatotroph cell gene expression and GH synthesis are determined by the POU1F1 transcription factor. Net GH secretion is determined by integration of hypothalamic, nutritional, hormonal, and intrapituitary signals. GH synthesis and secretion are induced by hypothalamic GHRH and gut-derived ghrelin. GHRH may also act as a coagonist for the ghrelin receptor. Hypothalamic SRIF suppresses GH secretion mainly by high-affinity binding to somatotroph SSTR2 and SSTR5 receptor subtypes. SRLs signal through SSTR2 and SSTR5 to control GH hypersecretion and shrink the tumor mass. GH secretion patterns in a normal subject and in acromegaly are depicted in the insets. Source: From Melmed S. New therapeutic agents for acromegaly. Nat Rev Endocrinol 2016;12(2):90 8 [7].

gene exhibit selective organomegaly without a profound increase in longitudinal skeletal growth [12]. As these mice had suppressed endogenous GH levels, GH and IGF-1 therefore appear to act both independently and synergistically in inducing clinical hypersomatotrophism. Both GH and GHRH transgenic mice also display renal glomerulosclerosis, which is not observed in animals bearing the IGF-1 gene, suggesting that GH acts directly to cause renal mesangial changes. Interestingly, IGF-1, when administered to

Over 95% of patients with acromegaly harbor a specific pituitary adenoma type responsible for unrestrained GH secretion, which are classified according to hormone gene expression, ultrastructural features, and cytogenesis [22,23]. These tumors, accounting for B60% of GH-secreting adenomas, contain either densely or sparsely staining cytoplasmic GH granules, and are either slow (densely granulated) or rapidly growing (sparsely granulated) [24]. Mixed GH-cell and prolactin (PRL)-cell adenomas are composed of two distinct cell types, somatotrophs expressing GH and lactotrophs expressing PRL [25,26]. Bimorphous tumors cause acromegaly with moderately elevated serum PRL levels. Acidophil stem cell adenomas are monomorphous tumors arising from the common GH and PRL stem cell and expressing both hormones [27], and often contain giant mitochondria and misplaced exocytosis of GH granules. They are often rapidly growing and invasive, and hyperprolactinemia rather than acromegaly may be the predominant presenting feature. In contrast, monomorphous mammosomatotroph cell adenomas consist of a single mature cell expressing both GH and PRL. Serum PRL levels are usually normal or moderately elevated. Plurihormonal tumors, which are either monomorphous or plurimorphous, may express GH with any combination of PRL, thyroid-stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), or α-subunit [28,29]. Often, little correlation exists between specific hormone staining of the tumor and peripheral hormone levels. These patients may present with clinical features of acromegaly as well as the effects of the respective elevation of other pituitary trophic hormones. GH-cell carcinomas with well-documented distant metastases

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FIGURE 15.2 Causes of acromegaly. Acromegaly is caused by excessive production of GH or GHRH. Rarely, the disease is associated with familial syndromes, including MEN-1, McCune Albright syndrome, familial acromegaly, and Carney’s complex. GHS, growth hormone secretagogues; PRL, prolactin. Source: Adapted from Melmed S. Medical progress: acromegaly. N Engl J Med 2006;355(24):2558 73.

are exceedingly rare [30]. Despite exhibiting hypercellularity, necrosis, nuclear pleomorphism, and mitotic figures, they very rarely metastasize. Although locally invasive somatotrophinomas are occasionally aggressive and rapidly growing, they should not be classified as malignant unless definitive proof of distant metastases is present [31]. GH-cell hyperplasia is difficult to distinguish histologically from a GH-cell adenoma. Hyperplasias usually consist of more than one cell type and silver staining reveals the presence of a well-preserved reticulin network without a surrounding pseudocapsule. The rigid morphologic diagnosis of GH-cell hyperplasia has usually been associated with extrapituitary stimulation by GHRH from an extrapituitary tumor causing acromegaly [32]. Silent somatotroph tumors [28], while staining positively for the presence of GH, are clinically nonfunctional. Features of acromegaly are absent, although

GH and/or PRL levels may in fact be elevated in some of these patients [33]. Cluster analysis of 242 acromegaly patients yielded three subtypes. Type 1, the most commonly encountered, comprised older patients with small, densely granulated microadenomas and abundant somatostatin receptor (SSTR) 2 and p21 expression. Type 2 tumors, accounting for B20%, are densely or sparsely granulated. Type 3 tumors, B30% of the total, occur in younger patients and are sparsely granulated, larger and invasive microadenomas with low SSTR2 and p21 expression and more adverse therapeutic outcomes [34]. T2-weighted magnetic resonance imaging (MRI) may also yield noninvasive assessment of tumor responsiveness [35]. Thus, distinct patterns of tumor aggressiveness may yield a personalized, more focused approach to determine therapeutic outcome as well as the cost benefit of therapeutic options [36 39].

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Pathogenesis of Somatotroph Cell Adenomas Both the hypothalamus and the pituitary contribute to development of acromegaly. Disordered secretion of GHRH or somatotrophin-release inhibitory factor (SRIF), with or without an intrinsic pituitary cellular defect, may result in adenoma formation [31,40]. Disordered GHRH secretion or action. GHRH induces GH gene expression and also induces somatotroph DNA synthesis, cell replication, and c-fos expression [41]. These growth-promoting actions of GHRH are prevented by SRIF. Somatotroph GHRH action is mediated by activation of adenylate cyclase and increased cyclic adenosine monophosphate (cAMP) levels, which stimulate both GH synthesis and adenoma formation. Somatotroph hyperplasia, increased GH secretion, and gigantism were observed in mice expressing a pituitary-directed cholera toxin trans gene that induced intracellular cAMP [42]. In contrast, mice with inactivated cAMP responses developed dwarfism and low GH secretion [43]. Excess GHRH production by functional hypothalamic tumors or by abdominal or chest neuroendocrine tumors causes somatotroph hyperplasia and occasionally adenoma with resultant unrestrained GH secretion [44], implying hypothalamic hormone involvement in the pathogenesis of GH-cell adenomas and acromegaly. However, histology of most GH-cell adenoma tissue specimens does not reveal hyperplastic somatotroph tissue surrounding the adenoma [45], implying no exogenous hypothalamic overstimulation of the pituitary. GHRH production by extrapituitary tumors causing acromegaly is usually associated with somatotroph cell hyperplasia and elevated GH levels, as well as paradoxical responses of GH to glucose, thyrotrophin-releasing hormone (TRH), and dopamine [46]. These biochemical perturbations revert to normal when the ectopic source of GHRH is removed, suggesting that exposure to high levels of GHRH alters the somatotroph response to other factors regulating GH secretion. Expression of adenoma GHRH receptors, and the failure of GH downregulation during prolonged GHRH stimulation, also points to a possible role for GHRH in maintaining persistent GH hypersecretion. Intrapituitary and adenoma-derived GHRH correlates with tumor size and activity, implying a paracrine role for GHRH in mediating adenoma pathogenesis [47]. GHRH also modestly stimulates PRL secretion in most acromegaly patients [46]. These observations, coupled with the fact that up to 40% of these patients also have hyperprolactinemia, imply a role for GHRH in the pathogenesis of the disorder. Although SRIF secretion may theoretically be attenuated, thus giving rise to unrestrained GH secretion, TSH responses to TRH in acromegaly are either

normal or in fact blunted, suggesting intact SRIF activity. Alternatively, high GH levels in these patients may abnormally autoregulate the somatotroph. Surgical responses. Postoperative GH testing often remains disordered after initial pituitary tumor resection, suggesting that the hypothalamus is primarily responsible for altered GH secretion and tumor development. However, surgical resection of well-defined GH-secreting tumors (,5 mm) results in a definitive cure of excess hormone secretion in most patients [48,49]. Low postoperative tumor recurrence rates, together with restoration of most dynamic GH responses after surgery, is strongly suggestive of intact hypothalamic function in these patients. GH secretory patterns. Although basal GH levels are usually high in acromegaly, episodic pulsatile patterns of GH release may also be apparent, and nocturnal GH surges are also preserved [50]. Patients receiving long-acting somatostatin receptor ligands (SRLs), also retain GH pulsatility, suggesting persistent GHRH secretion [51]. Paradoxical GH responses to glucose, dopamine, and TRH, and loss of pituitary desensitization to hypothalamic GHRH, however, point to an intrinsic somatotroph abnormality. Disordered somatotroph cell function. In vitro responses of somatotroph tumor cell cultures exposed to physiologic levels of GHRH, SRIF, and IGF-1 are similar to those observed in the limited number of similar studies in normal cultured human pituitary tissue [52]. GH gene expression is stimulated in vitro by GHRH and inhibited by IGF-1, as evidenced by changes in adenoma cells’ GH mRNA content [53]. Adenoma tissue also expresses receptors for GHRH, ghrelin [54], and SRIF [55], but activating mutations of either the GHRH or SRIF receptor have not been reported. Ghrelin also induces proliferation of rat somatotroph tumor cells [56]. These apparently physiologic responses imply intact control of GH gene expression in tumor cells and favor disordered hypothalamic etiology for clinically abnormal GH hypersecretion. A preexisting somatotroph cell mutation may be a prerequisite for the abnormal growth response to disordered GHRH secretion or action (Table 15.1). The monoclonal origin of somatotroph adenomas as determined by X-chromosome inactivation analysis of somatotroph tumor DNA [57] suggests that a somatic somatotroph mutation leads to clonal expansion and tumor formation. Autonomous GH secretion by the transformed somatotroph likely ensues as a result of several pathogenetic mechanisms, including intrinsic cell-cycle dysfunction, altered hormonal and paracrine factors regulating both GH gene expression and secretion, and somatotroph cell growth [20]. An altered

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TABLE 15.1

Genes That Contribute to the Molecular Pathogenesis of GH-Secreting Adenomas Mode of activation/ inactivation

Clinical context

Specificity for GH-secreting pituitary adenoma

Gene

Function

GNAS

Oncogene

Activating, imprinting

Nonfamilial, syndromic, or sporadic

Relatively specific

CREB

Transcription factor

Constitutive phosphorylation

Sporadic

Relatively specific

AIP

Tumor suppressor

Inactivating

Familial, syndromic

Relatively specific

MEN1

Tumor suppressor

Inactivating

Familial, syndromic

Not specific

PRKAR1A

Tumor suppressor

Inactivating

Familial, syndromic

Not specific

H-RAS

Oncogene

Activating

Invasive or malignant

Not specific

CCNB2

Cyclin

Induced by HMGA

Sporadic

Not specific

CCND1

Oncogene

Overexpression

Sporadic

Not specific

HMGA2

Oncogene

Overexpression

Sporadic

Not specific

FGFR4

Oncogene

Alternative transcription

Sporadic

Not specific

PTTG

Securin

Overexpression

Sporadic

Not specific

Rb

Tumor suppressor

Epigenetic silencing

Sporadic

Not specific

CDKN1B

CDK inhibitor

Nonsense mutation

Sporadic

Not specific

GADD45G

Proliferation inhibitor

Epigenetic silencing

Sporadic

Not specific

MEG3

Proliferation inhibitor

Epigenetic silencing

Sporadic

Not specific

Adapted from Melmed S. Acromegaly pathogenesis and treatment. J Clin Invest 2009;119(11):3189 202.

Gs(α) protein identified in a subset of GH-secreting pituitary adenomas is characterized by high levels of intracellular cAMP and GH hypersecretion [58]. Point mutations in two critical sites, Arg201, the site for ADPribosylation, and Gly227, the GTP-binding domain of Gs(α) proteins, prevent GTPase activity and result in constitutive adenylyl cyclase activation. The tumor contains a dominant mutant Gs(α), termed gsp, which recapitulates effects of GHRH and results in elevated cAMP levels [58]. These activating gsp mutations are present in about 30% of GH-secreting tumors with enhanced tumor adenylyl cyclase activity, and result in lower GH levels than seen in nonmutant-bearing tumors. Although the somatotroph is clearly transformed, the sequence of events leading to clonal expansion is multifactorial. A putative activated oncogene(s) may be required for initiating tumorigenesis, while promotion of tumor growth may require permissive GHRH and other growth factor (e.g., basic fibroblast growth factor (bFGF)) stimulation. Dysregulated pituitary signaling or expression of neurotransmitters and growth

factors, including FGF, dopamine, estrogen, and nerve growth factor, have been identified in pituitary adenomas, but no uniform mutation has been identified [20,59]. Overexpressed oncogenes, inactivated tumor suppressor genes, or epigenetic changes associated with GH-secreting adenomas are depicted in Table 15.1. Pituitary tumor transforming gene (PTTG) [60,61] is overexpressed in GH-secreting and other functional pituitary tumors, and its abundance correlates with tumor size and invasiveness [60]. PTTG is the index securin protein, regulating sister chromatid separation during the cell cycle [61], and its overexpression leads to aneuploidy. In contrast, expression of a proapoptotic and growth arrest factor, GADD45γ, is lost in GH-secreting adenomas [62]. Thus, a spectrum of genetic events appears to culminate in somatotroph transformation and adenoma pathogenesis. Nevertheless, GH-secreting tumors rarely transform to true malignancy, and their growth restraint is likely reflective of lineage-specific pituitary cell growth arrest and senescence mediated by p21, a cyclin-dependent kinase (CDK) inhibitor [63].

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Extrapituitary Acromegaly The source of excessive GH secretion in acromegaly may not necessarily be pituitary in origin [20]. Patients with extrapituitary acromegaly include those with excess ectopic GHRH or GH secretion, and very rarely a putative growth factor disorder termed acromegaloidism (Fig. 15.2).

biosynthesis, and polypeptide immunoreactivity. Patients with nonconventional biochemical, imaging, or clinical features of pituitary acromegaly may inadvertently be diagnosed as harboring a nonpituitary source of excess GHRH or GH secretion, and be inappropriately treated. A definitive diagnosis of the etiology of hypersomatotrophism should therefore be made prior to instituting acromegaly therapy.

Criteria for Diagnosis of Ectopic Acromegaly

GHRH Hypersecretion

Because ectopic acromegaly requires a different management approach than that recommended for classic pituitary GH hypersecretion, stringent clinical and biochemical criteria should be fulfilled to confirm this diagnosis [64]. These include the demonstration of elevated circulating GHRH or GH levels in the absence of a primary lesion of the pituitary gland, as well as a significant arteriovenous hormone gradient across the ectopic tumor source (Fig. 15.3). Excision or functional ablation of the ectopic hormone-producing tumor source should ideally result in biochemical and clinical cure of acromegaly. Tumor tissue should also be shown to express the GHRH or GH gene product by demonstrating specific mRNA expression, hormone

Hypothalamic. Hypothalamic GHRH is secreted into the portal system, impinges upon the somatotroph cells, binds to specific surface receptors, and elicits intracellular signals that modulate pituitary GH synthesis and/or secretion [66]. Hypothalamic tumors, including hamartomas, choristomas, gliomas, and gangliocytomas may produce excessive GHRH with subsequent GH hypersecretion and resultant acromegaly [45]. These patients may have somatotroph hyperplasia, or very rarely a pituitary GH-cell adenoma, supporting the notion that excess hypothalamic GHRH leads to pituitary hyperplasia and subsequent adenoma formation. As patients reported with hypothalamic acromegaly have all usually undergone

FIGURE 15.3 Somatostatin receptor scan for diagnosis of ectopic acromegaly. Radiolabeled octreoscan reveals metastatic neck GH-secreting carcinoma that expresses SSTR2, SSTR3, and SSTR5 receptors. Mass resection normalizes GH levels within 3 h. Source: From Greenman Y, Woolf P, Coniglio J, et al. Remission of acromegaly caused by pituitary carcinoma after surgical excision of growth hormone-secreting metastasis detected by 111-indium pentetreotide scan. J Clin Endocrinol Metab 1996;81(4):1628 33 [65].

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resection of the adenomatous or hyperplastic pituitary, definitive proof of a primary hypothalamic tumor causing acromegaly may be elusive. Pituitary mammosomatotroph hyperplasia with no evidence for pituitary adenoma or an extrapituitary tumor source of GHRH has been described in a young child with gigantism [67]. Peripheral. GHRH is synthesized and expressed in multiple extrapituitary tissues [68,69]. Excessive peripheral production of GHRH by a tumor source would therefore be expected to cause somatotroph cell hyperstimulation and increased GH secretion. The structure of hypothalamic GHRH was in fact elucidated from material extracted from pancreatic GHRH-secreting tumors in two patients with acromegaly [32,70]. Immunoreactive GHRH is present in several tumors, including carcinoid tumors, pancreatic cell tumors, small-cell lung cancers, adrenal adenomas, and pheochromocytomas, which have been reported to secrete GHRH [32,71 77]. Acromegaly in these patients, however, is uncommon. In a retrospective survey of 177 patients with acromegaly, only a single patient was identified with elevated plasma GHRH levels [78]. The association of acromegaly with carcinoid tumors had been recognized prior to the characterization of hypothalamic GHRH [79,80]. Carcinoid tumors comprise most of the tumors associated with ectopic GHRH secretion, the majority bronchial in origin [71 74,81,82]. Although most patients with carcinoid do not exhibit clinical features of acromegaly, many of these tumors express immunoreactive GHRH and manifest abnormal GH-secretory dynamics [75]. The observed high incidence of GHRH expression and low incidence of acromegaly in these patients may be due to disordered GHRH tissue processing, or to impaired GHRH bioactivity. Most carcinoid tumors are slow-growing, with insidious development of acromegaly. These patients present with features of classical acromegaly, accompanied by elevated circulating GH and IGF-1 levels. Patients also often experience systemic effects, obvious metastatic disease, or other humoral effects of the carcinoid syndrome [73]. Following surgical removal, GH levels fall and soft tissue signs of acromegaly regress. The pituitary often shows evidence of somatotroph hyperplasia with preserved reticulin network. A true GH-cell adenoma with distorted reticulin network may also occasionally be present. Treatment. Surgical resection of the tumor secreting ectopic GHRH should reverse GH hypersecretion, and pituitary surgery should not be required in these patients. Nonresectable, disseminated, or recurrent carcinoid syndrome with ectopic GHRH secretion can also be managed medically with long-acting SRLs [83]. Administration of the SRL lowers circulating GH

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and IGF-1 levels, and also suppresses ectopic tumor elaboration of GHRH [71 73,84 86]. The drug suppresses both pituitary GH as well as the peripheral tumor source of GHRH, thus attenuating the deleterious effects of chronic hypersomatotrophism [87]. GH Hypersecretion Ectopic pituitary adenomas. Embryonal pituitary development involves dorsal migration of fetal adenohypophyseal cells. Functional pituitary adenomas secreting GH may arise from ectopic pituitary remnants in the sphenoid sinus and wing, petrous temporal bone, and nasopharyngeal cavity [88,89]. Residual tumor cells may also be dislodged after neurosurgical resection of invasive pituitary adenomas and give rise to subsequent recurrent ectopic adenomas. Very rarely, pituitary carcinoma may spread to the meninges, cerebrospinal fluid, or cervical lymph nodes, resulting in functional GHsecreting metastases that may also be diagnosed by radiolabeled octreotide imaging (Fig. 15.3) [65]. Peripheral GH-secreting tumors. Immunoreactive GH has been identified in normal human tissues, including liver, kidney, lung, colon, stomach, and brain [90]. Extracts of lung adenocarcinoma, breast cancer, and ovarian tissues also contain immunoreactive GH without clinical evidence of acromegaly [91]. A GHsecreting intramesenteric pancreatic islet cell tumor was associated with acromegaly [64], as was a nonHodgkin lymphoma [92]. Ectopic GH secretion by the pancreatic tumor was unambiguously confirmed by high arteriovenous tumor gradient of GH, normalization of GH and IGF-1 after tumor resection, positive GH immunoperoxidase staining, demonstration of in vitro GH synthesis and release, and expression of GH mRNA. Postoperative GH suppression after glucose and stimulation by GHRH were intact. Based on the features of this unique case, the very rare patients with ectopic GH secretion would be expected to exhibit a normal-sized or small pituitary gland and normal GHRH levels. Acromegaloidism Patients who manifest clinical features of acromegaly but do not harbor a demonstrable pituitary or extrapituitary tumor have been termed “acromegaloid.” An exhaustive evaluation for acromegaly should be undertaken prior to patients being diagnosed as acromegaloid. These patients exhibit soft tissue and skin changes usually associated with acromegaly and some may even have bony features of the disorder, and occasionally hyperglycemia. GH and IGF-1 levels are apparently normal and respond appropriately to dynamic pituitary testing. Pachydermoperiostosis should be considered in the differential diagnosis. Unique growth factor activity, partially characterized

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by bioassay, patients [93], IGF-1 binding two patients nigricans [94].

was and were with

described in the sera of these insulin resistance and defective demonstrated in cells derived from acromegaloidism and acanthosis

GENETIC SYNDROMES Several genetic syndromes associated with acromegaly or gigantism have been described (Table 15.2) [95] (see also chapter: Genetics of Pituitary Tumor Syndromes).

McCune Albright Syndrome Polyostotic fibrous dysplasia, cutaneous cafe-au-lait pigmentation, sexual precocity, hyperthyroidism, hypercortisolism, hyperprolactinemia, and acromegaly comprise a rare hypersecretory endocrinopathy, McCune Albright syndrome (OMIM 1748000) [96]. Postzygotic GNAS mutations lead to a mosaic tissuespecificity that results in GH (and rarely TSH) hypersecretion due mainly to diffuse somatotroph cell hyperplasia [97]. Few of these patients exhibit definitive evidence for a pituitary adenoma, although most dynamic GH responses are indistinguishable from patients harboring GH-secreting somatotroph adenomas. In four of eight patients, Gs(α) mutations were detected in both endocrine and nonendocrine organs [98]. Surgical management of GH hypersecretion in these patients is challenging due to skull base fibrous dysplasia. Medical treatment with SRLs controls about

30% of cases, and pegvisomant appears to be effective in most patients [96].

Multiple Endocrine Neoplasia GH-cell pituitary adenoma causing acromegaly is a well-documented component of the autosomal dominant multiple endocrine neoplasia 1 (MEN-1) syndrome, which also includes parathyroid and pancreatic tumors (OMIM 131100). The disorder is associated with germ cell inactivation of the MENIN tumor suppressor gene, which maps to chromosome 11q13 [99], leading to development of endocrine tumors. About 40% of these patients harbor pituitary tumors, including prolactinomas or GH-secreting adenomas, and rarely ACTH- or TSH-secreting adenomas and other neuroendocrine tumors [100]. Rarely, functional pancreatic tumors in MEN-1 also express excess circulating GHRH [101]. The disorder is associated with decreased life expectancy, with larger, more aggressive multisite tumors; genetic screening and subsequent counseling is recommended. A rare germline mutation in the CDKN1B inhibitor encoding p27 has been described, with features of a recessive MEN-4 phenotype [102], which includes acromegaly and parathyroid adenomas.

Carney Complex This syndrome, which comprises spotty skin pigmentation, mucosal and cardiac myxomas, acromegaly, and adrenal hyperplasia (OMIM 160908), is associated with

TABLE 15.2

Inherited Pituitary Tumor Syndromes

Syndrome

Gene (locus)

Most frequent mutation(S)

Pituitary features

Other key features

MEN-1

MEN1 (11q13)

c.249-252delGTCT, an exon 2 predicted frameshift, in 4.5%

Pituitary adenoma in 30 40% (PRL 60%, NFA 15% GH, 10%, ACTH 5%, TSH rare)

Primary hyperparathyroidism, pancreatic tumors, foregut carcinoid tumors, adrenocrotical tumors (usually nonfunctional), rarely pheochromocytomas, skin lesions (facial angiomas, collagenomas, and lipomas)

MEN-1-like (MEN-4)

CDKN1B (12p13)

Only two reported casesa

Pituitary adenoma

Primary hyperparathyroidism, and single cases reported of renal angiomyolipoma, neuroendocrine cervical carcinoma

Carney complex

PRKAR1A (17q23-24)

c.491-492delTG in exon Pituitary hyperplasia in most 5 patients, adenoma in B10% (GH and PRL)

Familial, isolated pituitary adenomas

AIPb (11q13.3)

Gln14X nonsense mutationc

Pituitary adenoma (majority GH, PRL, or mixed GH and PRL)

a

Atrial myxomas, lentigines, Schwann-cell tumors, adrenal hyperplasia NR

Only two reported cases to date: one GH-secreting and one ACTH-secreting adenoma. AIP mutations reported in 20% of individuals with familial isolated pituitary adenoma and 50% of those with isolated familial somatotrophinomas. This is the most commonly identified mutation, but is likely to be overrepresented secondary to a Finnish founder effect. Adapted from Elston MS, McDonald KL, Clifton-Bligh RJ, Robinson BG. Familial pituitary tumor syndromes. Nat Rev Endocrinol 2009;5(8):453 61. b c

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defective somatotroph protein kinase A levels [103]. These patients usually harbor an inactivating PRKARIA mutation coding for type 1 α-subunit of protein kinase A.

Familial Acromegaly In rarely encountered families with familial isolated pituitary adenomas, gigantism or acromegaly may affect young patients with mostly macroadenomas [95,104] (Table 15.2). In B20% of such families predisposed to isolated cases of acromegaly and/or gigantism, a mutation in chromosome 11q13 has been mapped to the aryl hydrocarbon receptor-interacting protein (AIP) gene [105,106]. AIP, a chaperone protein, acts to stabilize the dioxin (aryl hydrocarbon) receptor, and the mutated protein leads to disrupted cell cycle signaling pathways, although mechanisms for selective somatotroph expression remain enigmatic [105,106]. In AIP-mutated individuals, adenomas are larger and present at a younger age, often as gigantism [107]. Other non-GH-secreting tumors are infrequently encountered in these families. Because of the very low penetrance, the value of follow-up of carriers without overt disease is not resolved [108,109]. Prevalence of the mutation is very low (B5%) in apparently sporadic tumors and is negligible after the age of 40 years [110].

Gigantism The diagnosis of pituitary gigantism should be considered in children who are .3 standard deviations (SDs) above normal mean height for age, or .2 SDs over their adjusted mean parental height. Clinical features of excess GH include tall stature, acral changes, increased appetite, coarsened facial features, sleep apnea, excessive perspiration, and widening of incisor gaps [111]. The biochemical diagnosis is similar to that for acromegaly, i.e., GH levels are in excess of 1 ng/mL after a glucose load and serum IGF-1 concentrations are elevated. Gigantism may be caused by a variety of conditions [112] (Table 15.3). Familial tall stature, redundancy of Y chromosomes, Marfan syndrome, and homocystinuria should be excluded prior to considering the endocrine causes of tall stature. About 20% of patients with gigantism are associated with the McCune Albright syndrome, usually associated with somatotroph hyperplasia. Somatotroph hyperplasia and acidophilic stem cell adenomas have been reported in cases of gigantism beginning in infancy or early childhood, suggesting early hypersecretion of GHRH or disordered pituicyte cell differentiation accounting for hypersomatotrophism [67,112]. In children undergoing pubertal growth spurts, however, GH response to glucose may be paradoxical

TABLE 15.3

Causes of Tall Stature

Endocrine—metabolic Growth hormone-secreting pituitary adenomas or hyperplasia Known genetic mutations: AIP MEN-1 Carney complex X-LAG McCune Albright syndrome MEN-4 Hyperinsulinism Lipoatrophic diabetes Hyperthyroidism Prepubertal sex steroid excess Nonpituitary Familial Marfan syndrome Homocystinuria Neurofibromatosis Unclassified Cerebral gigantism

and serum IGF-1 concentrations are often physiologically elevated. Early-onset rapid growth acceleration (,2 years of age), with GH hypersecretion and features of gigantism manifest by 5 years, may be associated with microduplications on chromosome Xq26.3 [113]. As pituitary tumor or hyperplasia tissue derived from these patients over-expresses GPR101, a GHsecretagogue receptor, this syndrome has been termed X-linked acrogigantism (X-LAG). If pituitary imaging reveals the presence of an adenoma, surgical resection should be attempted, but these are usually large tumors, and as B80% are invasive, they are difficult to resect. SRLs with or without dopamine agonists and pegvisomant have successfully been employed in treating these children or young adults [111,114 116]. Radiation therapy should be considered for failed responses to surgery and medical treatment.

CLINICAL FEATURES OF ACROMEGALY Acromegaly manifestations may be due to central pressure effects of the pituitary mass or peripheral actions of excess GH and IGF-1 (Table 15.4). Central features of the expanding pituitary mass are common to all pituitary masses. They include headache, visual dysfunction due to chiasmal compression, and rarely hypothalamic and frontal lobe dysfunction. The headache is often severe and sometimes debilitating. Lateral extension may impinge upon cranial nerves III, IV, and VI with diplopia, or nerve V leading to facial

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432 TABLE 15.4

15. ACROMEGALY

Clinical Features of Acromegaly

Local tumor effects Pituitary enlargement Visual-field defects Cranial nerve palsy Headache Somatic systems Acral enlargement, including thickness of soft tissue of hands and feet Musculoskeletal system Gigantism Prognathism Jaw malocclusion Arthralgias and arthritis Carpal tunnel syndrome Acroparesthesia Proximal myopathy Hypertrophy of frontal bones Skin and gastrointestinal system Hyperhidrosis Oily texture Skin tags Colon polyps Cardiovascular system Left ventricular hypertrophy Asymmetric septal hypertrophy Cardiomyopathy Hypertension Congestive heart failure Pulmonary system Sleep disturbances Sleep apnea (central and obstructive) Narcolepsy

TABLE 15.5

Visceromegaly Tongue Thyroid gland Salivary glands Liver Spleen Kidney Prostate Endocrine—metabolic Reproduction Menstrual abnormalities Galactorrhea Decreased libido, impotence, low sex hormone-binding globulin MEN-1 Hyperparathyroidism Pancreatic islet cell tumors Carbohydrate Impaired glucose tolerance Insulin resistance and hyperinsulinemia Diabetes mellitus Lipid Hypertriglyceridemia Mineral Hypercalciuria, increased levels of 25-hydroxyvitamin D3 Urinary hydroxyproline Electrolyte Low renin levels Increased aldosterone Thyroid Low thyroxine-binding globulin levels Goiter

Adapted from Melmed S. Medical progress: acromegaly. N Engl J Med 2006;355(24): 2558 73.

pain; temporal lobe invasion may also occur. Inferior extension of the mass may cause cerebrospinal fluid rhinorrhea and nasopharyngeal sinus invasion. These local signs are especially important in acromegaly, as most series report a relatively higher preponderance of macroadenomas (.65%) in acromegaly [117].

GH Action in Acromegaly High GH levels observed in acromegaly lead to complex gene expression patterns controlling cell proliferation, glucose metabolism, and growth factor functions. An in-frame deletion of exon 3 in the GH receptor (d3-GHR) has been reported to enable increased intracellular signaling and accelerated growth ensuing from enhanced GH responsiveness. Acromegaly patients harboring this polymorphism may exhibit a more florid clinical phenotype with increased prevalence of

Presentation of Acromegaly

Presenting chief complaint

Frequency (%)

Menstrual disturbance

13

Change in appearance/acral growth

11

Headaches

8

Paresthesias/carpal tunnel syndrome

6

Diabetes mellitus/impaired glucose tolerance

5

Heart disease

3

Visual impairment

3

Decreased libido/impotence

3

Arthopathy

3

Thyroid disorder

2

Hypertension

1

Gigantism

1

Fatigue

0.3

Hyperhidrosis

0.3

Somnolence

0.3

Other

5

Chance (detected by unrelated physical or dental examination or X-ray)

40

Based on 310 patients. From Molitch ME. Clinical manifestations of acromegaly. Endocrinol Metab Clin North Am 1992;21(3):597 614.

osteoarthritis and colon polyps and persistent biochemical resistance to pituitary-directed treatment [118]. Enhanced responsiveness to GH-receptor antagonists has not been uniformly observed [119].

Effects of Excessive GH Secretion The protean clinical manifestations of hypersomatotrophism are caused by elevated GH and/or IGF-1 levels (Table 15.4). Effects of hypersomatotrophism on acral and soft tissue growth, as well as metabolic function, may occur insidiously over several years [120,121] (Table 15.5, Figs. 15.4 15.6). The overall clinical and biochemical features of acromegaly, as well as the pituitary tumor characteristics of patients presenting with acromegaly, appears to be unchanged from 1981 to 2006 [126]. These findings underscore the elusiveness of the symptomatology, which often results in the disease being diagnosed only when patients seek care for dental, orthopedic, or rheumatologic disorders. Only 13% of 256 acromegalic patients diagnosed during a 20-year period presented with primary symptoms of altered facial appearance or enlargement of

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CLINICAL FEATURES OF ACROMEGALY

(A)

(B)

(C)

(D)

(E)

(F)

433

FIGURE 15.4 Clinical signs of acromegaly. (A) Original figure depicting earliest illustration of clinical features of acromegaly by Minkowski in 1887. Note acromegalic facies, fleshy fingers and toes, and frontal bossing. (B) Acromegaly in a young male with active perspiration, oily skin, acne, and widened tooth gap. (C) Prominent skin tags may be associated with the presence of colon polyps. (D) Jaw overbite and widening of spaces between incisors due to mandibular growth in acromegaly. (E) X-ray image of bony “tufting” seen at ends of terminal phalanges indicates bony overgrowth. (F) Increased heel pad thickness. Source: (D) and (F) from Melmed SB, Braunstein GD. Disorders of the Hypothalamus and Anterior Pituitary. 5th ed. St. Louis: Mosby Publishing; 1998 [122].

extremities [127]. In a review of several hundred patients worldwide, 98% were reported with acral enlargement, while hyperhidrosis was prominent in 70% [120]. Moreover, the time between onset of symptoms and diagnosis of acromegaly ranges from 6.6 to 10.2 years, with a mean delay of almost 9 years [128]. The latency period of time to diagnosis appears to have shortened, likely reflective of enhanced physician awareness, availability of more sensitive diagnostic tools, and increased use of MRI, which unmasks incidentally discovered pituitary adenomas [121]. Generalized visceromegaly occurs with enlargement of the tongue, bones, salivary glands, thyroid, heart, liver, and spleen [129]. Patients have characteristic facial features, large fleshy nose, spade-like hands, and frontal bossing. Some patients, if presenting early, may have subtle facial and peripheral features. Serial review of old photographs often accentuates the

progress of these subtle physical changes [130]. Increase in shoe, ring, or hat size is commonly reported. Although skeletal muscle mass is largely unchanged in acromegaly, the nonsmooth muscle lean compartment is increased [131]. Mechanisms underlying metabolic adaptation to maintaining steady-state protein mass include enhanced protein breakdownsynthesis rates, with intact protein oxidation [132]. Skeletal Changes Progressive acral changes if untreated, lead to severe facial and skeletal disfigurement, especially if the excess GH secretion begins prior to closure of the epiphyses (Fig. 15.6) [125,133,134]. Periosteal new bone formation in response to IGF-1 [135] results in skeletal overgrowth leading to mandibular overgrowth with prognathism, maxillary widening, teeth separation, frontal bossing, jaw malocclusion

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15. ACROMEGALY

(A)

(B)

(C)

(E)

(D)

(F)

FIGURE 15.5 Clinical signs of acromegaly. (A) MRI of GH-secreting pituitary macroadenoma depicting lateral tumor extension into cavernous sinus, and dorsal elevation of optic chiasm (coronal image). (B) Limestone portrait of Egyptian Akhenaten ca. 1365 BC showing jaw prognathism and thickened lips. (C) Jaw prognathism and mandibular overbite and (D) widened incisor tooth gap in two acromegaly patients. (E) Governor Pio Pico of California in 1858. Note acromegaly facial features and mild left proptosis consistent with cavernous sinus tumor invasion [123]. (F) Dolicomegacolon in acromegaly as visualized by CT colonography. Source: (B) Reproduced with permission from http:// commons.wikimedia.org/wiki/file:reliefportraitofakhenaten01.png and Staatliche Museen Zu Berlin-Preufsicher Kulturbesitz, Agyptisches Museum. (F) Reproduced with permission from Resmini E, Tagliafico A, Bacigalupo L, et al. Computed tomography colonography in acromegaly. J Clin Endocrinol Metab 2009;94(1):218 22 [124] and Melmed S. Acromegaly pathogenesis and treatment. J Clin Invest 2009;119(11):3189 02.

FIGURE 15.6 Severe skeletal disfigurement in three patients (A, B, C) with growth hormone-secreting pituitary tumors. Source: From Whitehead EM, Shalet SM, Davies D, Enoch BA, Price DA, Beardwell CG. Pituitary gigantism: a disabling condition. Clin Endocrinol (Oxf) 1982;17 (3):271 7 [125].

and overbite, and nasal bone hypertrophy. Characteristic voice deepening with a sonorous resonance occurs because of laryngeal hypertrophy and enlarged paranasal sinuses. A meta-analysis showed

that acromegaly is associated with increased bone formation and resorption, higher femoral neck bone mineral density, and increased frequency of vertebral fractures (odds ratio 8.26; 95% CI 2.91 23.34;

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p , 0.0001). Associated hypogonadism, male gender, and higher IGF-1 levels appear to predict vertebral fractures [136]. Arthropathy occurs in about 70% of patients, most of whom exhibit objective signs of joint swelling, hypermobility, and cartilaginous thickening [137,138]. Up to half of patients experience joint symptoms severe enough to limit or impair daily activities [139,140]. Severe joint pain unusually signifies irreversible joint degeneration. Knees, hips, shoulders, lumbosacral joints, elbows, and ankles are affected in decreasing order of frequency. Joint involvement may be mono- or polyarticular, and although crepitus, stiffness, tenderness, and hypermobility are common, joint effusions are rarely encountered [141]. Local periarticular fibrous tissue thickening may cause subsequent joint stiffening, deformities, and nerve entrapment. Neural enlargement, local fluid retention, and swelling of wrist soft tissues may lead to carpal tunnel syndrome, a painful edematous entrapment median neuropathy, which occurs in up to half of all patients. This condition generally resolves early after treatment [142]. In patients with uncontrolled GH and IGF-1 levels, spinal involvement including osteophytosis, disk space widening, and increased anteroposterior vertebral length may lead to dorsal kyphosis scoliosis and vertebral fractures [143]. About 70% of patients exhibit large-joint and axial arthropathy, including synovitis and periarticular calcifications [138,144,145]. Pathology of arthropathy. Uneven chondrocyte proliferation with subsequent increased joint space occurs early in response to increased GH and IGF-1 levels. Ulcerations and fissures on the weight-bearing areas of new cartilage are often accompanied by new bone formation. This process eventually results in debilitating osteoarthritis associated with bone remodeling, osteophyte formation, subchondral cysts, narrowed joint spaces, and lax periarticular ligaments. Osteophytes are seen at the tufts of the phalanges and over the anterior aspects of spinal vertebrae. Ossification of ligaments and periarticular calcium pyrophosphate deposition are also found [138]. The duration of hypersomatotrophism appears to directly correlate with the severity of the joint changes, and responses to therapy (see below) will usually depend on the degree of irreversible cartilage degeneration already in place. Skin Changes Hyperhidrosis and oily skin with an unpleasant odor are common early signs, occurring in up to 70% of patients. Patients often relate the need to increase their use of deodorant or cosmetic powders. Facial wrinkles, nasolabial folds, and heel pads are increased in thickness, and body hair may become coarsened [146]. These

435

effects may correlate with IGF-1 levels, and improve after treatment. Thickening of the skin has been attributed to glycosaminoglycan deposition [147], while connective tissue collagen production is also increased [148]. Skin tags are common and may be important markers for the concomitant presence of adenomatous colonic polyps [149]. Raynaud’s phenomenon may also be present in up to one-third of patients. Cardiovascular Complications Cardiovascular disease is a major cause of morbidity and mortality [150], with symptomatic cardiac disease present in up to 60% of patients. Arrhythmias, hypertension, valvular disease, and sodium and fluid retention leading to expanded extracellular fluid volume are common manifestations. Hypertension (systolic and diastolic), diabetes mellitus, elevated HbA1c, decreased low density lipoprotein, and elevated cholesterol all lead to increased Framingham risk score in acromegaly versus a control population (p , 0.0001); normalization of IGF-1 is associated with several of these adverse risk factors and decreased Framingham risk score [151]. Overall, the constellation of glucose intolerance, hypertension, arrhythmias, and diastolic overload may lead to intractable heart failure, especially if rigorous biochemical acromegaly control is not achieved [20]. About half of all patients are at “intermediate-to-high” risk for coronary atherosclerosis [152,153]. Nevertheless, in a 5-year prospective report of 52 patients, calcium scores and results of myocardial SPECT perfusion indicated no major coronary arterial changes from expected controls. Furthermore, acute ischemic events were not observed in this cohort [154]. About half of patients with active acromegaly have hypertension, and 50% of these have evidence of left ventricular dysfunction. Interestingly, left ventricular hypertrophy is also reported in 20% of young normotensive patients, and in up to 90% of those with long-standing disease. Increased postexercise ventricular ejection fraction is observed in B70% of patients [150,155]. Aortic root ectasia (73.8 cm) was also observed in about 25% of patients [156]. Asymmetric septal hypertrophy is common and concentric myocardial hypertrophy develops, with associated diastolic heart failure if GH levels are not controlled [157]. Subclinical left ventricular diastolic dysfunction is consistent with unique pathologic findings, including myocardial hypertrophy, interstitial fibrosis, and lymphocytic myocardial infiltrates. Electrocardiograms are abnormal in about 50% of patients, with S-T segment, T-wave abnormalities, conduction defects, and arrhythmias accounting for most changes. Hypertension has been ascribed to plasma volume expansion and

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15. ACROMEGALY

increased cardiac output [158], and GH also exerts direct antinatriuretic effects. GH acts to induce transepithelial sodium transport at the aldosterone-sensitive distal nephron, and induces transcription of the cortical collecting duct epithelial sodium channel α-subunit [159]. Although heart failure is usually reversible with SRL treatment, aortic and mitral valve regurgitation and hypertension usually persist despite biochemical disease control [160]. Cardiovascular disease is the most important cause of mortality in acromegaly, accounting for B60% of deaths. The presence of cardiovascular disease at the time of diagnosis is associated with high mortality rates, and effective control of GH and IGF-1 levels improves cardiac function [151,155].

may occur, and this rare mixed motor and sensory impairment should be distinguished from diabetic neuropathy that may occur secondarily to acromegaly. Pathologic features of median neuropathy have been ascribed to increased edema, rather than extrinsic compression [142]. About half of all patients develop proximal myopathy, which may be accompanied by myalgias and cramps, nonspecific electromyogram myopathic changes with hypertrophy and necrosis of muscle fiber, and elevated creatine phosphokinase levels [167]. Open-angle glaucoma may also result from impaired aqueous filtration through hypertrophied tissue surrounding the canal of Schlemm.

Respiratory Complications

Self-esteem may diminish with progressive facial and bodily disfigurement. It is unclear whether reported depression, mood swings, and apathy result from these physical effects or whether they are intrinsic central effects inherent to high GH levels.

Prognathism, thick lips, macroglossia, and hypertrophied nasal structures may result in significant airway obstruction [161]. Additional clinical features of acromegaly contribute to impaired upper respiratory function. Irregular hypertrophy of laryngeal mucosa and cartilage may lead to unilateral or bilateral vocal cord fixation or laryngeal stenosis with accompanying voice changes [161]. Tracheal calcification and cricoarytenoid joint arthropathy may also be present. These obstructive features may necessitate tracheostomy either to maintain adequate baseline airway function, or especially at the time of surgical anesthesia. Difficulty in tracheal intubation is often encountered in patients undergoing anesthesia. Central respiratory center depression as well as upper airway obstruction may contribute to the development of paroxysmal daytime sleep (narcolepsy), sleep apnea, and habitual excessive snoring. Obstructive sleep apnea, characterized by excessive daytime sleepiness with at least five nocturnal episodes of obstructive apnea per hour, has been documented in .50% of patients [162]. These patients may also have a ventilation perfusion defect with hypoxemia. The sleep apnea of acromegaly may be due to obstruction of the respiratory tract, or central in origin [163,164]. Interestingly, the central form of sleep apnea is associated with higher GH and IGF-1 levels, possibly reflecting a loss of central somatostatin tone accounting for the disorder [165].

Neuromuscular Changes Peripheral acroparesthesias occur in almost half of all patients. Synovial edema and hyperplastic wrist ligaments and tendons contribute to painful median nerve compression with the resultant carpal tunnel syndrome [166]. Symmetrical peripheral neuropathy

Psychologic Changes

Development of Neoplasms Several benign and malignant tumors have been reported in association with acromegaly and retrospective studies have suggested increased risk for gastrointestinal malignancies [168]. However, a compelling cause effect relationship of acromegaly with cancer has not been established [169 172], except for thyroid cancer [173]. In the German acromegaly registry of 446 patients with 6656 person-years of follow-up since diagnosis, no increased cancer incidence was found [174]. Although coexistence of acromegaly and meningioma has been reported, meningiomas are known to develop at sites of previous head trauma, inflammation, or irradiation [175]. No association has been reported between acromegaly and other intracranial neoplasms. Reports of high prevalence of colonic polyps in acromegaly may reflect increased physician awareness in screening for these tumors, as well as the use of diagnostic colonoscopy. Prospectively, B45% of patients with acromegaly harbor colonic polyps, but a controlled study in 161 patients revealed no increased polyp incidence in acromegaly [171]. Acrochordons (skin tags) have been noted in most patients found to harbor colonic lesions [149]. Hypertrophic mucosal folds, colonic hypertrophy, dolichocolon, and slow colonic transit times are commonly encountered, and intestinal bacterial overgrowth has been attributed to autonomic dysfunction [176]. The prevalence of colonic diverticula is also increased (odds ratio 3.6; 95% CI 1.4 5.7) [177]. Colonoscopy is warranted in these

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TABLE 15.6 • • • • • • • • •

Predictors of Mortality

TABLE 15.7 Acromegaly

GH level .2.5 ng/mL Elevated IGF-1 level Hypertension Age Time of delay from diagnosis Male gender Prior pituitary radiotherapy ACTH-dependent adrenal insufficiency Treatment with hydrocortisone .25 mg per day

Disease-Specific Mortality in Patients With N

Deaths (n) SMR

Wright (1970)

194

55

Overall: 1.8

Alexander (1980)

164

45

Overall: 3.3 (male, 4.8; female, 2.6)

Nabarro (1987)

256

47

Overall: 1.3 (,55 years, 1.9; female, 1.7)

Cardio/cerebrovascular disease: 1.3

Adapted from Ben-Shlomo A. Pituitary gland: predictors of acromegaly-associated mortality. Nat Rev Endocrinol 2010;6(2):67 9 [178].

patients once every 3 5 years after diagnosis, depending on the presence of other risk factors. Timely diagnosis and resection of premalignant polyps is prudent for improved morbidity in this relatively high-risk group of patients. On repeat colonoscopy, colon polyp prevalence correlated with IGF-1 levels [172]. CT has also been advocated in light of the technical challenges posed by colonoscopy in acromegaly [124]. The body of evidence suggests that mortality in patients with acromegaly is largely related to GH levels, rather than any observed enhanced incidence of cancer (Tables 15.6 and 15.7). In a large yet uncontrolled survey of 1362 patients in the United Kingdom, cancer incidence was in fact lower than expected, and the observed enhanced colon cancer mortality in acromegaly correlated with GH levels [180]. As patients with acromegaly are living longer due to improved biochemical control, it is apparent that long-term prospective controlled studies are required to resolve this question, as the incidence of malignancy increases with aging.

Endocrine Complications

Bengtsson (1998)

62

Overall: 3.2

Cardio/cerebrovascular disease: 3.6 Malignancy: 2.7 Rajasoorya (1994)

151

32

Overall: 3.0

Cardiovascular disease: 3.0 Cerebrovascular disease: 3.0 Malignancy: 1.0 Etxabe (1993)

74

10

Overall: 3.2 (male, 7.0; female, 1.4)

Bates (1993)

79

28

Overall: 2.63

Orme (1998)

1362 366

Overall: 1.60

Vascular disease: 1.76 Cerebrovascular disease: 2.06 Respiratory disease: 1.85 Malignancy: 1.16 Swearingen (1998)

149

12

Overall: 1.16

Abosch (1998)

254

29

Overall: 1.28

Beauregard (2003)

103

18

Overall: 2.14

Arita (2003)

154

11

Overall: 1.17

Biermasz (2004)

164

28

Overall: 1.33

Holdaway (2004)

208

72

Overall: 1.22

Ayuk (2004)

419

95

Overall: 1.26

Cardiovascular disease: 1.37 Cerebrovascular disease: 2.68 Respiratory disease: 1.52 Malignancy: 0.91 Mestron (2004)

Elevated serum PRL levels, with or without galactorrhea, occur in about one-third of patients, some of whom present with PRL levels .100 ng/mL [117,181]. Several mechanisms may underlie hyperprolactinemia in acromegaly. Functional pituitary stalk compression by an adenoma may prevent hypothalamic dopamine from impinging upon pituitary lactotrophs, resulting in release from tonic hypothalamic inhibition [181]. GH-secreting adenoma types may also concomitantly secrete PRL, including mixed GH-cell and PRL-cell plurihormonal adenomas, monomorphous mammosomatotroph adenomas, and acidophilic stem cell adenomas [28]. In patients with galactorrhea and normal PRL levels, elevated GH concentrations may crossreact and behave as an agonist for PRL-binding sites in the breast. Hypopituitarism, which develops as a result of the tumor mass compressing surrounding normal

166

1219 56

Overall: NA

In remission: 1.3 Persistent disease: 1.38 Kauppinen-Makelin (2005)

334

56

Overall: 1.16

Trepp (2005)

94

13

Overall: 1.34

Sherlock (2009)

501

162

Overall: 1.7

Cardiovascular disease: 1.9 Cerebrovascular disease: 2.7 Respiratory disease: 1.8 Malignancy: 1.2 NA 5 not available; SMR 5 standardized mortality ratio.

Adapted from Sherlock M, Ayuk J, Tomlinson JW, et al. Mortality in patients with pituitary disease. Endocr Rev 2010;31(3):301 42 [179].

pituitary tissue, leads to amenorrhea or impotence [117,182], while up to 20% of patients may also have secondary thyroid or adrenal failure. Gonadal function

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15. ACROMEGALY

is also an important determinant of bone density in these patients. Carbohydrate intolerance is caused by direct antiinsulin effects of GH, and patients may develop insulin-requiring diabetes mellitus or microalbuminuria [183]. Insulin resistance is most strongly associated with IGF-1 levels, and is a significant cardiovascular risk factor [184]. Carbohydrate intolerance and insulin requirements improve remarkably after lowering of GH by surgery or SRL therapy. Hypertriglyceridemia (type IV), hypercalciuria, and hypercalcemia are commonly found. Pituitary hypersecretion of associated hormones by mixed somatotroph tumors commonly result in hyperprolactinemia, and rarely in Cushing disease (ACTH hypersecretion) or hyperthyroidism (TSH hypersecretion). IGF-1 is a determinant of thyroid cell growth, leading to diffuse or nodular toxic or nontoxic goiter, or Graves disease. Associated manifestations of MEN-1 may be present in affected individuals. These include hypercalcemia with hyperparathyroidism or pancreatic tumors. Benign prostatic hypertrophy has been documented with no apparent increase in prostate cancer rates [185].

Effects on Morbidity and Mortality Increased acromegaly mortality is mostly attributed to cardiovascular, cerebrovascular, and respiratory abnormalities [179,186 195]. Reduced life expectancy in a series of 194 patients was due to cardiovascular disorders in 24% of deaths, followed by respiratory (18%), and cerebrovascular disease (14%) [187]. Other reports confirming these findings showed a two- to threefold increase in mortality primarily due to cardio/cerebrovascular and respiratory disease, with some series also showing differences in mortality in age- and sex-specific subgroups (Table 15.7, Fig. 15.7) [179,186]. Significant independent determinants of mortality include serum GH levels .2.5 ng/mL, elevated age-matched serum IGF-1 levels, and the presence of preexisting heart disease [191]. As achieving optimal biochemical control may be challenging, rigorous diagnosis and treatment of comorbidities that contribute to mortality is important. Hypertension and diabetes mellitus are amenable to early interventions, which have likely contributed to the observed trend of reduction in mortality rates more recently reported [190]. Other variables that increase mortality include hypertension, age at diagnosis, time delay for diagnosis, male gender, previous pituitary irradiation, and the presence of pituitary failure, especially ACTH insufficiency. In the West Midlands acromegaly database, mortality rates were increased in 226 men and 275 women with acromegaly over a 14-year median

FIGURE 15.7 Pooled standardized mortality ratios (SMRs) in studies of acromegaly. Data are given as SMR (95% CI). Source: From Holdaway IM, Bolland MJ, Gamble GD. A meta-analysis of the effect of lowering serum levels of GH and IGF-1 on mortality in acromegaly. Eur J Endocrinol 2008;159(2):89 95.

follow-up period (SMR 1.7; 95%; CI 1.4 2.0, p , 0.001) as compared with the general population [191]. Mortality rates were also higher in patients who had received radiotherapy (SMR 2.1; 95% CI 1.7 2.6; p 5 0.006). While cardiovascular and cerebrovascular diseases contributed to overall increased mortality, cerebrovascular disease was the major cause of death in patients who had undergone radiotherapy (Table 15.8). In another study, 211 acromegaly patients receiving pituitary-directed radiotherapy were shown to exhibit high mortality rates (SMR 1.58; 95% CI 1.22 2.04; p 5 0.005), predominately as a result of cerebrovascular disease (SMR 4.42; 95% CI 2.71 7.22; p 5 0.005) [192]. Most published studies on mortality outcomes have employed measuring last available biochemical values, leading to a potential confounding selection bias, which overestimates risk of an elevated value [193,194]. Substituting time-dependent cumulative hormone values, only GH levels .5 ng/mL were predictive of increased mortality [193]. Thus, multiple factors independently increasing mortality rates (Table 15.6) should be considered at diagnosis and follow-up of a patient with acromegaly. Early diagnosis of comorbidities, tight biochemical control of

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439

DIAGNOSIS

TABLE 15.8 Mortality in Patients With Acromegaly Undergoing Pituitary Radiotherapy Mortality

RT

SMR

95% CI; p Value

Overall

No

1.4

1.7 2.6; p 5 0.006

Yes

2.1

No

1.1

Yes

2.4

No

1.7

Yes

2.2

No

1.7

0.8 3.3

Yes

4.1

2.3 6.6; p 5 0.034

Malignancy

Cardiovascular disease

Cerebrovascular disease

TABLE 15.9

Posttreatment GH (ng/mL) ,2.5 (n 5 541)

2.5 9.9 (n 5 493)

.10 (n 5 207)

Overall

1.10 (0.89 1.15)

1.41 (1.16 1.69)

2.12 (1.70 2.62)

,0.0001

Cancerrelated

0.96 (0.63 1.41)

0.81 (0.50 1.24)

1.81 (1.13 2.74)

,0.05

0.8 2.2; p 5 0.442

1.6 3.1; p 5 0.247

RT 5 radiotherapy. Adapted from Sherlock M, Reulen RC, Alonso AA, et al. ACTH deficiency, higher doses of hydrocortisone replacement, and radiotherapy are independent predictors of mortality in patients with acromegaly. J Clin Endocrinol Metab 2009;94(11): 4216 23 [191].

Post-treatment GH Levels and Mortality

p

Standardized mortality ratios and 95% confidence intervals for overall mortality and cancer-related mortality. Adapted from Orme S, McNally RJQ, Cartwright RA, Belchetz PE. Mortality and cancer incidence in acromegaly: a retrospective cohort study. J Clin Endo Metab 1998;83:2730 4 [180].

TABLE 15.10

Diagnosis of Acromegaly Elevated age-matched IGF-1 level AND GH nadir during OGTT .1 ng/mL

growth hormone and IGF-1 levels, normalization of blood pressure, and early detection and treatment of adrenal insufficiency with doses of hydrocortisone ,25 mg per day should contribute to reduced mortality rates. Cerebrovascular and cardiovascular disorders and diabetes mellitus should be addressed early and aggressively. Control of GH levels to ,2.5 ng/mL after surgery or medical treatments appears to generally reverse adverse mortality rates [188,195] and tight biochemical control significantly reduces both morbidity and mortality (Table 15.9).

DIAGNOSIS The biochemical diagnosis of acromegaly requires demonstration of central autonomous GH hypersecretion, as well as elevated IGF-1 levels reflecting systemic peripheral exposure to tonically elevated GH levels [196]. Basal morning and random GH levels are elevated in acromegaly [197,198]. Because of the episodic nature of GH secretion, however, serum concentrations may normally fluctuate from “undetectable” up to 30 ng/mL [199]. When GH is sampled every 5 minutes in nonacromegaly individuals, GH levels are undetectable in about half of samples collected over 24 hours [198,200]. In acromegaly, however, samples collected over 24 h contain detectable levels of GH ( . 2 ng/ml) [187,201], while mean 24-hour integrated GH levels ,2.5 ng/mL usually exclude the diagnosis of acromegaly [202]. Strikingly, a random GH value of 2 ng/mL may be associated with a mean 24-hour GH concentration ranging from 1 10 ng/mL [50].

From Katznelson L, Laws Jr ER, Melmed S, et al. Acromegaly: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2014;99(11):3933 51.

Although episodic basal GH secretion patterns are sustained in acromegaly, normal diurnal variation is absent with a loss of sleep-related rise in GH [203]. These patients exhibit a higher episodic GH pulse frequency that often persists after surgical adenoma resection. In acromegaly, serum GH levels invariably do not suppress to ,1 ng/mL within 1 2 hours of an oral glucose (75 g) load; glucose may actually stimulate GH secretion in about 10% of patients [204]. Thus, biochemical exclusion of acromegaly requires a GH nadir during oral glucose tolerance test (OGTT) ,1 ng/mL with measured normal IGF-1 levels [205] (Table 15.10). Single random GH levels are not recommended for diagnosis of acromegaly because of the episodic nature of GH secretion [203,205 207]. Using ultrasensitive GH assays, nadir GH levels ,0.3 ng/mL after a glucose load accurately distinguish patients with active acromegaly from those controlled, or those without disease [208,209]. With these highly sensitive assays, random GH levels in patients with acromegaly may be ,1.0 ng/mL, and even as low as 0.37 ng/mL when IGF-1 levels are still elevated postoperatively [210] (Fig. 15.8). Serum IGF-1 levels are invariably high in acromegaly [211], reflective of a biomarker for integrated GH secretion. Age-matched IGF-1 elevations may persist for several months postoperatively when GH levels are apparently controlled [212]. As pregnancy and late puberty are associated with elevated IGF-1 levels, a high IGF-1 value is highly specific

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440

15. ACROMEGALY

FIGURE 15.8 Ultrasensitive GH immunoradiometric assay distinguishes acromegaly status in postoperative patients. Source: Adapted from Freda PU, Post KD, Powell JS, Wardlaw SL. Evaluation of disease status with sensitive measures of growth hormone secretion in 60 postoperative patients with acromegaly. J Clin Endocrinol Metab 1998;83 (11):3808 16.

for acromegaly in the nonpregnant adult and correlates with clinical indices of disease activity [211] (Figs. 15.9 and 15.10). IGFBP-3 levels are usually elevated, but provide little added diagnostic value [213,214]. In summary, the challenges of accurate biochemical diagnosis include the pulsatile pattern of GH secretion, sleep-related GH elevations, and age, body mass index, and nutritional-related changes in GH levels. Patients with malnutrition, liver disease, renal failure, or uncontrolled diabetes may fail to suppress GH after a glucose load. GH and IGF-1 measurements are also beset by poor reproducibility and standardization [215] (Table 15.11). These challenges were exemplified in a study that reported nadir GH levels after OGTT varied widely by B50%, with acromegaly diagnosis in 30% of subjects inconsistently validated [216]. Once the biochemical diagnosis of autonomous GH hypersecretion has been established, a pituitary MRI with administration of contrast material should be performed. Relatively accurate acromegaly diagnosis has been enabled by utilizing three-dimensional facial classification software, or by cephalometry [217,218].

FIGURE 15.9 Circulating IGF-1 levels in acromegaly. Source: From Clemmons DR, Van Wyk JJ, Ridgway EC, Kliman B, Kjellberg RN, Underwood LE. Evaluation of acromegaly by radioimmunoassay of somatomedin-C. N Engl J Med 1979;301(21):1138 42.

FIGURE 15.10 IGF-1 levels correlate with indices of clinical activity in acromegaly. Source: From Clemmons DR, Van Wyk JJ, Ridgway EC, Kliman B, Kjellberg RN, Underwood LE. Evaluation of acromegaly by radioimmunoassay of somatomedin-C. N Engl J Med 1979;301(21):1138 42.

III. PITUITARY TUMORS

DIAGNOSIS

TABLE 15.11 Factors Resulting in Discordant Circulating IGF-1 and GH Values • Unreliable or imprecise definition of “normal” GH values • Delayed normalization of IGF-1 levels following therapeutic intervention • GH secretory pattern that more effectively stimulates IGF-1 production • Persistently elevated and erratic GH pulse frequency following treatment • Contribution of local IGF-1 production to circulating IGF-1 levels • Variable sensitivity and reproducibility of assays employed Adapted from Drange MR. IGFs in the evaluation of acromegaly. In: Rosenfeld RG, Roberts CT, editors. Contemporary endocrinology the IGF system: molecular biology, physiology, and clinical applications. Humana Press; 1999. p. 699 720.

441

Differential Diagnosis The approach to diagnosis of the various forms of acromegaly [21] is outlined in Fig. 15.11. Over 95% of patients harbor a GH-secreting pituitary adenoma [21]. The very rare diagnosis of extrapituitary acromegaly should only be considered in a small number of patients, but is important in planning effective management. Regardless of the cause, GH and IGF-1 levels are invariably elevated and GH levels fail to suppress (,1 ng/mL) after an oral glucose load in all forms of acromegaly [219]. Patients with clinical features of acromegaly, normal GH and IGF-1 levels, and no evidence for extrapituitary tumor likely represent “burned out” acromegaly associated with an infarcted pituitary

FIGURE 15.11 Diagnosis and treatment of acromegaly. The oral glucose tolerance test is performed with 75 g of glucose and GH is measured over a period of 2 h. Disease control implies a GH nadir level ,1 ng/mL after glucose tolerance test and an age-adjusted normal IGF-1 level. GHRa, GH-receptor antagonist. Source: From Melmed S. Medical progress: acromegaly. N Engl J Med 2006;355(24):2558 73.

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442

15. ACROMEGALY

adenoma, often with resultant empty sella. Discordant circulating IGF-1 and GH levels may be encountered (Table 15.11), and these may reflect poor assay precision, or persistently elevated bioactive GH levels despite apparent appropriate glucose suppression [206]. Dynamic pituitary tests are not helpful in distinguishing GH-secreting pituitary tumors from extrapituitary tumors [220]. However, measuring GHRH plasma is precise and cost-effective for the diagnosis of ectopic acromegaly. Unique and unexpected clinical features in acromegaly, including respiratory wheezing or dyspnea, facial flushing, peptic ulcers, or renal stones will sometimes be helpful in alerting the physician to diagnosing associated nonpituitary endocrine tumors. Specific biochemical markers of an underlying ectopic tumor (including hypoglycemia, hyperinsulinemia, hypergastrinemia, and rarely hypercortisolism) are not usually encountered in pituitary acromegaly, and their presence should alert the physician to search for an extrapituitary source of GH excess. Anatomic localization of the pituitary or extrapituitary tumor is achieved using imaging techniques, including MRI and CT scanning. As routine abdominal or chest imaging will yield a very low incidence of true positive cases of ectopic tumor, such screening of these patients is not recommended as being cost-effective. Elevated circulating GHRH levels, a normal or small-sized pituitary gland, or clinical and biochemical features of other tumors known to be associated with extrapituitary acromegaly are indications for extrapituitary imaging. An enlarged pituitary is, however, often found on MRI of patients with peripheral GHRH-secreting tumors, and the radiologic diagnosis of a pituitary adenoma may be difficult to exclude. Exceedingly rare miscellaneous conditions associated with acromegaly, including acromegaloidism and the McCune Albright syndrome, should be considered

(A)

only after definitive exclusion of pituitary and extrapituitary tumors.

TREATMENT OF ACROMEGALY Aims A strategy for managing patients with acromegaly should aim to comprehensively manage the pituitary mass, suppress hypersecretion of GH and IGF-1, and prevent long-term sequelae of hypersomatotrophism [219] (Table 15.12). The mortality associated with untreated or partially treated acromegaly is about double the expected mortality rate of age-matched healthy subjects, and it is therefore important to achieve optimally effective GH control. Elevated GH levels are associated with increased morbidity and account for the single most important determinant of mortality [180,188] (Figs. 15.12 and 15.13). Goals of Therapy An effective management strategy for patients with acromegaly should address the comprehensive goals of eliminating morbidity and reducing mortality rates to those expected for age- and sex-adjusted control populations (Table 15.12). TABLE 15.12

Suppress autonomous GH secretion to ,1 ng/mL after glucose load Normalize IGF-1 levels to age- and gender-matched controls Remove or reduce pituitary tumor mass Correct visual and neurologic defects Preserve pituitary trophic hormone function Treat acral, cardiovascular, pulmonary, and metabolic complications Prevent systemic sequelae of long-term hypersomatotrophism Prevent biochemical or local recurrence Restore mortality rates to expected age-matched controls

(B)

1.0

1.0 Matched population

0.8

Survival probability

Survival probability

Goals of Therapy in Patients With Acromegaly

0.6 p = .001 0.4

Acromegaly

0.2 0

0

5

10

15

20

25

30

35

40

0.8

GH 50

Follow-up GH mg/L

FIGURE 15.13

Death rates per 1000 in patients with acromegaly related to posttreatment GH levels. Source: Adapted from Ayuk J, Clayton RN, Holder G, Sheppard MC, Stewart PM, Bates AS. Growth hormone and pituitary radiotherapy, but not serum insulin-like growth factor-I concentrations, predict excess mortality in patients with acromegaly. J Clin Endocrinol Metab 2004;89(4):1613 7 [192].

1. Selective resection or shrinkage of the pituitary tumor should be accompanied by correction of associated parasellar local pressure effects, and growth recurrence of the pituitary mass should ideally be prevented. 2. Anatomic or functional ablation of the disordered pituitary mass should not compromise residual anterior pituitary trophic function, especially the adrenal, thyroid, and gonadal axes. 3. The morbid effects of hypersomatotrophism, including glucose intolerance, hypertension, soft tissue swellings, nerve entrapments, and arthritis should be ameliorated or reversed. These disorders are often neglected in the context of specialized neurosurgical or endocrine management, which tends to rely on objective radioimaging or hormone assay criteria. As patients with acromegaly are living longer, their metabolic, acral, and soft tissue manifestations require rigorous diagnosis and management, if they cannot in fact be prevented. 4. Integrated 24-hour GH secretion and IGF-1 levels should be normalized, and postoperatively, serum GH levels should be suppressed to ,1 ng/mL after an oral glucose load. Ideally, a “cured” patient should have “normal” 24-hour integrated GH secretion of GH, restored circadian rhythm, and exhibit appropriate responses of GH to provocative stimuli. Long-term adverse implications of mildly elevated (1 2.5 ng/mL) integrated GH levels are presently unclear. 5. Long-term follow-up should ideally be aimed at preventing both biochemical and anatomic recurrences. Early detection of undesirable late sequelae of hypersomatotrophism is essential to

Surgical Management Selective transsphenoidal surgical resection, often with minimally invasive endoscopic techniques, is the indicated treatment for well-circumscribed somatotroph cell adenomas [223] (Fig. 15.14). The uses of the operative microscope, microinstrumentation, sophisticated head immobilization techniques, and accurate MRI localization have all combined to achieve a high level of expert success with this procedure [224 226]. Residual pituitary function is usually intact after resection of well-encapsulated tumors that are totally confined within the pituitary fossa. These resections are technically challenging, compounded by microscopic lesions situated in anatomically inaccessible confined sellar spaces, the proximity of vital vascular and brain structures, and the location of dural tumor microfoci that persistently hypersecrete GH. Safe surgical access may also be impeded by internal carotid artery tortuosity or microaneurysms. Surgery reverses the signs of preoperative compression and the compromised trophic hormone secretion is often restored. The success of surgery is largely dependent on the expertise and experience of the neurosurgeon. The skilled surgeon will balance the extent of maximal tumor tissue removal with the need to preserve anterior pituitary function, especially when resecting large invasive tumors. Metabolic dysfunction and soft tissue swelling start improving almost immediately, as GH levels return to normal levels within hours of successful tumor resection. Surgical outcome can usually be correlated with the size of the adenoma and preoperative serum GH level. In patients with tumors less than 5 mm in diameter and totally confined to the sella, and in whom preoperative serum GH levels are ,40 ng/mL, a favorable surgical response is portended. One series showed that, overall, long-term biochemical control is achieved in 60% of all patients [227]. In other series, up to 87% of patients with well-circumscribed microadenomas ,10 mm in diameter achieved control [225,228]. In contrast, ,50% of all-sized macroadenomas had postoperative GH levels ,2 ng/mL after glucose [49,223]

III. PITUITARY TUMORS

444 TABLE 15.13

15. ACROMEGALY

Acromegaly Management

Mode:

Surgery

Radiotherapy

SRL

GH antagonist

Transsphenoidal resection

Noninvasive

Monthly injection

Daily injection

B50% in 10 years

B60%

0

, 30%

B60%

.90%

Effects: Biochemical control • GH , 2.5 ng/mL Macroadenomas ,50% Microadenomas .80% • IGF-1 normalized

Macroadenomas ,50% Microadenomas .80%

Onset

Rapid

Slow (years)

Rapid

Rapid

Tumor mass

Debulked or resected

Ablated

Growth constrained or tumor shrinks B50%

Unknown

Hypopituitarism

B10%

.50%

Very rare

Low IGF-1 possible

Other

Tumor persistence or recurrence, diabetes insipidus, local complications

Local nerve damage, second brain tumor, visual and CNS disorders, cerebrovascular risk

Gallstones, nausea, diarrhea

Elevated liver enzymes

Disadvantages:

Goals of Acromegaly Management Include: (1) control of GH and IGF-1 secretion and tumor growth; (2) relief of compressive effects on central nervous system and vascular structures, if present; (3) preservation or restoration of pituitary hormone reserve function; and (4) treatment of comorbidities and normalization of mortality rates. From Melmed S. Medical progress: acromegaly. N Engl J Med 2006;355(24):2558 73, Melmed S. Acromegaly pathogenesis and treatment. J Clin Invest 2009;119 (11):3189 202, and Carmichael JD, Bonert VS, Nuno M, Ly D, Melmed S. Acromegaly clinical trial methodology impact on reported biochemical efficacy rates of somatostatin receptor ligand treatments: a meta-analysis. J Clin Endocrinol Metab 2014;99(5):1825 33 [222].

(Table 15.14). Unique surgical problems encountered in acromegaly include difficulties in endotracheal intubation due to macroglossia and/or kyphosis [229]. Rarely, tracheostomy may be required for anesthesia. Long-term surgical remission can often be predicted by measuring postoperative GH levels 24 hours after surgery (Fig. 15.15). GH levels ,1 ng/mL 1 week after surgery reflect 89% probability for remission with 95% specificity [230]. Although GH levels may be suppressed, normalization of IGF-1 levels may require a longer latency period, and about 25% of IGF-1 measurements require 12 months or more to normalize [231]. Surgery may also predispose to enhanced subsequent medical therapy efficacy [232]. Side Effects of Surgery New hypopituitarism develops in B30% of patients undergoing endoscopic transsphenoidal surgery, reflecting operative damage to the surrounding normal pituitary tissue [233]. Although often transient, these complications may require lifelong pituitary hormone replacement. Permanent diabetes insipidus, cerebrospinal fluid leaks, hemorrhage, and meningitis occur in up to 10% of patients. Secondary empty sella may also develop postoperatively. The incidence of local

complications depends on the size of the tumor and the extent of local invasiveness. Experienced pituitary surgeons report significantly lower postoperative complication rates [224 226]. Recurrence (B7% over 10 years) or persistence of acromegaly after surgery usually indicates incomplete surgical removal of adenomatous tissue, inaccessible cavernous sinus tissue, or nesting of functional tumor tissue within the dural sellar lining, which is difficult to visualize and resect. Rarely, persistent or recurrent postoperative GH hypersecretion may require reoperation.

Radiation Treatment Conventional external deep X-ray therapy as well as heavy-particle (proton-beam) irradiation is employed as primary or adjuvant therapies for acromegaly. High-energy ionizing radiation can be delivered to the pituitary tumor by megavoltage radiation sources [234 236]. Factors important in balancing maximal tumor radiation with minimal soft tissue damage include precise MR image localization, effective simulation and isocentral rotational techniques, and high-voltage (6 15 MeV) delivery. Indications for use of radiation as primary therapy are a highly

III. PITUITARY TUMORS

TREATMENT OF ACROMEGALY

445

FIGURE 15.14 Transsphenoidal surgery in acromegaly. (A) Depiction of standard transsphenoidal midline approach to secreting pituitary tumors; (B) schematic depiction of midline tumor curettage; (C) schematic depiction of lateral parasellar curettage. Source: From Fahlbusch R, Honegger J, Buchfelder M. Surgical management of acromegaly. Endocrinol Metab Clin North Am 1992;21(3):669 92.

individualized choice, depending on the expertise and experience of the treating radiotherapist, as well as the willingness of the patient to choose the benefits of the therapy versus its potential risks. Patients undergoing conventional radiation therapy are administered up to 5000 rads in split doses of 180 rad fractions divided over 6 weeks. Tumor growth is invariably arrested and most pituitary adenomas shrink [236]. GH levels begin falling gradually during the first year after treatment. After 10 years, B50% of patients having undergone conventional radiation achieve GH levels ,2 ng/mL and normalized IGF-1 levels. Biochemical response to radiation correlates with pretreatment GH levels, and when these are .100 ng/mL, B60% of patients exhibit GH ,5 ng/mL after 18 years. This slow rate of biochemical response is the major disadvantage of this form of treatment. During the initial years after irradiation, over half of all patients may continue to be

exposed to unacceptably high levels of circulating GH and IGF-1. During the first 7 years after irradiation, few patients normalize IGF-1 levels, and B70% of patients exhibit normal IGF-1 levels when tested during longer follow-up [234] (Fig. 15.16). Stereotactic Radiosurgery Focused gamma radiation derived from a 60Cobalt source can be delivered with stereotactic precision and is especially useful for microadenomas ,3 cm in diameter and distant from the optic tracts. Five years after gamma irradiation, about half of 82 acromegaly patients in one series exhibited post-OGTT GH levels ,1 ng/mL, while pituitary failure developed in about a quarter of all patients [237]. In another series of 136 patients followed for a mean of 61.5 months, 65% of patients achieved GH ,1 ng/mL after a glucose load or a normalized IGF-1 level [238]. Favorable response determinants

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446 TABLE 15.14

15. ACROMEGALY

Surgical Remission Rates in Acromegaly Patients Remission rate n

Size (micro-/macroadenoma)

Overall

Macroadenoma

Microadenoma

SERIES USING 2010 CONSENSUS CRITERIA Microscopic Starke 2013

43

11/32

70%

82%

66%

Endoscopic Wang 2012

43

13/30

67%

77%

63%

Hofstetter 2010

24

4/20

38%

Starke 2013

72

13/59

71%

88%

66%

SERIES USING 2000 CONSENSUS CRITERIA Microscopic Kim 2009

42

12/30

64%

67%

60%

Ludecke 2006

147

21/126

72%

95%

68%

Trepp 2005

69

5/64

42%

80%

39%

Nomikos 2005

506

142/364

57%

75%

50%

Esposito 2004

67

13/54

57%

77%

52%

De 2003

90

29/61

63%

79%

56%

Beauregard 2003

103

22/81

52%

82%

47%

Kaltsas 2001

67

17/42

34%

59%

26%

Shimon 2001

88

44/44

74%

84%

64%

Kreutzer 2001

57

19/38

70%

40

0/40

56%

Gondim 2010

67

14/53

75%

86%

72%

Campbell 2010

26

4/22

58%

75%

55%

Tabaee 2009

6

83%

Yano 2009

31

71%

Dehdashti 2008

34

8/26

71%

83%

65%

Frank 2006

83

24/59

70%

83%

65%

Rudnik 2005

12

4/8

83%

Cappabianca 2002

36

6/30

64%

83%

60%

Cappabianca 2002

23

3/20

57%

67%

55%

Endoscopic Wagenmakers 2011

56%

From Starke RM, Raper DM, Payne SC, Vance ML, Oldfield EH, Jane Jr JA, Endoscopic vs microsurgical transsphenoidal surgery for acromegaly: outcomes in a concurrent series of patients using modern criteria for remission. J Clin Endocrinol Metab 2013;98(8):3190 8.

include higher margin radiation dose, higher maximum dose, and lower initial IGF-1 levels (Fig. 15.17). Side Effects of Radiotherapy About 50% of all patients receiving radiotherapy develop pituitary trophic hormone disruption within 10 years of treatment, and this incidence increases annually thereafter. After stereotactic radiotherapy, about

5 30% of patients develop pituitary deficits within 5 years [238,240]. Replacement of gonadal steroids, thyroid hormone, and/or cortisone is necessary in these patients. Side effects of conventional radiation include hair loss, cranial nerve palsies, tumor necrosis with hemorrhage, and rarely loss of vision due to optic nerve damage or pituitary apoplexy. These effects have been documented in 1 2% of patients [241 243]. Lethargy, impaired memory, and personality changes may also

III. PITUITARY TUMORS

TREATMENT OF ACROMEGALY

447

FIGURE 15.15 GH as a postoperative biomarker of remission. (A) Mean level of immediate postoperative GH is consistently lower through 72 h after surgery in the remission group than in the nonremission group. (B) Probability of remission 1 week after surgery. When nadir GH level on 1-week postoperative OGTT is approximately 1 ng/mL, probability of surgical remission is B90%. Source: From Kim EH, Oh MC, Lee EJ, Kim SH. Predicting long-term remission by measuring immediate postoperative growth hormone levels and oral glucose tolerance test in acromegaly. Neurosurgery 2012;70(5):1106 13; discussion 1 [230].

FIGURE 15.16

(A) Long-term effect of radiation therapy on GH secretion using a GH nadir after oral glucose load below ng/mL as the cure criterion and the probability of not being cured with time after radiotherapy. The numbers of patients not cured at 5, 10, and 20 years after pituitary irradiation are indicated in parentheses. Each step represents one cure; each cross (1) denotes a patient not cured at the latest follow-up. (B) Percentage of patients who were not receiving medical therapy with a normal IGF-1 level according to years after RT. n, the total number of patients who had IGF-1 measured in that time interval after RT. Source: (A) From Barrande G, Pittino-Lungo M, Coste J, et al. Hormonal and metabolic effects of radiotherapy in acromegaly: long-term results in 128 patients followed in a single center. J Clin Endocrinol Metab 2000;85(10):3779 85; (B) From Powell JS, Wardlaw SL, Post KD, Freda PU. Outcome of radiotherapy for acromegaly using normalization of insulin- like growth factor I to define cure. J Clin Endocrinol Metab 2000;85(5):2068 71.

occur. The incidence of local complications has been markedly diminished by the use of highly reproducible simulators; precise rotational isocentric arc capability and doses of ,5000 rad. Proton-beam therapy [244] is performed in a limited number of specialized centers

FIGURE 15.17 Acromegaly response to radiosurgery. Biochemical remission rate after radiosurgery for 46 patients with GH-producing pituitary adenomas. Source: Adapted from Pollock BE, Jacob JT, Brown PD, Nippoldt TB. Radiosurgery of growth hormoneproducing pituitary adenomas: factors associated with biochemical remission. J Neurosurg 2007;106(5):833 8 [239].

and is contraindicated in patients with suprasellar extension of their tumors due to exposure of the optic tracts to the radiation field. After stereotactic ablation of pituitary tumors by gamma irradiation in one report of 1567 patients, 13 developed cerebral radionecrosis [245]. Secondary brain neoplasms occurring following conventional radiation are very rare [246] and arise within the radiation field region at a cumulative risk frequency of 1.9% over 20 years. Pituitary radiation has also been associated with increased risk for strokerelated mortality [247]. Use of lowest effective radiation doses and careful replacement of hormone deficiencies may mitigate the stroke risk. Radiation therapy is highly effective in shrinking most GH-cell adenomas and in effectively lowering

III. PITUITARY TUMORS

448 TABLE 15.15

15. ACROMEGALY

Acromegaly Medical Treatments

Drug SRL Octreotide Lanreotide autogel Octreotide LAR Pasireotide LAR GH antagonist Pegvisomant Dopamine agonist Cabergoline

Primary target

Dose

SSTR2 SSTR2 SSTR2 SSTR5 . SSTR2 . SSTR3 . SSTR1

50 60 10 40

GH receptor

10 40 mg SC daily

D2 receptor

1 4 mg PO weekly

GH levels over 20 years in most patients. Overall, determinants of radiation-induced hypopituitarism include prior surgery, the precision of stereotactic tumor focus, dose used, and pituitary stalk exposure to the radiation field [20]. Because of the relatively commonly observed side effects, radiation is usually employed as adjuvant therapy for patients not clinically and biochemically controlled by surgical and/or medical management, or for those who decline surgery or medical therapy.

Medical Treatment Several medications are available to treat acromegaly (Table 15.15). These molecules are largely developed based on understanding receptor signaling and their respective dysfunctions in the disorder (Fig. 15.18).

Dopamine Agonists Dopamine acutely inhibits GH secretion in up to one-third of patients with acromegaly. The dopamine agonists bromocriptine and cabergoline have been used as either a primary or adjuvant therapy for acromegaly [248]. The high dose requirement usually precludes routine use. Up to 20 mg/day bromocriptine is required to suppress GH in these patients, a dose higher than required to suppress PRL in patients harboring prolactinomas [249]. Side effects of bromocriptine, especially with the high doses required, include gastrointestinal upset, transient nausea and vomiting, headache, transient postural hypotension with dizziness, nasal stuffiness and, rarely, cold-induced peripheral vasospasm. In a meta-analysis, cabergoline, a long-acting dopamine agonist, given alone or in combination with an SRL, was reported to suppress GH to ,2 ng/mL, and normalize IGF-1 in up to a third of patients, especially in those with lower baseline IGF-1 levels [250]. In another study of 198 acromegaly patients receiving dopamine agonists, GH and IGF levels were reduced by B30%, and this effect appeared to correlate with

400 μg SC every 8 h 120 mg deep SC every 4 weeks 40 mg IM every 4 weeks 60 mg IM every 4 weeks

prior radiotherapy, but not with pretreatment prolactin levels [248]. Cabergoline administered in combination with SRLs improves biochemical outcomes in resistant patients [251]. Although cabergoline monotherapy did not alter IGF-1 levels in 24 patients studied for 18 weeks, subsequent addition of 10 mg daily pegvisomant achieved IGF-1 normalization in 68% of patients [252]. Side effects of cabergoline include gastrointestinal symptoms, dizziness, headache, and mood disorders. High doses of cabergoline used in patients with Parkinson disease have been associated with cardiac valvular dysfunctions [253]. Somatostatin Receptor Ligands Endogenous SRIF inhibits pituitary GH secretion, attenuates insulin secretion, and regulates multiple gastrointestinal secretions and functions [130]. SRIF action is mediated by five receptor subtypes expressed in a cell- and tissue-specific pattern that confers both functional and therapeutic ligand specificity [254,255]. The SSTR2 and SSTR5 subtypes are preferentially expressed on somatotroph and thyrotroph cell surfaces and mediate GH, TSH, and ACTH secretion by suppressing intracellular cAMP levels [256 259]. As most GH-secreting adenomas abundantly express SSTR2 and SSTR5, SRLs have been successfully employed for treating acromegaly. Both octreotide and lanreotide preparations exhibit selective affinity for SSTR2 and SSTR5 and have proven safe and effective for long-term acromegaly treatment. Octreotide, an octapeptide SRIF analogue [260,261], inhibits GH secretion with a potency 45 times greater than native SRIF, while its potency for inhibiting insulin release is only 1.3-fold that of SRIF. Because of its relative resistance to enzymatic degradation, the in vivo half-life of the analogue is prolonged (up to 2 hours) after subcutaneous injection [262]. Lantreotide is an 8-amino-acid cyclic peptide [263], and drug responsiveness correlates with GH-secreting adenoma SSTR2 expression [264]. Rebound GH hypersecretion seen following SRIF infusion does not

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FIGURE 15.18 Receptor targets for treatment of acromegaly. Pituitary somatostatin receptor subtypes and D2 receptors and peripheral GH receptors are targets for therapeutic ligands. Clinically approved and investigational drugs with ligand affinities for human somatostatin receptors are dually selective (octreotide and lanreotide), panselective (pasireotide), or monoselective, or are chimeric for the D2 dopamine receptors. SRL suppresses levels of both GH and IGF-1, constrains tumor growth, and inhibits hepatic GH-receptor binding and action. GHreceptor antagonists prevent GH-receptor signaling, which attenuates peripheral IGF-1 levels. ALS, acid-labile subunit; IGFBP-3, insulin-like growth factor-binding protein 3; IRS, insulin receptor substrate; JAK2, Janus kinase 2; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3 kinase; PL-C, phospholipase C; PTP, protein tyrosine phosphatase; STAT, signal transducers and activators of transcription. Source: From Melmed S. Medical progress: acromegaly. N Engl J Med 2006;355(24):2558 73.

occur after octreotide or lanreotide administration. These pharmacologic differences provide unique advantages for using SRLs in long-term acromegaly therapy [265,266]. Pasireotide exhibits a distinctive

receptor-binding profile, with a preferential high affinity to SSTR5, 39-fold higher than the octreotide ligand, and also binds to SSTR1, SSTR2, and SSTR3 (Table 15.15). When 358 patients were randomized to

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FIGURE 15.19 Effect of octreotide on hourly growth hormone levels in acromegaly. Mean percentage changes (6SE of basal values) of serum growth hormone (GH) concentrations in patients with acromegaly treated with 100 μg octreotide subcutaneously every 8 h (n 5 52). Blood was sampled before an injection and every hour for 8 subsequent hours before treatment (“baseline”), at the end of weeks 2 and 4 of treatment, and 4 weeks after discontinuation of treatment (“washout”). Octreotide was administered just after the 0-h sampling. Source: From Ezzat S, Snyder PJ, Young WF, et al. Octreotide treatment of acromegaly. A randomized, multicenter study. Ann Intern Med 1992;117(9):711 8.

FIGURE 15.20 Meta-analysis of SRL responsiveness in acromegaly. Source: From Carmichael JD, Bonert VS, Nuno M, Ly D, Melmed S. Acromegaly clinical trial methodology impact on reported biochemical efficacy rates of somatostatin receptor ligand treatments: a meta-analysis. J Clin Endocrinol Metab 2014;99(5):1825 33.

receive monthly 40 mg pasireotide LAR or 20 mg octreotide LAR, biochemical control was achieved after 12 months in 31% versus 19% of subjects, respectively [267]. In a subsequent trial, pasireotide efficacy was tested in patients resistant to maximal doses of other SRLs; 15% and 20% of these resistant patients were controlled on 40 mg and 60 mg pasireotide, respectively [268]. Effects of SRLs on biochemical control. A single subcutaneous administration of 50 or 100 μg subcutaneous octreotide suppresses both basal and stimulated GH secretion for up to 5 hours [261]. In a double-blind,

placebo-controlled trial subcutaneous octreotide administered as 8-hourly injections significantly attenuated GH and IGF-1 levels in over 90% of patients [269] (Fig. 15.19) and the medication normalizes IGF-1 levels in about 70% of patients. In patients with GHsecreting microadenomas, control of integrated GH and pooled IGF-1 levels is far more favorable than in patients with larger tumors [269]. Overall, a global meta-analysis showed 55% control rates for both GH and IGF-1 (Fig. 15.20). Later year of publication, study duration, and prior SRL use are important determinants of efficacy [222].

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1200

42

900 8 600

6 4

GH